Lecture No. 1 (Unit I)
Scope of Classical Biological Control
Biological Control
Definition : “The study and utilization of Parasitoids, predators and pathogens for the
regulation of pest population densities”.
Biological control is often shortened to biocontrol. In biocontrol, pest population is
reduced to a level, that no longer of economic or health concern, yet leaves sufficient pest to
allow survival of the control organisms. The biocontrol organism maintain its own
population and prevents the pest from reaching to damaging level.
History of Biological Control
In China use of ant, Monomorium Pharaonis against pests :

First use of insect predators was in 900 A.D. when Chinese citrus growers used red
ants (Oecophylla smaragdina) on citrus trees to control citrus leaf chewing insects.

Aldroyandi an Italian first noted the hymenopteran parasite in 1602. Apanteles
glomeratus laying eggs on pupae of cabbage butterfly, Pieres brassicae.

Earliest introduction of natural enemy dates back to 1762, when Indian Mynah
bird, Gracula religiosa was exported from India to Mauritius to control red locust,
Nomadacris septemfasciata.

First success of insect control on large scale was in 1888, when cottony cushion
scale lcerya purchasi a pest on citrus was controlled in California by use of ladybird
beetle predator, Rodolia cardinalis.

Rodolia cardinalis was introduced into India in 1929 in Tamil Nadu for control of
cottony cushion scale, lcerya purchasi.

Aphelinus mali (Aphelinidae) was introduced in 1937 from North America into
Coonor (TN) forcontrol of apple wooly aphid, Eriosoma lanigerum.

Cryptolaemus montrouzieri (Coccinellidae) was introduced in 1898 from Australia
into south India for control of citrus mealy bug.
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
1

In 1960, a Tachinid, Spogossia bezziana was introduced from Srilanka into India for
control of coconut black headed caterpillar, Opisina arenosella.
Introduction of exotic species with hopes of controlling pest species
Pest – Cotton cushion scale, lcerya purchesi
Predator – Vedalia beetle, Rodolia cardinalis
Conservation consists of conserving existing agents by creating refugia that are protected
from pesticides and provide an alternative food source to its host. Eg.: Alfalfa provides a
reservoir for predators and parasitoids of key pests.
Augmentation is the process by which mass-reared agents are released to augment
existing populations in the field or for reintroduction into habitats where natural enemies
have been killed
Inundation is the process by which mass-reared agents are released to inundate the pest
population, to control pest populations within the first generation of release. Eg. :
Greenhouses.
Biological Control
Biological control is a component of an integrated pest management strategy. It is
defined as the reduction of pest populations by natural enemies and typically involves an
active human role keep in mind that all insect species are also suppressed by naturally
occurring organisms and environmental factors, with no human input. This is frequently
referred to as natural control. Natural enemies of insect pests, also known as biological
control agents, include predators, parasitoids, and pathogens. Biological control of weeds
includes insects and pathogens. Biological control agents of plant diseases are most often
referred to as antagonists.
Left : A wasp parasitoid of stink bug eggs. J.K. Clark, University of California Statewide IPM Project
Center : Hippodamia glacialls, a predator of aphids. J. Ogrodnick
Right : Aphid killed by fungus. J.Ogradnick
Predators, such as lady beetles and lacewings, are mainly free-living species that
consume a large number of prey during their lifetime. Parasitoids are species whose
immature stage develops on or within a single insect host, ultimately killing the host. Many
species of wasps and some flies are parasitoids. Pathogens are disease-causing organisms
2
including bacteria, fungi, and viruses. They kill or debilitate their host and are relatively
specific to certain insect groups.
The behaviors and life cycles of natural enemies can be relatively simple or
extraordinarily complex, and not all natural enemies of insects are beneficial to crop
production. For example, hyperparasitoids are parasitoids of other parasitoids. In potatoes
grown in Maine, 22 parasitoids of aphids were identified; yet these were attacked by 18
additional species of hyperparasitoids.
A successful natural enemy should have a high reproductive rate, good searching
ability, host specificity, be adaptable to different environmental conditions, and be
synchronized with its host (pest).
A high reproductive rate is important so that populations of the natural enemy can
rapidly increase when hosts are available. The natural enemy must be effective at searching
for its host and it should be searching for only one or a few host species. Spiders, for
example, feed on many different hosts including other natural enemies. It is also very
important that the natural enemy occur at the same time as its host. For example, if the
natural enemy is an egg parasitoid, it must be present when host eggs are available. No
natural enemy has all these attributes, but those with several characteristics will be more
important in helping maintain pest populations.
There are three broad and somewhat overlapping types of biological control;
conservation, classical biological control (introduction of natural enemies to a new locale),
and augmentation.
Conservation
The conservation of natural enemies is probably the most important and readily
available biological control practice available to growers. Natural enemies occur in all
production systems, from the backyard garden to the commercial field. They are adapted to
the local environment and to the target pest, and their conservation is generally simple and
cost-effective. Lacewings, lady beetles, hover fly larvae, and parasitized aphid mummies are
almost always present in aphid colonies. Fungus-infected adult flies are often common
following periods of high humidity. These natural controls are important and need to be
conserved and considered when making pest management decisions. In many instances the
importance of natural enemies has not been adequately studied or does not become
apparent until insecticide use is stopped or reduced. Often the best we can do is to
recognize that these factors are present and minimize negative impacts on them. If an
insecticide is needed, every effort should be made to use a selective material in a selective
manner.
3
Examples of classical bioligical control :
Left : An egg parasitoid introduced from Europe for biological control of southern green stink bug.
J.K. Clark, Uniersity of California Statewide IPM Project
Center : A european weevil improted to attack purple loosestrife. B.Blossey
Right : A successfully introduced lady beetle. J. Ogrodnick
Photo Classical biological control
In many instances the complex of natural enemies associated with an insect pest
may be inadequate. This is especially evident when an insect pest is accidentally introduced
into a new geographic area without its associated natural enemies. These introduced pests
are referred to as exotics and comprise about 40% of the insect pests in the United States.
Examples of introduced vegetable pests include the European corn borer, one of the most
destructive insects in North America. To obtain the needed natural enemies, we turn to
classical biological control. This is the practice of importing, and releasing for
establishment, natural enemies to control an introduced (exotic) pest, although it is also
practiced against native insect pests. The first step in the process is to determine the origin
of the introduced pest and then collect appropriate natural enemies (from that location or
similar locations) associated with the pest or closely related species. The natural enemy is
then passed through a rigorous quarantine process, to ensure that no unwanted organisms
(such as hyperparasitoids) are introduced, then reared, ideally in large numbers, and
released. Follow-up studies are conducted to determine if the natural enemy successfully
established at the site of release, and to assess the long-term benefit of its presence.
There are many examples of successful classical biological control programs. One of
the earliest successes was with the cottony cushion scale, a pest that was devastating the
California citrus industry in the late 1800s. A predatory insect, the vedalia beetle, and a
parasitoid fly were introduced from Australia. Within a few years the cottony cushion
scale was completely controlled by these introduced natural enemies. Damage from the
alfalfa weevil, a serious introduced pest of forage, was substantially reduced by the
introduction of several natural enemies. About 20 years after their introduction, the alfalfa
acreage treated for alfalfa weevil in the northeastern United States was reduced by 75
percent. A small wasp, Trichogramma ostriniae, introduced from China to help control
4
the European corn borer, is a recent example of a long history of classical biological
control efforts for this major pest. Many classical biological control programs for insect
pests and weeds are under way across the United States and Canada.
Recent example of classical biological control is papaya mealy bug Paracoccus
Marginatus by parasite spalgis epius from Pune, Maharasthtra in July – Aug 2010.
Classical biological control is long lasting and inexpensive. Other than the initial
costs of collection, importation, and rearing, little expense is incurred. When a natural
enemy is successfully established it rarely requires additional input and it continues to kill
the pest with no direct help from humans and at no cost. Unfortunately, classical biological
control does not always work. It is usually most effective against exotic pests and less so
against native insect pests. The reasons for failure are often not known, but may include
the release of too few individuals, poor adaptation of the natural enemy to environmental
conditions at the release location, and lack of synchrony between the life cycle of the
natural enemy and host pest.
Purchase and Release of Natural Enemies
Many commercial insectaries rear and market a variety of natural enemies including
predaceous mites, lady beetles, lacewings, praying mantids, and several species of
parasitoids. Success with such releases requires appropriate timing (the host must be
present or the natural enemy will simply die or leave the area) and release of the correct
number of natural enemies per unit area (release rate). In many cases, the most effective
release rate has not been identified as it will vary depending on crop type and target host
density. Success also requires a healthy and robust natural enemy.
Figs. A) Baculovirus particles, or polyhedra; B) Cross-section of a polyhedron, C) Diagram of polyhedron
cross-section. Electron micrographs (A and B) by Jean Adams, graphic (C) by V.D’Amico.
5
Baculoviruses (Baculoviridae)
Baculoviruses are pathogens that attack insects and other arthropods. Like some
human viruses, they are usually extremely small (less than a thousandth of a millimeter
across), and are composed primarily of double-stranded DNA that codes for genes needed
for virus establishment and reproduction. Because this genetic material is easily destroyed
by exposure to sunlight or by conditions in the host’s gut, an infective baculovirus particle
(virion) is protected by protein cost called a polyhedron (plural polyhedra : see Figs. A, B,
and C). Most insect baculoviruses must be eaten by the host to produce an infection, which
is typically fatal to the insect.
The majority of baculoviruses used as biological control agents are in the genus
Nucleopholyhedrovirus,
so
“baculvoirus”
or
“virus”
will
hereafter
refer
to
nucleopolyhedroviruses. These viruses are excellent candidates for species-specific, narrow
spectrum insecticidal applications. They have been shown to have no negative impacts on
plants, mammals, birds, fish, or even on non-target insects. This is especially desirable
when beneficial insects are being conserved to aid in an overall IPM program, or when an
ecologically sensitive area is being treated. The USDA Forest Service currently uses the
gypsy moth nuclear polyhedrosis virus (LdNPV) to aerially spray thousands of acres of
forest each year. This product, registered as Gypchek, is effective against gypsy moths but
leaves all other animals unharmed (Reardon et al. 1996).
On the other hand, the high specificity of baculoviruses is also cited as a weakness
for agricultural uses, since growers may want one product to use against a variety of pests.
Currently, researchers are attempting to use genetic engineering techniques to expand
virus host ranges to the desired pest species. Releases of such genetically-engineered
baculoviruses have been made by researchers in the U.K. and the United States and show
promise, although the cost of commercial production of these agents must be reduced if
they are to be competitive. Compaines like Dupont, biosys, American Cyanamid, and
Agrivirion (to name a few) have continued to explore the expansion and development of
agricultural-use viral insecticides. Recently, biosys has released two baculovirus-based
products, Spod-X for beet armyworm and Gemstar LC for tobacco budworm and cotton
bollworm.
Life Cycle
Viruses are unable to reproduce without a host they are obligate parasites.
Baculovirses are no exception. The cells of the host’s body are taken over by the genetic
message carried within each virion (Fig. C), and forced to produce more virus particles until
the cell, and ultimately the insect, dies. Most baculoviruses cause the host insect to die in a
way that will maximize the chance that other insects will come in contact with the virus
and become infected in turn (above and Fig. D).
6
As seen in the animation on the right,
infection by baculovirus begins when an insect eats
virus particles on a plant – perhaps from a sprayed
treatment. The infected insect dies and “melts” or
falls apart on foliage, releasing more virus. This
additional infective material can infect more insects,
continuing the cycle.
Fig. D Representative baculovirus life cycle. Graphic by
V.D’Amico.
Relative Effectiveness
It is widely acknowledged that baculoviruses can be as effective as chemical
pesticides in controlling specific insect pests. However, the expense of treating a hectare of
land with a baculovirus product invariably costs more than an equally efficacious chemical
treatment. This difference in price is due primarily to the labor intensive nature of
baculvirus production. Some viruses can be produced in vitro (within cell cultures in the
laboratory, not requiring whole, living insects). These are less expensive than those that can
only be produced in vivo, that is, inside of living insects. The cost of rearing live hosts adds
greatly to the final cost of the product. It is to be hoped that insect cell culture systems
currently being developed for other uses may ultimately make viral pesticides more costeffective.
Appearance
Insects killed by baculoviruses have a characteristic
shiny-oily appearance, and are often seen hanging limply from
vegetation (Fig. E). They are extremely fragile to the touch,
rupturing to release fluid filled with infective virus particles. This
tendency to remain attached to foliage and then rupture is an
important aspect of the virus life-cycle. Infection of other insects
will only occur if they eat foliage that has been contaminated by
virus-killed larvae.
Fig. E Virus-killed caterpillar.
Photo by Roger T. Zerillo.
Animation by Vince D’ Amico
It is interesting to note that most baculoviruses, unlike many other viruses, can be
seen with a light microscope. The polyhedra of many viruses look like clear, irregular
7
crystals of salt or sand when viewed at 400x or 1000 x. The fluid inside a dead insect is
composed largely of virus polyhedra – many billions are produced inside of one cadaver.
Habitat
Baculoviruses can be found wherever insects exist. Because rain and wind readily
carry baculoviruses from place to place, it is likely that every piece of land and body of
water contains some virus particles. It is widely accepted by researchers that most produce
currently on the shelves is “contaminated” by baculovirus particles (Heimpel et al., 1973).
In fact, the pervasiveness of baculovirus particles, along with the results of tests performed
in conjunction with registration, may be considered both indirect and direct evidence for
the safety of these agents.
Pests Attacked
Like most viruses, baculoviruses tend to be species or genus specific, although there
are some exceptions to this rule, notably the Autographa californica nuclear polyhedrosis
virus. Much of the genetics work currently being done to improve baculvirus-based
pesticides is concentrated in the area of the virus genome controlling its host range.
CURRENT USE OF BACULOVIRUSES AS BIOLOGICAL INSECTICIDES
Below is a list of currently registered baculvirus products as of October 1997.
TABLE 1
Commodity
Apple, pear, walnut
Insect pest
Codling moth
and plum
Virus used
Virus product
Codling moth
Cyd-X
granulosis virus
(3)
Cabbage, tomatoes,
Cabbage moth,
Cabbage army worm
Mamestrin*
cotton, (and see
American bollworm,
nuclear polyhedrosis
(5)
pests in next column
diamondback moth,
virus
potato tuber moth, and
grape berry moth
Cotton, corn,
Spodoptera littoralis
tomatoes
Spodoptera littoralis
Spodopterin*
nuclear polyhedrosis
(5)
virus
Cotton, and
Tobacco budworm
Helicoverpa zeae
Gemstar LC,
vegetables
Helicoverpa zea, and
nuclear polyhedrosis
Biotrol, Elcar
Cotton bollworm
virus
(3)
Heliothis virescens
Vegetable crops,
Beet armyworm
Spodoptera exigua
Spod-X
greenhouse flowers
(Spodoptera exigua)
nuclear polyhedrosis
(3)
virus
8
Vegetables
Celery looper
Anagrapha falcifera
(Anagrapha falcifera)
nuclear polyhedrosis
none at present
virus
Alfalfa and other
Alfalfa looper
Autographa
Gusano
crops
(Autographa californica)
californica nuclear
Biological
polyhedrosis virus
Pesticide (3)
Forest Habitat,
Douglas fir tussock
Orgyia
TM Biocontrol
Lumber
moth
psuedotsugata
(2)
(Orgyia psuedotsugata)
nuclear polyhedosis
virus
Forest Habitat,
Gypsy moth
Lymantria dispar
Gypchek
Lumber
(Lymantria dispar)
nuclear polyhedrosis
(1)
virus
*These products are not produced or registered in the U.S. at the present time.
Pesticide Compatibility
Viruses particles per se are generally unaffected by pesticides, although some
chlorine compounds should be expected to damage or destroy viruses if applied at the same
time. Baculovirus efficacy, however, can be altered in many ways by the effects of chemical
pesticides on the host insect. A review by Jacques and Morris (1981) showed that of 10
pesticide-virus combinations, 9 resulted in an additive effect on insect mortality. However,
some of the pesticides included in that review have since been banned, and this study is of
limited use at present. More work is needed to explore the effectiveness of insecticide
“cocktails’ consisting of enviromentally friendly chemical agents and baculoviruses.
6. Steps involved in classical biological control

Conservation and encouragement of indigenous natural enemies

Importation of exotic natural enemies

Augmentation (mass rearing and release)
Biological control methods
1. Conservation of bioagents :
Sometimes it happens that the indigenous or exotic natural enemies feed and
reproduce on a noxious plant host yet do not provide effective biological control. The
conservation approach employs environmental manipulation to enhance the effect of
existing native or exotic control organisms. For example, DDT has been used selectively to
eliminate Exochomus sp., a coccinellid beetle predator that restricted the potential of an
introduced cochineal insect, Dactylopius opuntiae (Cockerell), to control the prickly pear
cacti, Opuntia ficus indica (L.) Miller and O. tardispina Griffiths in South Africa Reducing
9
the coccinellid predators allowed the cochineal insects to increase in number and the cacti
were effectively controlled (Moran and Zimmerman 1984).
2. Augmentation of bioagents
Inundative releases of a natural enemy may increases its abundance and time its
impact against a noxious plant. The impact of the moth, Bactra verutana Zeller could be
enhanced by supplementing existing field populations with large numbers of this inset
against purple nutsedge, Cyperus rotundus L. in cotton Although experiental control was
demonstrated, the method proved too expensive to be practical. (Frick and Chandler, 1978).
3. Introduction of bioagents (Classical biological control)
Naturalized weeds often have some host specific natural enemies which effectively
regulate their abundance. More species of natural enemies may be imported from the
plant’s native range and introduced to the problem areas. This approach is common
worldwide, and has led to the introduction of numerous plant-feeding insects and mites,
and recently, plant pathogens and nematodes (Julien, 1987).
Finding
and
introducing
phytophagous
organisms
requires
thorough
pre-
introduction studies to assure that control can be achieved and that economically and
ecologically important plants will not be adversely affected. The steps as mentioned by
Goeden and Andres (1999) in a classical biological control programme are : i) project
selection, ii) search for natural enemies, iii) host range and biological studies, iv) summary
of host range studies, v) importation and release, and vi) evaluation. These steps can be
broadly grouped into three following parts :
There are different ways in which a biological control agent can be used;
namely introduction, inoculation, inundation, augmentation and conservation.
Introduction : When a pest organism is introduced from another country, it is
usually not accompanied by its natural enemies. Classical biological control involves the
visiting of the country of origin to obtain specimens of natural enemies, which, after
screening is introduced into the country to ‘control’ the new pests and to maintain the
reduction so that pest is no longer causing the damage. It normally aims at permanent
solution of the problem.
Inoculation involves the seasonal release of the control agent. The method is useful
when the introduction method fails due to control agent dying out after reduction in pest
numbers. The aim is to establish control over a shorter period of time than in case of
introduction, such as seasonal requirements e.g. against glass house pests such as redspider mite and whitefly.
Inundation. Involves the mass release of the control agent to control a particular
pest outbreak at a particular time. It is once and for all treatments. There is no possibility
10
of establishment of control agent, the function is like a pesticide. This method is most
commonly used with viruses and bacteria e.g. Bacillus thuringensis.
Augmentation. Under certain conditions natural control agents, although present,
achieve control too late in the growing season, because of slow population build up.
Therefore, by adding other control agents early in the season control is achieved in time to
prevent pest populations which are maintained at low level until the natural enemy
populations are sufficient to take overcontrol.
Conservation – involves the main manipulation of the agro ecosystem to make it
more beneficial to control agents, and enable them to perform better e.g. reduce the use of
pesticides to the minimum.
Pre-introduction studies :
The process of conducting a weed biological control project has a number of discrete
steps that must be followed if a project is to succeed. The project is initiated by learning
what is known from the literature about the weed pest, and if it has any known natural
enemies. The target species is confirmed with the regulatory authorities. This is to resolve
conflicts of interests like ecological, economical and aesthetic and ensure there are no
compelling reasons to avoid such a project.
Introduction and host specificity tests
The natural enemies are imported in India through Project Directorate of biological
Control, Bangalore after taking permission form Plant Protection Advisor to Government of
India. Host specificity tests are conducted in the quarantine laboratory in Indian Institute
of Horticulture Research, Bangalore to determine if introduced organisms are likely to
become a pest in their own right.
Host specificity means that the agent can feed on, infect or parasitize only one
species of host. To be effective, a weed agent must accept the weed as food, develop on it,
and sustain populations over several generations. To be safe, the agent must not cause
significant damage to non-target species.
It is absolutely crucial to know the biology of the weed agent, and the biology of the
weed itself. The testing starts with the target species (even several populations of the same
species), then the closest related species (of the same genus), then related genera of plants
(related to the target), the move toward more distantly related species in related plant
families. Then plants that may be less related, but occur in the same habitat, are tested.
And finally, we must test agronomic plant species (maize, paddy, wheat cotton etc.) to
ensure there is no potential effect on those non-targets. The result of the testing will
provide the basis for making a decision as to how safe the introduced natural enemy will
be. Three necessary actions by the natural energy must occur for a non-target species to be
affected at the population level. i) Adultsmust feed on the plants, ii) the females must lay
11
eggs on the plants, and iii) the larvae must be able to complete development on the plant. If
one or two of those steps do not occur, the natural enemy cannot harm that plant species
at the population level. While an individual plant may be fed on, the overall population will
not be at risk.
Two kinds of tests exist for herbivores i.e. choice and no-choice tests. A no-choice
test means the agent is placed with the test plant and kept with it until the agent feeds (or
oviposits) or it dies. Negative feeding in a no-choice test is very convincing but the agent
may have fed or oviposited only because there was no choice. In the real would, no-choice
situations do not occur. So, non-feeding is convincing, whereas feeding means further
(choice) testing. No-choice is especially important in case of those bioagents which are
dispersed by wind and land on any plant species (e.g. mites and fungi).
Choice tests are more realistic and are similar to the real world. In this case, the
agent is exposed to several species of hosts i.e. the target species as well as non-targets, all
at once. The agent has a choice to make. If it feeds on non-target plants then these plants
can be in the potential host range of the biological control agent. However, the agent must
feed on the plant, lay eggs on the plant, and the offspring must be able to feed on and
complete development on the plant, for it to be at risk. Further, the non-target plant must
occur in the same area and at the same time to be at risk. For some agents, that have very
finite periods of emergence and development or short periods when they are active there is
reduced risk. Other agents may live and be active for an entire season (or multiple years),
hence would offer different risks. The point of conducting rational host-specificity testing is
to avoid risks when possible and manage those risks wisely. Biological control of weeds has
a very good track record on safety.
Releases, monitoring and evaluation
Permission is taken from Plant Protection Advisor to Government of India or field
releases if the host specificity tests reveal potential biocontrol agents. The bioagent is
reared in large numbers and released in the field. Release site should be most favourable to
the natural enemy. Release sites should have less interference from grazing animals, farm
implements and other weed control operations in order to enhance chances for
establishment especially if the bioagent has a long life cycle.
Often forgotten or overlooked, but absolutely critical, is the evaluation of a release,
monitoring the site for changes as a result of the release, and the subsequent redistribution
of the agent to other sites. Observations are recorded on the population buildup, dispersal
from release area, adaptability to local climatic conditions and effect of local natural
enemies. Evaluations are done on success of establishment, field reproduction and damage
inflicted against the target plant.
12
Biological control work in India
Biological control work began rather by accident. The earliest record of the biological
control of a noxious plant involved the intentional introduction of the cochineal insect,
Dactylopius ceylonicus (Green) to northern India from Brazil in 1795 in the false belief
that it was. D. coccus Costa, a species cultured commercially as a source of carmine dye.
Instead of reproducing well on the cultivated, spineless prickly pear cactus, Opuntia ficus
indica (L.) Miller, D. ceylonicus readily transferred to its natural host plant, O. vulgaris
Miller, that had become widespread in India when it escaped cultivation in the absence of
its South American natural enemies. Once the value of D. ceylonicus as a biological
control agent was recognized, it was introduced in 1836 – 1838 to southern India, where it
brought about the first successful, intentional use of an insect to control a noxious plant.
Shortly before 1865, D. ceylonicus also was transferred from India to Sri Lanka which
resulted in the successful control of O. vulgaris throughout the Island (Moran and
Zimmerman 1984).
Planned and organised biological control work was started after the inception of the
All India Coordinated Research Project on Biological Control of Crop Pests and Weeds in
1977. Successes were achieved in biological control of aquatic weeds like water hyacinth by
weevil, Neochetina eichhorniae Warner and N. bruchi Hustachi and leaf mining mite,
Orthogalumna terebrantis Wallwork and water fern weed by weevil, Cyrtobagous
salviniae Calder and Sands (Joy et al., 1985a; Jayanth, 1987a; Jayanth, 1988).
Among the terrestrial weeds, good progress has been made for the control of
Chromoleana odorata by releasing Pareuchaetes pseudoinsulata Rego Barros (Joy et
al., 1985b). Parthemium weed, Parthenium hysterophorus L. in an important alien weed
in India which was first observed in Pune in 1955 (Rao, 1956) and since then it has spread
throughout the country infesting about 5 million hectares of pastures, waste lands and
agricultural fields (Gidwani, 1975).
Several species of insects have been reported to feed on parthenium plant in India.
Most of these insects are polyphagous and attack the weed in the absence of other hosts in
the off season.
In the weed’s native range in North America 144 insect species feed on the weed at
some stage of their life cycle (McClay et al., 1995). The order of their abundance was
Coleoptera (33.2%) > Homoptera (22.9%) > Lepidoptera (20.2%) > Hemiptera (18.3%).
Owing to lack of effective indigenous insects, efforts were initiated by the Indian
Institute of Horticulatural Research, Bangalore, to try biological control of the weed by
introducing the leaf-feeding beetle, Zygogramma bicolorata Pallister (Coleoptera :
Chrysomelidae), the flower-feeding weevil, Smicronyx lutulentus Dietz (Coleoptera :
Curculionidae) and the stem-boring moth, Epiblema strenuana (Walker) (Lepidoptera :
13
Tortricidae). The cultures of these insects were imported from the Mexican sub-station of
the Commonwealth Institute of Biological Control in 1983. The culture of S. lutulentus
could not be initiated as only dead insects were received in the shipment where as the
culture of E. strenuana was terminated because it was found capable of multiplying on an
important oilseed plant, Guizotia abyssinica L., in the quarantine studies (Jayanth,
1987b). Detailed host specificity tests of Z. bicolorata in Mexico (McClay, 1980), Australia
(McFadyen, 1980) and India (Jayanth and Nagarkatti, 1987) revealed that this isnect is
incapable of attacking any cultivated plant.
Field releases of Z. bicolorata were thus initiated in Bangalore in 1984 (Jayanth,
1987a). The insect established readily and become widespread in and around Bangalore,
causing large-scale defoliation of the weed indicating its potential to permanently reduce
weed density (Jayanth, 1991). Dhileepan et al. (2000) also observed Z. bicolorata as an
efficient biocontrol agent in Australia that can have significant negative impact on the
growth and reproduction of parthenium. Thus, inoculative releases of Z. bicolorata (100
parts of adults/acre) may be an important component of integrated parthenium weed
management (IPWM).
The beetle was involved in a controversy when adults of Z. bicolorata were
observed to feed on tender leaves of sunflower, Helianthus annuus L., in Kolar district in
Karnataka State. Jayanth et
al. (1993)
conducted laboratory
studies
to
isolate
phagostimulants from parthenium weed and concluded that parthenium pollen, which
contains parthenin, on sunflower leaves growing in close proximity to the weed stand, could
have induced feeding by Z. bicolorata on sunflower leaves. Based on their studies and
other information available in the literature they concluded that members of genus.
Zygogramma exhibit host-specificity and, therefore, chances of Z. bicolorata becoming a
pest of sunflower appear very remote.
The adults and larvae of Z. bicolorata feed on parthenium leaves. Females lay eggs
singly or in groups of up to 5, mostly on the undersurface of the leaves. The eggs hatch in 4
– 6 days. The early stage larvae feed on the terminal and axillary buds and move on to the
leaf blades as they grow. The full grown larvae enter the soil and pupate. The larval and
pupal periods range from 10 – 14 days and 8 – 12 days, respectively, under laboratory
conditions. The adults live for 3 – 4 months. The females lay up to 1786 eggs (mean
836.13). The insect remains active during rainy season (July to October). It undergoes
diapauses within the soil during the dry months of the year (November to May) and emerges
with the onset of heavy rains. The insect completes four generations in a year (Jayanth,
1987a).
The management of parthenium weed through use of insects is challenging because
of its regeneration capacity, large seed production ability (25,000 seeds/plant), germination
14
ability throughout the year and extreme adaptability of parthenium in a wide range of
ecosystem. Balyan et al. (1997) suspected the usefulness of the biocontrol agents due to
variable climatic conditions in India and difficulty in multiplying and producing huge
quantity of bioagents under laboratory conditions besides restriction of their host
specificity. The beetle Z. biocolorata multiply in large numbers only during rainy season
(July to October) whereas parthenium can germinate throughout the year. Long dry periods
reduce the insect population to very low levels thus giving inadequate control of the weed
which germinates after rain. Z. bicolorata grubs and adults feed on the leaves causing
indirect damage to the plant and low insect population can not prevent plant from
producing flowers. Thus, more searches need to be conducted in the native of the weed to
find insects which are active during most part of the year. The insects feeding on roots,
stem and flowers of the plant should be preferred over leaf-feeding insects.
Attempts of Classical Biological Control
In India, several attempts have been made to introduce exotic natural enemies for
classical biological control of sugarcane pests. However, none of these parasites or
predators have got permanently established and / or successfully controlled sugarcane
pests in India. This may probably be due to the fact that most of the sugarcane pests are
native to the Indian subcontinent and finding suitable exotic species for the control may be
difficult. Moreover, most of the natural enemies introduced in eariler years were those
which were readily available with little or no cost or effort. Furthermore, the number of
individuals released and frequency of release was generally low and limited to one or two
areas in the country. Planned introductions and repeated releases of a large number of
individuals of each species may enhance the scope of establishment of exotic natural
enemies.
For protection of natural enemies crop management and pest management practices
to be followed fall into three categories :
1) Developing appropriate crop management practices e.g. strip cropping, poly cultures and
alternate harvesting.
2) Providing required resources for natural enemies e.g. habitat provision and management,
host provision and provision of non-host foods.
3) Reducing interference with or mortality of natural enemies e.g. reducing the enemies of
natural enemies, reducing the negative impacts of cultural practices, host plant effects and
reducing pesticidal impacts.
The periodic release of natural enemies :
Natural enemies do not always occur in adequate numbers to provide satisfactory
pest control because of both natural environmental factors and agricultural practices. One
15
possible control method is to increase the natural enemy population by periodically
releasing additional natural cnemies. These may be the same species that exist at the site3
or they may be different species or strains. This method of biological control is called
augmentation of natural enemies. Many companies produce insect pathogens as microbial
insecticides, and other companies, called insectaries, mass-produce predatory and parasitic
insects and mites.
The two general approaches to augmentation are inundation and inoculation
inundation involves releasing large numbers of natural enemies for immediate reduction of
a damaging or near-damaging pest population. It is analogous to a corrective insecticide
application; the expected outcome is immediate pest control. The use of microbial
insecticides (such as those containing Bacillus thuringiensis) is a common inundative
release of a natural enemy. The inundative release of predaceous and parasitic insects is
recommended only in certain situations because of the expense and the nature of their
activity. One parasite that has been effective in mass releases is Trichogramma for
controlling the eggs of various moths.
Inoculation involves releasing small numbers of natural enemies at prescribed
intervals throughout the pest period, starting when the pest population is low. The natural
enemies are expected to reproduce themselves to provide more long-term control. The goal
of inoculative releases is to keep the pest at low numbers, never allowing it to approach an
economic injury level.
Augmentative natural enemy releases are neither low-input nor sustainable. They
require a relatively high input of time, labor, and money and usually must be repeated at
least annually and often several times per growing season. However, when compared to the
use of broad-spectrum insecticides, augmentative biological control has certain advantages,
such as reduced hazard to people and the environment and reduced harm to other
beneficial organisms. If you are considering purchasing and releasing natural enemies, you
must evaluate both the constraints and the benefits to determine if, augmentative biological
control is appropriate for your needs.
Examples of successful biological control in India
1) Control of apple woolly aphid Eriosoma lanigerum in Assam using parasitoid
Aphelinus mali fom England (1920).
2) Control of Cottony cushion scale Icerya purchasi of wattle tree in Nilgiris and Kodaikanal
using predator Vedalia beetle Rodolia cardinalis from USA (1929-1931).
3) Control of Apple San jose scale Quadraspidiotus perniciosus using parasitoid
Prospatella perniciosi from China (1958 – 1960) and another parasitoid Aphytis diaspidis
from USA (1960).
16
4) Control of castor semilooper Achaea janata using parasitoid Telenomus spp. from
New Guinea (1964).
5) Control of coconut rhinoceus beetle Oryctes rhinoceros using a predator Platymeris
laevicollis from Zanzibar (1965).
Successful classical examples of Biological control :
In all total 327 successful examples in pest control and 58 in weed control have
been achieved in the world due to use of parasites, predators and pathogens.
The cottony cushion scale (Icerya purchasi) a serious pest of citrus was introduced
in California which destrolyed thousands of citrus trees. The native place of this pest was
New Zealand and Australia. The predatory ladybird beetle Rodolia cardinalis was introduced
in California and within a short period i.e. in 1 1/2 years they multiplied enormosuly and
controlled the pest successfully. These predators were also introduced in India in 1929 in
the Nilgiri and within two years destroyed the citurs scales completely from region.
Biological control in India :
Some of the important successful biological control programmes conducted in India
and the parasites sent to other countries for control of pests are described below.
1)
A parasite Aphelinus mali was introduced in most of the apple growing areas in
India which kept check the apple wooly aphids.
2)
San Jose scale a serious pest of deciduous fruit trees has been controlled by
introducing the strains of parasite Prospaltella perniciosi in Kumaon in UP, HP and
Kashmir from china, USA and USSR in 1967-68. Aphytis diaspides another parasite
introduced from California controlled the pest successfully.
3)
Trichogramma minutum (a minute egg parasite) has been extensively used for the
control of sugarcane borer. Another egg parasite Talenomus electo was introduced
from Columbia for the control of sugarcane borer and gave 60% parasitism at
plassey in West Bengal.
4)
Another highly destructive sugarcane borer (Bissetia steiliella) commonly called
Gurdaspur borer was controlled by Trichogramma perkinsi at Ludhiana.
5)
In 1959 Neodusmetia sangwani a parasitic wasp was introduced in to texas, USA
from India to control Antonina graminis the grass scale that controlled this pest
within five years.
6)
Orgilus lepidus and Apanteles subanding were introduced in Chikkaballapur near
Bangalore to control potato tuber moth which controlled the incidence very
effectively.
7)
The employment of Microbracon trichospillus and Perisiorola for the control of
Nephantis serinopa (black headed caterpillar) of coconut.
8)
Apanteles angaleti larval parasite of pink bollworm.
17
Biological control by exotic parasites :
1) Copidosoma koetileri : egg larval parasite of potato tubeworm.
2) Bracon kirkpalricki : larval parasite of bollworms.
3) Chelonus blackbumi : egg larval parasite of bollworm and potato tubermoth.
4) Trichgramma brasiliensis : Egg parasite of cotton bollworms.
5) T. japonicum / T. fasciatum | Egg parasite on jowar, sugarcane and paddy
T. australicum / stem borer.
6) Epiricania melanoleuca : egg parasite of sugarcane pyrilla on sugarcane in Maharashtra.
Examples of successful biological control in abroad
Korea :
Control of Cottony cushion scale I. purchasi on citrus using vedialia beetle R. cardinalis
from Australia (1930).
Control of apple woolly aphid E. lanigerum using a parasitoid A. mali from Japan (1934).
Biological suppression of Weeds
Weeds are said to be undersirable plants grown out of place, harmful to the
cultivated crops, forest and other environmental vegetation, which also create health
hazards to human beings and animals. Losses in crop yield due to weeds are mainly
attributed to the competitive effect of these plants. World-wise, in cereals alone this
represents a loss of more than 155.5 billion tonnes. There is of course considerable
variation between different countries, between different crops and even between the same
crops in different situations. World-wide there is a 10.6%, loss in rice, 13% in maize, 9.8%
loss in wheat, 15.1% loss in sugarcane and 5.85% loss in cotton. Pathak et al. (1976) have
calculated that yield losses due to weeds in rice range from 10 to 50% in transplanted rice
and from 50 to 90% on upland fields.
Basic considerations for the development of classical biological weed control
Schrocder 1983 summarised the following steps in this direction :
1. The selection of suitable target weed species.
2. The selection of suitable bio-control agents.
a) The selection of survey areas for biocontrol agents.
b) The selection of effective biocontrol agents.
c) Host specificity determination of biocontrol agents.
3. Introduction, liberation and establishment of control agents with permission of the
competent authority.
Biological control of weeds in India
Work on Opuntia spp., Lantana camara, Parthenium hysterophours and a few others
are classical examples of biological control of weeds.
18
A] Suppression of terrestrial weeds
Prickly pear, Opuntia spp. (family : Cactaceae)
Prickly pear : Opuntia spp. vulgaris was introduced into India somewhere about 1780, to
have been used in the culture of commercial dye, produced by cochineal insects (Ayyar,
Ramakrishna, 1931). The two other species viz. O. dillenii (Kergawl) Mill and O. eletior Mill
were in all probability introduced before O. vulgaris. In due course of time Opuntia escaped
cultivation and for more than a century and half was recognised weed of importance (David
and Muthukrishnan, 1953). The cochincal insect, Dactylopius ceylonicus Green (=indicus
Green) (Hemiptera: Dactylopidae) which was mistaken for the true cochineal, D. coccus
Costa and introduced from Brazil about 1795 or carlier (Green 1922). The multiplication of
the coccid was started at Calcutta Botanical garden and at Madras, but its propagation was
not encouraged for a long as it was found much inferior to D. coccus in dye production. The
cochineal though was not of much importance in commercial dye-production, was found of
great value in controlling O. vulgaris. Its releases in field were made considered by Rao. et
al. (1917) Rao (1931) and Tryon (1910) Goeden (1978, it become established on wild O.
vulgaris and by the middle of 19th century it had become wide spread in the North and
Central India. It gave effective control in North and Cetral India and practically
exterminated this species in Sourth India (Ayyar, Ramakrishna, 1931, and Beeson, 1940).
During 1926 a North American Cochineal insect Dactylopius opuntiae (Cockerell)
(=tomentosus Lamarck) was introduced via Australia via Sri Lanka privately by merchants
into Sourth India (Goeden 1978, Tryon 1910). It soon become established dispersed to as
far north as Delhi (Beeson, 1940).
Lantana camara Linnaeus (Family : Verbenaceae)
Lantana camara was introduced into India as in ornamental plant in 1809. It is a
native of Central America. By its unrestricted spread, prolific seed production it escaped
cultivation and become a serious weed in pasture, wasteland, roadsides and forests,
replacing local vegetation in most parts of India. The birds like Indian Myna which feed the
fruits are responsible for far off dispersal. The weed forms good fencing shelter surrounding
kitchen garden, orchards and farms, thus man is also responsible for its spread. The
Lantana, has been considered as one of the ten worst weeds in the world (Holm and
Herberger, 1969), it has been reported as a symptomless carrier of scandal spike disease in
India (Mayar and Srimath 1968).
19
Lecture No 2 (Unit I)
Augmentative Bio-cointrol
Augmentation
This third type of biological control involves the supplemental release of natural
enemies. Relatively few natural enemies may be released at a critical time of the season
(inoculative release) or literally millions may be released (inundative release). Additionally,
the cropping system may be modified to favor or augment the natural enemies. This latter
practice is frequently referred to as habitat manipulation.
01]
An example of inoculative release occurs in greenhouse production of several crops.
Periodic releases of the parasitoid, Encarsia formosa, are used to control greenhouse
whitefly, and the predaceous mite, Phytoseiulus persimilis, is used for control of the
two spotted spider mite.
02]
Lady beetles, lacewings, or parasitoids such as Trichogramma are frequently
released in large numbers (inundative release). Recommended release rates for
Trichogramma in vegetable or field crops range from 5,000 to 200,000 per acre per
week depending on level of pest infestation. Similarly, entomopathogenic nematodes
are released at rates of millions and even billions per acre for control of certain soildwelling insect pests.
03]
Habitat or environmental manipulation is another form of augmentation. This
tactic involves altering the cropping system to augment or enhance the effectiveness
of a natural enemy. Many adult parasitoids and predators benefit from sources of
nectar and the protection provided by refuges such a hedgerows, cover crops, and
weedy borders.
04]
Mixed plantings and the provision of flowering borders can increase the diversity
of habitats and provide shelter and alternative food sources. They are easily
incorporated into home gardens and even small-scale commercial plantings, but are
more difficult to accommodate in large-scale crop production. There may also be
some conflict with pest control for the large producer because of the difficulty of
targeting the pest species and the use of refuges by the pest insects as well as
natural enemies.
05]
Examples of habitat manipulation include growing flowering plants (pollen and
nectar sources) near crops to attract and maintain populations of natural enemies.
For example, hover fly adults can be attracted to umbelliferous plants in bloom.
06]
Recent work in California has demonstrated that planting prune trees in grape
vineyards provides an improved overwintering habitat or refuge for a key grape pest
parasitoid. The prune trees harbor an alternate host for the parasitoid, which could
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
20
previously overwinter only at great distances from most vineyards. Caution should
be used with this tactic because some plants attractive to natural enemies may also
be hosts for certain plant diseases, especially plant viruses that could be vectored
by insect pests to the crop. Although the tactic appears to hold much promise, only
a few examples have been adequately researched and developed.
Harmonious, Co-ordinated and Fortuitous Biological Insect
Pest Suppression.
(A) Harmonius Biological Insect Pest Suppression (HBIPS) :
When two or more heneficial biotic agents are introduced, estabilished, and together
provide insect pest suppression, they are said to be working in harmony with one another.
Diprion (= Gilpinia) hercyniae (Hartig) (Hymenoptera : Diprionidae
The European spruce sawfly. D herecyniae was reproted in epizootic form by
foresters in the Gaspe Peninsula of Quebec in 1930. Continuing severe defoliation and
mortality of spurce stands posed a major threat to the forest economy of eastern Canada
Timber loss in the Gaspe between 1930 and 1945, when the infestation collapsed, was
estimated at over 11 million cords of spruce, plus additional loss from reduced tree growth
(Reeks and Barter, 1951). Native benefical organisms were in nsufficient to prevent the
sawfly from increasing and accounted for only 0.02% sawfly mortality (Recks 1938). Balch
and Bird (1944) estimated that a 97.8% mortality would be required in each generation to
maintain the sawfly population at enzootic levels. A massive beneficial insect importation
program was initiated and from 1933 – 1951, over 27 species of exotic parasitoids were
introduced and released in eastern Canada (MeGugan and Coppel, 1962). A cocoon
parasitoid D. fuscipennis became firmly estabilished in high host populations and exerted
considerable suppression (Reeks 1953), however, the cool weather patterns of eastern
Canada initially limited it overall effectiveness (MeGugan and Coppel, 1962). Mass
propagation of parasitoids was undertaken at the Dominion Parasite Laboratory, and
annual releases of all species increased from 2.5 million in 1935 to over 18 million in 1936.
In 1936, a new development manifested itself, and altered the course of events in
the years to follow. That development consisted of the laboratory observation of mortality of
sawfly larvae caused by a virus. By 1938, the disease was reported in the field, and it soon
became difficult to rear disease-free larvae. Prior to the appearance of the virus, beneficial
biological agents were causing 85 – 95% mortality in each sawfly generation in the field
(Bird and Elgee, 1957). The addition of the virus tipped the balance in favor of the biotic
agents, and the European spruce sawfly was no longer an economic pest by 1945. The
disease was caused by a nucleopolyhedrosis virus (NPV) which multiplies only in the nuclei
of the digestive cells of the larval sawfly midgut epithelium (Balch and Bird, 1944; Bird,
21
1949, 1952, 1953; Bird and Whalen, 1954). The origin of the virus in unknown, but it is
generally accepted that it was interoduced from Europe along with parasitoid material.
Key factor status in maintaining D Hercyniae at low levels since the initial pest
population collapse is attributed to the harmonious relationship between the virus and two
parasitoids, Drino bohemica Mesnil and Exenterus vellicatus Cushman (Neilson and Morris,
1964). Both parasitoids are effective at low host densities, whereas the virus is effective at
high host densities. Some of the parasitoids established early in the program have now
apparently been naturally replaced by species more effective at low population levels.Bird
and Burk (1961) stated that the virus alone is capable of reducing sawfly populations,
although the resultant host density is always several times higher than when both virus
and parasitoids are present.
Coleophora laricella (Hubner) (Lepidoptera : Coleophoridae)
The larch casebearer, C laricella, has gradually dispersed in a westerly direction and
caused serious damage to tamarack and western larch stands from eastern North America
to Idaho by 1957 (Denton, 1965), and into Oregon shortly thereafter. It reached British
Columbia by 1966 (Webb and Quednau, 1971). The severe outbreaks in eastern North
America from the 1930s through the early 1950s stimulated a biological insect suppression
program through which several parasitoids were introduced from Europe. A. pumila and
C. laricinellae were successfully established in all areas of release (MeGugan and Coppel,
1962) and the casebearer was relegated to enzootic status. The release of only A. pumila in
Idaho was apparently unsuccessful in reducing high populations of the casebearer (Denton,
1965).
Of particular interest here, is the complex relationship which exists between the
multivoltine C. laricinellae and the univoltine A. pumila. Early studies in Ontario (1940)
revealed that casebearer larvae parasitized by A. pumila were slowed in their development
by at least two weeks. This delay allowed second and third generation C. laricinellae to act
as multiparasitoids on larvae previously parasitized by A. pumila. Chrysocharis was always
the successful competitor and was able to maintain a higher population throughout the
year by utilizing this later developing host material (Graham, 1958a). Quedanau (1970),
using a life table approach, concluded that despite the intrinsic competition which destroys
a portion of the A. pumila population, host population regulation resulted from the
combination of prasitoids. Agathis, which does not appear to be host regulative by itself in
eastern Candada, synergizes C. laricinellae to suppress the casebearer. The extrinisic
superiority of A. pumila at low host densities precludes competitive displacement by C.
laricinellae. In additon, being more widely and uniformly distributed, A. pumila is able to
move more easily with the advancing host front. Even with the competitive relationship
22
manifested by C. laricinellae, the two species are able to work harmoniously to suppress
casebearer populations.
Operophtera brumata (L.) (Lepidoptera : Geometridae)
The European winter moth. O. brumata, suspected of having been introduced into
Nova Scotia around 1930, was first reported there in 1949. It attacks fruit orchards and
hardwoods, principally red oak, and in a ten-year period in two counties in Nova Scotia, it
destroyed about 26000 cords of wood annually, valued at two million dollars (DeBach,
1974).
A program for the introduction of beneficial parasitoids was initiated in 1954 from
the Dominion Parasite Laboratory, and six species were released between 1954 and 1958
(McGugan and Coppel. 1962). Two of the most numerous parasitoids enconuntered in
collections from Europe were established readily. These were the tachinid, Cyzenis albicans
(Fallen), and the inchneumonid, Agrypon flaveolatum (Gravely) (Embree, 1966). The two
species are completely compatible with one another (Mesnil, 1967). C. albicans is the more
efficient at high host densities, whereas A. flareolatum is regulative at low host densities
(Embree 1971). Though Varley and Gradwell (1968) predicted strong fluctuations between
host and parasitoid propulations leading to winter moth outbreaks every 9 – 10 years, these
have not occurred in Canada thus far. In addition to the parasitoids, a virus was recorded
in field populations as early as 1961. It has apparently been an important factor
contributing to the collapse of two infestations (Embree, 1971). However, the successful
establishment within six years after initial release, and the spread of these two compatible
species of parasitoids is sufficient to maintain winter moth populations at low levels.
B) Coordinated Biological Insect Pest Suppression (Co-ordinated BIPS)
Combinations of biotic agents may enhance the success of the classical approach to
biological insect pest suppression. Similarly, it is possible to coordinate the use of various
beneficial agents with natural products, such as pheromones, hormones, or antifeedants,
or with other biological methods, such as host resistance, environmental manipulation, or
autocidal techniques.
This type of co-ordinated use falls within the sphere of bidogical insect pest
suppression and though of a higher order than the harmonius type is of lower order than
our interpretation of integrated pest suppresion.
Choristoneura fumiferana (lemens) (Lepidopetera : Tortricidae)
The spruce budworm C. fumiferana, caused serious tree mortality in eastern
Canada from 1940 through part of the 1950 s. Though the outbreak subsided by 1959, a
resurgence in the mid-1960s necessitated the use of DDT via aerial spraying to preserve the
forests (Blais, 1968). Since 1968, budworm populations have been increasing in Ontario
and Quebec, and all avenues of possible pest suppression are being studied. The failure of
23
introduced parasitoids from Europe and western Canada, the failure of native parasitoids
(Miller, 1963), and the failure of viral diseases to effect any significant degree of host
regulation, turned efforts towards the possible use of the bacterium, B. thuringiesis
Berliner, as a suppressive agent.
Bacillus thurnigiensis had been tried against the budworm in New Brunswick
(Mott et al., 1961), in Quebec (Smirnoff, 1963 a, 1965), and in Ontario (Blais, 1973), with
some suppressive effect, but not to subeconomic levels. In a new development, Smirnoff
(1971) and Smirnoff et al. (1973 a, b) demonstrated that a combination of B. thuringiensis
plus the enzyme chitinase is very effective, both in the laboratory and in the field. Chitinase
is easy to procure from the gastiric juices of slaughtered chickens, and enhances the
effectiveness of B. thuringiensis by hydrolizing the protective chitin layer of the insect gut to
allow entrance of the bacterium into the budworm hemolymph (Smirnoff, 1974). After the
bacterium reaches the hemolymph, a fatal septicemia is soon produced. The successful
treatment of over 4000 ha of budworm-infested softwood forest with the bacteriumchitinase combination demonstrates this exciting new suppression concept.
Anthonomus grandis Boheman (Coleoptera : Curculionidae)
The boll weevil, A. qrandis, continues as a major threat to cotton production in the
southern USA. Originating from populations in Mexico, it entered the USA around 1800,
and has since spread over most cotton-growing areas. Nearly one third of the chemical
insecticides used on crops in the USA are applied against boll weevil (Cross 1973).
Resistance to insecticides, destruction of beneficial species by them, and far-reaching
environmental contamination stimulated investigations into alternative methods for cotton
pest suppression.
An interesting alternative to the use of chemical pesticides was demonstrated by
McLaughlin (1966, 1967), who utilized a pathogenic protozoan in conjunction with a
feeding stimulant. Boll weevils attracted to the feeding stimulant, ingested the protozoans,
and spread them through the population. The neogregarine protozoan. M. grandis
McLaughlin, was added to the bait formulation composed of natural oils and parts of the
cotton plant. A red dye additive marked those beetles ingesting protozoans, and also
allowed investigations of bait effectiveness (Daum et al. 1967). Later, the program was
expanded to include a second protozoan, the microsporidan, Glugea gasti McLaughlin
(McLaughlin et al., 1968, 1969).
Successful field infection of the weevil population by the two protozoans
incorporated into the feeding stimulant-bait suppressed the pest during the early spring.
This delayed the need for chemical treatment until late in the season (McLaughlin, 1973).
Infection rates of 50 – 70% were regularly achieved. Introduction of sporozoan pathogens
did not, by itself, suppress the boll weevil population below economic levels, however, the
24
coordinated use of the protozoan-bait stimulant principle could become an important
adjunct to the total suppression program.
Potential Coordinated Biological Insect Pest Suppression Techniques
The successful use of autocidal techniques against the codling moth, Laspeyresia
pomonella (L.), via the sterility principle (Proverbs et al., 1966, 1969; Butt, 1967), adds an
important factor to the apple orchard environment which may enhance the release of
Trichogramma spp. parasitoids. This factor is the additional sterile eggs available as hosts
for the parasitoids. At the current sterile-moth release rate for suppression programs, the
extra food source (sterile eggs) has been calculated at 5 – 10 million eggs/ha/season (Nagy,
1973). Theoretically, the resulting additional 2.5 – 5 million Trichogramma progeny/ha
might offer, with the reduced moth fertility, a complementary suppression system against
the codling moth. Additional benefit might also accrue from large numbers of Trichogramma
attacking various other important orchard pests in the family Tortricidae.
The hematophagous stable fly. Stomoxys calcitrans (L.), often becomes a serious
animal pest. The use of two juvenile hormone analogs in combination with the release of
the
hymenopterous
parasitoid,
Muscidifurax
raptor
Girault
and
Saunders,
was
demonstrated as a feasible technique by Wright and Spates (1972). The parasitoids develop
normally in JHA-treated fly pupae, with no effect on their reproductive capabilities. The two
approaches are compatible, and create a synergistic pressure against stable fly populations,
particularly since development of the latter is prolonged enough to allow M. raptor to
complete its own development.
The melon fly, Dacus cucurbitae Coquillett, is also a serious candidate for a
coordinated biological insect pest suppression program. Chambers et. al. (1972) proposed
the combination of sterile-male releases plus a male-annihilation program for its
suppression. They found that male melon flies, when exposed continuously to the male
attractant cue-lure for 4 – 5 days after emergence, were 3 – 10 times less responsive to field
traps baited with this substance. The males had become habituated to the attractant.
Thus, the proposal was to coordinate the release of habituated sterilized males along with
attractant baits incorporating a pathogenic microorganism. In this way it might be possible
to create a most effective suppression program because the baits would primarily attract
and infect wild males, while the released sterile males would have less competition for wild
females, and would not respond to the traps.
Along the same general lines, Burkholder and Boush (1974) proposed the use of sex
pheromones o stored product insets in an inoculation device containing a pathogenic
microorganism. They felt that males would be attracted to the pheromone trap, pick up the
pathogens (protozoans in this instance) on their bodies, and distribute them later to
females with which they mated, as well as to the medium in which they lived and fed.
25
C) Fortuitous Biological Insect Pest Suppression (BIPS)
Fortuitous biological insect pest suppression can best be defined as the chance
movement of exotic beneficial organisms to new areas and / or new pests, where pest
population suppression eventually results. Alternatively, it may also occur when indigenous
natural enemies successfully adapt to exotic pests. Though these happenstances are not
premeditated, humans may be indirectly involved by accidentally providing transportation
for the beneficial organisms, or by the serendipitous transfer of a purposely introduced
beneficial organism from its intended host to another host. Due to its accidental nature,
this type of suppression probably occurs in the field more frequently than it is ever noticed
or recorded. The following three examples briefly exemplify the fortuitous aspect of pest
suppression.
Coleophora malivorella Riley (Lepidoptera : Coleophoridae)
In 1943, the parasitoid, C. laricinellae, was imported from Europe and released in
Quebec for suppression of the larch case bearer (Graham, 1944). C. laricinellae was
recognized as one of two key factors reducing populations of the pistol casebearer. C.
malivorella, in Quebec apple orchards from epizootic levels in 1959 – 1960 to enzotic levels
by 1962. LeRoux et al. (1963) reported that the average mortality of summer pistol case
bearer larvae caused by C. laricinellae was 29%, whereas mortality of over wintering larvae,
attributed to both C. laricinellae and predaceous birds, was 2 – 99%. Thus, 18 years after
the successful establishment of C. laricinellae for larch casebearer suppression on forest
trees, the parasitoid appeared by chance in the apple orchards of southwestern Quebec,
where it was effective in suppressing the pistol casebearer as well.
Aonidiella aurantii (Maskell) (Homoptera : Diaspididae)
The hymenopterans parasitoid, A. lingnanensis Compere, was unintentionally
brought from the Orient to Mexico, perhaps via the Manila galleons. It eventually
distributed itself as far north as southern Texas. It went unnoticed in all areas for many
years, but apparently exerted effective suppression of the California red scale, A. aurantii,
where the two species occurred sympatrically. In 1948, this same parasitoid was purposely
introduced from southern China to California. It could have been collected only 480 km
from the San Diego citrus-growing areas with less trouble and cost had its existence there
been known.
Augmentation of Native Natural Enemies
In earlier years, it was thought that using indigenous natural enemies for biocontrol
may not yield any fruitful results. But this view has gradually undergone changes due to
rarity of success in classical biological control programmes especially against native
insects. Studies conducted in India and other countries (Ridgway and Vinson, 1976)
indicate that native natural enemies can be used profitably in pest management. In
26
sugarcane, some of the native natural enemies provided appreciable pest control when their
populations are conserved and/ or augmented.
Examples :
1) Trichogramma chilonis against Chilo infuscatellus, C. sacchariphagus indicus, C auricillus
and Trichogramma japonicum, against Scirpophaga excerptalis Walker in sugarcane.
2) Isotima javensis Rohw agaist S. excerptalis in sugarcane in Tamilnadu.
3) Epriricania melanolecuca F. against Leaf hopper, Pyrilla perpusilla in sugarcane.
4) Sturmiopsis inferens Townsend tachinid against C. infuscatellus, C. auricillus, Sesamia
inferens Walker.
5) Coccinellied predators
6) Granulosis virus
7) Other pathogens
27
Lecture No. 3 (Unit I)
Introduction of Natural Enemies
Present Status and Future Thrust
In sugarcane, biological control is attracting more attention in recent years than
ever before, as evidenced from increased demand for natural enemies. The main reason for
this is the increasing frequency of failure of chemical control methods, if not in sugarcane,
in other crops. In addition, non-availability of labour in time and increase in wages have
reduced the scope of adoption of cultural and mechanical control methods in sugarcane.
For practical biological control ready and timely availability of required quantities of
natural enemies is essential. At present, biocontrol laboratories in the country are at the
most equipped for mass production of Trichogramma and for redistribution of Epiricania.
According to Manjunath (1984), the demand for natural enemies is far in excess of the
production. Unless natural enemies are made as readily available as chemical pesticides,
biological control methods cannot reach the farmers in remote villages. This emphasises
the need to create additional facilities and to improve the existing facilities to make
biological control of sugarcane pests through augmentation of ntural enemies practical and
feasible. The facilities can be broadly ground into three :
01)
Research facilities to standardise and improve the various aspects of mass
production of promising natural enemies and their laboratory hosts and to improve
their quality and to determine dose, frequencies of release, methods of formulation,
and storage in working out the economics of biological control.
02]
Production facilities to mass produce and make readily available the required
quantity of quality natural enemies; and
03]
Distribution and advisory facilities to distribute the natural enemies to every needy
farmer in time as is done with chemical pesticides and to advise farmers about the
pest problems, kind of natural enemy needed, dosage and frequency of release.
Identification of insect parasitoids and predators
1. Parasitoids
Order : Hymenoptera
The ovipositor originates and protrudes ventrally from the abdomen and is used to
insert eggs into their hosts. There are three super families.
Super Family : Ichneumonoidea

Possess long and filiform antennae

Wings are veined
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
28
Family : Ichneumonidae

Eg. Eriborus trochanteratus, a larval parasitoid on coconut black headed caterpillar,
Opisina arenosella

Antennae longer with more than 16 segments

Trochanter two segmented

Possesses two recurrent veins and rarely one

Abdomn three times as long as the rest of the body

Ovipositor longer than the body

Large slender black, yellow or reddish yellow insects

Larvae are endo or ecto parasitic on many groups of insects and spiders.
Family : Braconidiae

Eg. Bracon brevicornis, a larval parasitoid on O. arenosella, Chelonus blackburni, egg
larval parasitoid on cotton spotted bollworms, Earias spp.

Adults are relatively small, more stout bodied than ichneumonids

Abdomen is about as long as the head and thorax combined

Not more than one recurrent vein

Adults not as bright as inchneumonids

Mostly endoparasitic on lepidopteran larvae
Super Family : Chalcidoidea

Mostly smallest parasitoids and gregarious

Antennae geniculate

Abdomen very short or globular with very slender propodeum

Wings without veins
Family : Chalcididae

Eg. Brachymeria nephantidis a larval parasitoid on O. arenosella

Minute insects

Abdomen humped

Hind femur enlarged and toothed

Wings are not folded longitudinally when at rest

Ovipositor straight and short

Parasitic on Lepidoptera, Diptera and Coleoptera
Family : Trichogrammatidae

Eg. Trichogramma chilonis, an egg parasitoid on many lepidopterous pests

Mostly egg parasitoids

Minute insects (0.3 to 1.0 mm long) with three segmented tarisi and broad and
elognated fore wingswith rows of microsocopic hairs on them
29

Hind wings reduced with hairs
Family : Eulphidae

Eg. Trichospilus pupivora and Tetrastichus israeli, pupal parasitoids on O. arenosella

Adults have four segmented tarsi

Many have brilliant metallic colouring

Males of many species have pectinate antennae

Mostly parasitic on aphids and scales and some are on pupae of Lepidoptera
Super family : Bethyloidea

Smaller than Ichneumrnonoidae and larger then Chalcidoidea
Family : Bethylodae

Eg. Parasierial (= Goniozus) nephantidis, a larval parasitoid on O. arenosella

Small to medium sized, usually dark coloured wasps

Females of many species are wingless and antilike in appearance

In a few species, both winged and wingless forms occur in each sex

Parasitic on Lepidoptera and Coleoptera.
Order : Diptera
Family : Tachinidae

Eg. Sturmiopsis inferens, a larval parasitoid on sugarcane shoot borer, Chilo
infuscatellus

Large bristle flies

Eggs may be macrotype or microtype

Macrotype eggs are laid directly on the host’s body usually attached to the neck
region by a glutinous secretion

Eg. Spoggosia bezziana on O. arenosella

Microtype eggs are laid on the host plant and the host larvae feeding on the plant
tissue ingest them
Order : Lepidoptera
Family : Epiricanidae

Eg. Epiricania melanoleuca

Parasitic on nymphs and adults of sugarcane leafhopper, Pyrilla perpusilla
2. Predators
Order
: Odonata
Sub order
: Anisoptera Eg. Dragon fly
Sub order
: Zygoptera Eg. Damsel fly

Relatively larger sized insects

Immature stages are aquatic (naiads) feeding on aquatic insects
30

In naiads, labium is modified into a prehensile organ called mask for catching the
prey

Adults feed on midges, mosquitoes, flies and small moths

Adults are capable of catching prey during flight with the help of basket shaped
legs.
Order : Dictyoptera
Family : Mantidae

Preying mantids are large elongate insects

Nymphs and adults are cryptically coloured with long of prehensilie raptorial
forcergas

Highly predaceopus feeding on variety of insects like flies, grasshopper and many
caterpillars Eg. Mantis religiosa
Order : Hemiptera
Family : Reduviidae

Assassin bugs or cone nose bugs or kissing bugs

Usually blackish or browish in colour

The beak or proboscis is short and three segmented

Most are predaceous and some are blood sucking

Both nymphs and adults are predaceous

Eg. Harpactor costalis on the red cotton bug Dysdercus cingulatus.
Family : Pentatomidae

Stink bugs

Bugs are shield shaped with 5 segemented antennae

Some of the species are predaceous on lepidopterous larvae

Both nymphs and adults are predaceous

Eg. Eucanthecona furcellata on the larvae of red hairy catepillan Amsacta albistriga
and gram caterpillar, Helicoverpa armigera
Family : Belostomatidae

Giant water bug

Elongate oval and somewhat flattened with reptorial forelegs

Feed on variety of aquatic insects.
Family : Miridae

Elongated soft bodied insects

A few species are predaceous
31

Eg. Green mirid bug, Cyrtorhinus lividipennis feeds mainly on the eggs and early
stage nymphs of green leaf hopper (GLH), brown plant hopper (BPH) and white
backed plant hopper (WBPH) in rice.
Veliidac :

Ripple bugs

Aquatic insects living on the surface of water.

Brown or black in colour

E.g. Microvelia atrolineata feeding on the first instar caterpillar of lepidopteran pess
and GLH, BPH and WBPH in rice ecosystem.
Order : Neuroptera
Family : Myrmeliontidae

Antions

Larvae construct pit falls and remain buried in the soil

Feed on the ants and other insects that fall into the pits

Feed by inserting the mandibulo- suctorial mouth parts into the prev and sucking
the internal contents
Family : Chrysopidae

Aphidwolfs, aphidlions or green lace wings

Adults are green in colour with golden or copper coloured eneys

Feed on more than 18 families of insects

The larvae are predaceous mainly on aphids and also on eggs of lepidopteran
insects, psyllids, coccids, thrips and mites

Larvae have sharp mandibles

The eggs of aphidions are stalked (pedicellate)
Order : Diptera
Family : Asilidae

Robber flies

Adults are mostly elongate with tapering abdomen

Body is covered with dense hairs

Legs are long, strong and well developed

Adults are predaceous and attack a variety of insects like wasps, bees,
grasshoppers, flies etc.

Mouth parts are pierciing type. They feed by sucking the body fluid of the prey
Family : Syrphidae

Hover fly adults are brightly coloured and resemble various bees and wasps

Good pollinators
32

Maggots are green in colour and feed on aphids by sucking their body fluids
Order : Coleoptera
Family : Coccinellidae

Lady bird beetles

Beetles are small, oval, convex and often brightly coloured

Grubs are elongate, somewhat flattened and covered with minute tubercles or
spines.

Adults and grubs feed on aphids, coccids, mealy bugs, whiteflies and other soft
bodied insects

Except one or two species in the family all are predaceous

Eg. Rodolia cardinalis on cottony cushion scale, Icerya purchasi
Family : Carabidae

Ground beetles

Dark in colour and shiny and some what flattened

Most of them feed on caterpillars

Eg. Anthia sexguttata, Ophionea indica
Family : Cicindelidae

Tiger beetles

Beetles are very active and brightly coloured

They run and fly rapidly

Both adults and grubs are predaceous

Adults capture the prey with sickle shaped mandibles

Eg. Cicindela spp.
Family : Staphylinidae

Rove beetles

Eg. : Paederus fuscipes feeds on rice leaf folder
Order : Hymenoptera
Family : Vespidae

Wasps collect various insects and feed their larvae with them

Mudwasps construct nests made of mud and provide caterpillars for the young ones
in the nest.
Family : Sphecidae

Digger wasps construct nests made of mud and feed its young ones with insect
caterpillars.
Family : Formicidae

About half the members of the family are predaceous upon insects.
33
Parasitoids
1. Definition : “A parasitoid is an insect parasite of an arthropod which is parasitic in
immature stages and adults are free living”.
Adult parasitoid seek out their host in the environment using a variety of olfactory
and tactile cues from the host and its habitat visual. The female lays eggs in, on or near the
host. Parasitoid larvae feed on host tissue, but may not kill their host until they develop to
adult stage. Most insects parasitic upon other insects are protelean parasites i.e. parasitic
only in their immature, larval stage and lead free life as adult. Adult parasitoid requires
food such as honeydew, nectar, pollen, host body fluids and water.
Parasitism : This is a kind of symbiosis in which parasitoid lives at the expense of the host
and killing the host in the process of development. Parasitization is an act of attack and
ovipositioning the eggs with the help of ovipositor by the parasitoid on the host.
2. Types of parasitism
Simple parasitism : A single attack of the parasitoid on the host irrespective ofthe number
of eggs laid.
eg. Goniozus nephantidis on larvae of coconut black headed caterpillar (CBHC)
Super parasitism : Many individuals of same species of parasitoid attack a single host at a
time.
e.g. Trichospilus pupivora on pupae of BHC
Multiparasitism : Parasitism by different species of parasitoids on the same host at a time.
e.g. Bracon brevicornis (Braconidae), Eriborus trochanteratus (Ichneumonidae) and Goniozus
nephantidis (Bethylidae) on larvae of BHC
Hyperparasitism : A parasitoid attacking another parasitoid
e.g. Pleurotropis sp. Hyperparasitoid on Bracon brevicornis a primary parasitoid
Asaphes sp. on Aphidius sp. (Parasitoid of aphids)
Autoparasitism (Adelphoparasitism) :
Female develops as a primary parasitoid, but the male is a secondary parasitoid
through females of its own species. This is also called as heteronomous hyperparasitoids.
eg. Encarsia formosa attacking scale insects and whiteflies.
Cleptoparasitism : A parasitoid attacking a host, already parasatized by another species of
parasitoid.
Pine shoot moth attacked by either Eurytoma pini or Rhyacionia buoliana one followed by
another.
Endoparasite : Parasitoid developing within the host body internally eg. Aphelinus mali on
wooly aphid, where only a single larvae completes its development is called Solitary
endoparasite eg. Itoplectis on pine sawfly and if many larvae develop to maturity in single
host is called Gregarious endoparasite e.g. Apanteles congregatus.
34
Ectoparasite : Parasitoid developing externally on host body.
eg. Epiricania meanoleuca on sugarcane pyrilla.
3. Parasitoids of agricultural importance
Parasitoids of agricultural importance mostly belongs to Hymenoptera (90%) and
Diptera (10%).
Hymenoptera
Families :
Braconidae
- Larval endoparasitoids
Ichneumonidae
- Larval parasitoids
Chalcidae
- Larval or pupal parasitoids
Eucyrtidae
- Endoparasitoids
Eulophidae
- Ectoparasitoids
Trichogrammatidae
- Egg parasitoids
Platygasteridae
- Larval parasitoids
Bethylidae
- Larval parasitoid
Aphelinidae
- Pupal parasitoid
Scelonidae
- Egg parasitoid.
Egg parasitoid
Trichogramma chilonis (Trichogrammatidae ) on the eggs of sugarcane internode
borer, cotton bollowrm and rice leaf folder.
T. japonicum (Trichogrammatidae) on the eggs of rice stem borer
Telenomus rowani (Scelonidae) on the eggs of rice stem borer
T. remus (Scelonidae) on the eggs of tobacco caterpillar
Egg-larval parasitoid
Chelonus blackburni : Braconidae on the eggs of cotton spotted bollworm.
Larval parasitoid
Bracon hebetor (Braconidae) on the larvae of coconut black headed caterpillar
B. brevicornis (Braconidae) on the larvae of coconut black headed caterpillar
Campoletis chloridae (Ichneumonidae) on the larvae of H. armigera
Cotesia plutella (Braconidae) on the larvae of diamondback moth
Eriborus trochanteratus (Ichneumonidae) on the larvae of coconut black headed
caterpillar
Goniozus nephantidis (Bethylidae) on the larvae of coconut black headed caterpillar
Platygaster oryzae (Platygasteridae) on the larvae of rice gall midge.
Larval-Pupal parasitoid
Isotima javensis (Ichneumonidae) on the pre-pupal stage of top shoot borer of
sugarcane
35
Pupal parasitoid
Brachymera nephantidis (Chalcidae) on the pupae of coconut black headed
caterpillar
Tetrastichus israeli Eulophidae on the pupae of coconut black headed caterpillar
Trichospilus pupivora (Eulophidae) on the pupae of coconut black headed caterpillar
Nymphal and adult parasitoid
Aphelinus mali (Aphelinidae) on the aphids
Encarsia formosa (Aphelinidae) on the cotton whitefly
Diptera
Family : Tachinidae (larval and larval-pupal parasitoid)
Larval parasitoid
Sturmiopsis inferens (Tachinidae) on the larvae of sugarcane early shoot borer
Spoggossia bassiana (Tachinide) on the larvae of coconut black headed caterpillar
Larval-pupal parasitoid
Eucelatoria bryani (Tachinidae) on the larvae of H. armigera
4. Idea qualities of a parasitoid

High host searching capacity

Having a narrowly limited host range

Having a life cycle shorter than that of pest

Potential rate of increase (high fecundity)

Able to survive in all habitats

Able to culture easily in the laboratory

Able to quickly reduce the pest population

Absence of superparasitism and multiparasitism.
5. Successful examples
Paddy :

7-9 release of Trichogramma chilonis and T. japonicum @1,00,000/- ha starting at 30
days after transplanting for stem borer and leaft folder.

Mirid bug @100 bugs or 50-75 eggs/m2 at 10 days intervals for BPH.
Sugarcane

Weekly releases of T. chilonis @ 1,25,000/ha from 4th to 11th week stage of crop for
internode borer.

Release of Epiricania melanoleuca @ 5000 cocoons/ha for pyrilla.
Cotton

Release of Trichogramma spp. @1,50,000/ha at weekly intervals for bollworm.
36

Release of Chrysopid grubs @ 1,00,000/ha at fortnightly intervals for aphids,
hoppers.
INSECT PARASITOIDS
ORDER : HYMENOPTERA
Egg parasitoids
1. Trichogrammatidae : Example are
Triochogramma chilonis against cotton bolloworm, sugarcane internode borer and
rice leaf folder.
Trichogramma japonicum agaisnt rice yellow stem borer
T. achea agaisnt Lepidopteran pests
2. Scleoniidae e.g., Telenomus remus against Tobacco caterpillar
3. Evaniidae e.g., Evania appendigaster targets ootheca cockroach
Egg larval parasitoids
1. Btraconidae e.g., Chelonus blackburni targets eggs of spotted bollworm
2. Encyrtidae e.g., Copidosoma koehleri targets Potato tuber moth.
Larval parasitoids
1. Ichneumonidae e.g.

Eriborus trochanteratus agaisnt coconut black headed caterpillar

Campoletis chloridae targets larvae of H. armigera
2. Braconidae e.g.

Bracon hebetor an Bracon brevicornis against coconut black headed caterpillar

Cotesia plutella against larvae of diamond back moth.
3. Bethylidae e.g. Goniozus nephantidis targets coconut black headed caterpillar
4. Platygasteridae e.g. Platygaster oryzae attacks larvae of rice gall midge.
Larval Pupal Parasitoids
1. Ichneumonidae e.g. Isotima javensis attacks pre-pupal parasite of top shoot
borer of sugarcane Xanthopimapla punctata targets pupae of coconut black
headed caterpillar.
2. Eulphidae e.g. Trichospilus pupivora and Tetrastichus israeli targets pupae
of coconut black headed caterpillar
3. Chalcidiae e.g. Brachymeria nephantidis attacks pupae of coconut black
headed caterpillar
Nymphal and Adult parasitoids
1. Aphelinidae e.g. Encarsia formosa attacking cotton whitefly Encarsia
perniciosi attacking Sanjose scale, Quadraspidiotus perniciosus Aphelinus mali
feeds on Apple wooly aphid.
37
2. Epiricandae e.g. Epiricamia melanoleuca parasitoid on Pyrilla perpusilla
Note : All the parasitoids attacking insect species belong to the order Hymenoptera
except Epiricania melanoleuca, which belongs to Lepidoptera order
Table 1 : Natural Hosts used for mass rearing of natural enemies
1. Aphids reared on cowpea
Ladybird beetles and Green Lace wing
2. Mealybugs on potatoes
Lady bird beetles
3. Corcyra cephalonica
Trichogramma spp; Apanteles flavipes
(Rice Meal Moth)
reared on broken grains of sorghum

A grub of ladybird beetle feeds on 400 – 500 nymphs of aphid (Aphis craccivora)
during its development.

The Black fungus beetle, Alphitobius diaperius is most injurious to Rice meal moth
when it is used for as factitious host for multiplication of Trichogramma spp in
laboratory.

Green lacewing – only grub is a predator whereas adults do not prey and they feed
on pollen grains or nectar of the flowers.

“Trichocard” (15 x 7.5 cm), 20,000 (1 CC) eggs are glued on this card using gum
acacia and the parasitoid completes its life cycle in 7 – 8 days at normal
temperature (270C) and humidity (75%).

The ratio used for parasitoid to host 1:6 i.e. for each parasitoid 6 eggs are provided.

Mite species, Phytosemilus persimilis is a predator on Tetranychus spp.
Predators
Pest targeted
Predator family
Order : Coleoptera
1. Coccinella septumpunctata
Aphids
2. Scymnus coccivora
Grapevine mealy bug
3. Cryptolaemus montrouzieri
Grapevine mealy bug
Coccinellidae
4. Rodolia cardinalis
Cottony cushion scale
bird beetles)
5. Menochilus sexmaculata
Mealybugs and scales
6. Chilocoris nigrita
Melanispis glomerata
7. Ground beetles
Coconut
BHC
and
rice
(lady
Carabidae
brown plant hopper
8. Tiger beetles
Small cterpillars
Cicindellidae
1. Platymeris laevicollis
Coconut Rhinoceros beetle
Reduviidae
2. Cyrtohinus lividipennis
Rice brown plant hopper
Miridae
3. Eucanthecona furcelleta
Red hairy caterpillar
Pentatomidae
Order : Hemiptera
38
Order : Neuroptera
1. Green lace wings or aphid lions.
Aphids, scales and mealy
Chrysopidae
Chrysoperla carnea
bugs and bollworms
2. Ant lions
Soft bodied insects
Myrmeliontidae
Caterpillars
-
Caterpillar and
Mantidae
Order : Odonata
1. Damselflies (Naiads and Adults)
Order : Mantodea
Preying
mantids
(Naiads
and
Adults)
grasshoppers
Order : Diptera
1. Robber flies (Adult)
Small insects
Asilidae
2. Hover flies(Adult)
Small insects
Syrphidae
Table 3 : Biotic Agents used for control of weeds : Terrestrial weeds
Weed
Scientific
Name
of
Biotic agent
Origin
Dactylopius opuntiae
USA
weed
1. Prickly pear
Opuntia dilleni
(Dactylopidae: Hemiptera)
2.
Congress
grass or carrot
Parthenium
Zygogramma bicolorata
hysterophorus
(Chrysomelidae : Coleoptera)
Lantana camera
Ophiomyia lantanae (Tortricidae
Mexico
weed
3. Lantana weed
Mexico
: Lepidoptera)
Teleonemia scrupulosa
(Tingidae : Hemiptera)
4. Siam weed
Chromolaena odorata
Pareuchaetes pseudoinsulata
West Indies
(Arctiidae : Lepidoptera)
5. Crofton weed
Eupatorium
Procecidochares utilis
adenophorum
(Trypetidae : Diptera)
Salvinia molesta
Crytobagus singularis
Mexico
Aquatic weeds
Water fern
Australia
(Curculionidae :Coleoptera)
Water hyacinth
Eucharnia crassipes
Neochetina eichorniae,
USA
N. bruchi (Curculionidae :
Coleoptera) Orthogalumna
terebrantis (Mite)
39
Microbes
Entomopathogenic virus
Baculoviridae are the one of the few virus families that are confined to invertebrate
species and this makes them particularly useful as insect pathogens, since the risk of
crossinfection in mammals is considered to be small. Baculoviruses are rod-shaped
particles which contain circular double stranded DNA genomes.

Symptoms of Baculvirus infections in insects were first recognised in silkworms in
16th century.

Bergold (1974) provided definitive evidence of the viral nature of the disease.
Classification of virus
Baculoviridae
Eubaculovirinae
(Occluded virus particles
NPV
(Nuclear Polyhedrosis Viruses
Nudibaculovirinae
(Non-occuluded virus particles)
GV
(Granulosis viruses)
Symptoms :
The insects that are infected stop feeding and the larvae turns into pinkish white on
the ventral side because of accumulation of polyhedral bodies. In advanced stage of the
larvae if the infction continues, larva become flaccid, skin becomes fragile and finally
ruptures. The diseased larvae in the field crawls to the tip of the plant and from that
position it hang upside down. This symptom is called as tree top disease.
Mode of action :
Occlusion bodies (Polyhedra for NPVs and Granules for GVs) are ingested by insect
larvae. In the higly alkaline pH of the midgut, the occlusion body protein dissolves and is
further degraded by host alkaline proteases. The virus particles are released from polyhedra
and subsequently attach to the peritrophic membrane lining the midgut. The lipoprotein
membrane surrounding the virus fuses with plasma membrane of the gut wall cells and
liberates nucleocapsids into the cytoplasm. The nucleotide transport virus DNA into the
nucleus of the cell and virus gene expression begins. The virus multiplies rapidly and
eventually fills the body of the host with the virus particles.

ELCAR® (H. zea NPV/SNPV), the first commercial viral pesticide which was
marketed by Sandoz Company in 1970s, was made as wettable powder spray.
40

4-6 days (Incubation period) laps between the time of insertion of virus and death of
the host.

UV protectants used to encapsulate baculoviruses are tinopal, aluminium powder or
egg albumin to ensure a longer field life.

The principal disadvantage of biocontrol agents is their slow speed of action in
comparison with chemicals.

Dose of virus is usually expressed as Larval equivalent (LE).

One LE is 6 x 109 POB. One larval equivalent is obtained from three matured virus
infected larvae.

The dose commonly recommended in the field for NPV is 250 – 500 LE/Ha
Table 1 : Commercially used viruses for insect control
NPV
Helicoverpa armigera NPV
Spodoptera litura NPV
Autographa californica (AcNPV)
CV
Chilo infuscatellus GV
Achaea janata GV
Phthorimaea opercullela GV
CPV
Helicoverpa armigera
Microbial control
1. Definition : “The control of pests by the use of microorgansims such as viruses,
bacteria, protozoa, fungi, rickettsia and nematodes or their by-products” is known as
microbial control. These microbes can be grouped as ingested microbes (bacteria,
viruses, rickettsia and protozoa) which enter insect body along with food (like stomach
insecticide) and penetrating microbes (nematode and fungi) that enter by penetrating the
integument (like contact insecticide).
2. Desirable attributes of Microbes

Pathogen should be highly virulent, able to cause disease in short period and
speread from one insect to another.

Host specificity.

Cost effective and economical.

Harmless to other forms of life (Safe to non-target organisms and man).

Rapid prevention of pest feeding.
3. VIRUSES : Viruses are submicroscopic, obligate, intracellular, pathogenic entities. These
are pathogenic to arthropods belongs to atleast eleven families. Viruses in the family
Baculoviridae are the best known of all the insect viruses because the disease symptoms
are easily recognized and they have the potential for development as microbial
41
insecticides. Baculoviruses are double stranded DNA viruses having baciliform or rod
shaped virions. Important sub groups within the family are Nuclear Polyhedrosis Viruses
(NPV) and the Granulosis Viruses (GV).
Nuclear Polyhedrosis Virus (NPV) : Improtant features of NPV are

Occluded (rod shaped) single or in groups in polyhedral (many sides) inclusion
bodies.

Site of multiplication is cell nucleus of epidermis, fat body, blood cells and trachea.

Wipfel Krankheit or tree top diseases : Diseased larvae not able to die. (eg.) NPV of
Spodoptera, Helicoverpa, RHC
Mode of entry : The virus should be ingested to produce the disease (Peros). Due to
alkaline gut juice, the virions are liberated from the polyhedral coat which attack nuclei of
cells of tissues viz., fat body tracheal matrix, heaemocytes, sarcolemma of muscles,
neurilemma and nerve cells of ganglion and brain.
Symptoms : Insects become dull in colour, feeding rate is reduced and larvae become
pinkish white especially in the ventral side due to accumulation of polyhedra. In advanced
stage larvae become flaccid, the skin becomes very fragile and eventually rupture. Diseased
larvae hang upside down from the plants. This is called tree top disease (or)
Wipfelkrankeit.
Incubation period : 4 – 6 days depending upon the stage of the infection, weather
conditions and dose of virus. Early instars are most susceptible to the virus.
01] Mass production of NPV of Spodoptera litura
S. litura can be mass cultured using the natural diet, viz., castor leaves under
laboratory condition in plastic buckets. The steps involved in the production of NPV are :
Pre starve 4th instar larva – over night

Prepare virus suspension containing 108POB/ml
in water containing 0.1% teepol

Dip clean castor leaves in virus suspension and shade dry

Allow the caterpillar to feed for 2 days and subsequently on untreated leaves

Collect the diseased larvae in distilled water

Allow to putrefy

42
5 days
Macerate in
Polyhedra settles at bottom as white layer
blender
as white layer
Decant
Supernatant
and discard
Filter through muslin cloth
Sediment contain POB

Suspend in distilled water

Centrifuge for 1 min at 500 RPM

Discard pellete (only tissue)
Supernatant containing POB’s

Centrifuge at 2500 RPM for 15 MIN

Discard supernatant
Collect pellet (POB’s)

Resuspend in distilled water

Repeat differential centrifugation

Pure POB’s
The dose of virus is expressed as larval equivalent (LE) and one LE is 6 x 10 9 POB. One LE
can be had from three fully grown up and virus infected larvae.
02] Mass production of Heliothis armigera
Pre starve fourth instar larvae for 8h

Dip the head of larvae in NPV suspension containing 108 POB/ml

Rear larvae individually in semi synthetic diet (or) water soaked bengal gram

Collect the diseased larvae in distilled water

Purification of virus as in S. litura.
43
Cytoplasmic Polyhedrosis Virus (CPV)

Spherical virions occluded singly in polyhedral inclusion bodies.

Site of multiplication is cytoplasm of midgut epithelium. (eg.) CPV of Cabbage looper
(Trichoplusia ni).
Granulosis virus (GV)

Virons (oval or egg shaped) are occluded singly in small inclusion bodies called
capsules.

Site of multiplication is either cytoplasm or nucleus of epidermis, trachea and fat
body.
(eg.) GV of early shoot borer of sugarcane. It is virulent and pathogenic to all larval
stages of the host insect. The virus is also transmitted to off springs through
diseased adults.
Symptoms : The larvae become sluggish, yellowish or pinkish in colour, swell and become
flaccid, integument become very fragile, and rupture to release the polyhedra. Dead larvae
are found hanging by their prolegs from the top of the host plant. Dead cadaver is black.
Mass production of GV
Collect fourth instar larvae

Prepare virus suspension at 107 – 108 inclusion bodies/ml

Feed the larvae with a drop of virus suspension through a pin head or by dipping
the head of the larvae in virus

Rear the larvae in sugarcane bits @ 3/plastic box (7.7 x 6.4 cm)

Collect the diseased larvae in distilled water (or) in 0.1%
Sodium Dodecyl Sulphate (SDS) and stored at 50C.
Purification
Macerate the infected larvae in distilled water (or) 0.1% SDS

Filter through muslin cloth

Centrifuge at 500 rpm for 2 min : Discard the sediment

Centrifuge the supernatant at 10,000 rpm for 30 min; Discard the supernatant

44
Resuspend the pellet in small volume of water

The virus can be stored by suspending in distilled water in amber coloured bottle
in a cool dark place.
Baculovirus preparations for pest control
Elcar
- NPV of Helicoverpa sp.
Gypcheck
- NPV of Gypsy moth
Agrovir
- GV of Agrotis segetum (Cutworm)
INSECT PATHOGENS
Entomopathogenic bacteria
Entomopathogenic bacteria
Spore producers
Non-spore producers
E.g., Pseudomonas spp.
Obligate spore producers
Facultative spore producers
e.g., Bacillus popillae
Crystelliferous
Non-Crystelliferous
E.g., Bacillus thruringiensis
E.g., Bacillus cereus
Bacterial pathogens used for insect control are spore forming, rod-shaped bacteria
in the genus Bacillus. Pathogenic bacteria of insects which have potential for use in
biological pesticides are limited to three species of spore forms, i.e., Bacillus thuringiensis,
B. sphaericus and B. popillae
The credit for discovering B. thuringiensis goes to a japanese scientist S. Ishiwata in
1901, who isolated the bacterium from silk worm (Bombyx mori) larvae suffering from a
disease called Flacherie. A similar organism was isolated later in Thuringe (Germany) by E.
Berliner in 1911 from a dise cased flour moth, Anagasta, kuehniella. He named it Bacillus
thuringiensis after the province of Thuringen.
Mode of Action
Life cycle of bacterium is considered in two stages. There is vegetative cell, division
in the manner of most bacterial species, when growth conditions are optimal. Sporulation
occurs when evironmental conditions change, resulting in low nutrient levels of desiccation.
It is during the process of sporulation that the -endotoxin is produced. Spore germinates
in response to the improved conditions in environment and initiates the process of
45
vegetative cell division once more. If the target insects to be effective, the insects must eat
bacterial insecticides, as they are not effective as contact poisons.
The crystal comprises a protoxin protein of 130 kilodaltons, which is solublized in
the alkaline pH of the larval midgut and subsequently cleaved enzymatically to active toxin
of 60 – 70 kDa. The toxin diffuses through the peritrophic membrane lining the gut and
binds to receptors present in midgut epithelium. The gut is paralysed and the insect stops
feeing. The toxin is thought to insert into the membrane with the resultant formation of the
pore. The pore interferes with the inward potassium in gradient and results in swelling of
microvilli.
These cells eventually lyse. The leakage of ions from the gut o haemolymph results
in ionic imbalance in haemolymph. Poisoned insects may die quickly from the activity of the
toxin or they may stop feeding and die within 2 or 3 days from the effects of septicemia
(blood-poisoning). The disruption to the gut may also permit invasion of the haemolymph
by the bacteria, finally resulting in death.

The anti-feeding effect of the activetoxin is an important-feature of Bt.

Majority of Bt. products are based on the sub-gspecies Kurstaki strain HD-I

First commercial microbial pesticide is Sporeine® containing Bacillus thuringiensis
in late 1930’s.

First Insect Pathology Laboratory was established in 1945 at the University of
California.

Commercial formulations of Bt. products are Dipel®. Thuricide®, Biobit®, Javelin®
and Halt® etc.

So far only one crop pest, the diamondback moth has evolved resistance to Bt. in
open field populations (Tabashnik, 1994).

Bacillus thuringiensis Var. kurstaki is only effective agaisnt caterpillars of butterfly
and moth

Bacillus popillae Var. popilliae (milky disease) kills Japanese beetle larvae

Bacillus thuringiensis Var. israelensis kills larvae of mosquitoes and certain flies

Bacillus sphaericus is especially active agaisnt larvae of mosquitoes

Bacillus thuringiensis Var. tenebrionis are toxic to few beetles
4. BACTERIA
Bacteria are single celled organisms that reproduce by fission. Those which affect
insects are broadly classified as spore formers and non-spore formers.
Spore forming (Obligate) : Bacillus popillae attack only beetles of family Scarabaediae. It
causes milky disease in Japanesebeetle, Popillia japonica. It produces endospores which
upon ingestion by the susceptible host, germinate in the gut and the vegetative cells invade
46
into the haemocoel where thy multiply and sporulate. At this point the blood becomes milky
white, hence the term “milky disease”. After the death, host disintegrates and spores are
released into the soil.
Commercial product : Doom a product promising against white grubs Holotrichia spp. in
groundnut.
Spore forming (Facultative) – Crystalliferous
In addition to endospores, produces a proteinaceous parasporal Crystal in the
Sporangium at the time of sporulation. The crystals contain an endotoxin capable of
paralyzing the gut of most lepidopteran larvae. The toxin is known as delta-endotoxin. It is
the most widely exploited microbial control agent. Lepidoteran larvae with a gut pH ranging
from 9.0 to 10.5 are most susceptible.
i)
B. thuringiensis var. israelensis is used against mosquitoes and black flies. The
commercial products available are Thurimos, Vectobac.
ii)
B. thuringiensis var. Kurstaki is used against caterpillar, looper and semilooper
pests. Products available are Delfin, Dipel. Thuricide, Bactucide, Bactospeine. Other
products and the pests targeted are
Halt :
- Cabbage DBM
Biolep, Bioasp- Cotton Amercian boll worm, pink boll worm and spiny and spotted
boll worms
Delifin WG
- Cotton prodenia
Biobit
- Lepidopteran caterpillars
iii)
B. thuringiensis var. thuringiensis used against Flies. Product available is Muscabac.
iv)
B. thuringiensis exotoxin used against mite and Dibeta.
v)
B. thuringiensis var. tenebrionis used against weevils as M-One.
vi)
B. sphaericus used agaisnt mosquitoes and blackflies which include the most
serious vectors of tropical diseases, marketed as Sphaerimos.
B.t. also produces other toxins viz.,
Beta-exotoxin
: Thermostable fly toxin
Alpha-exotoxin
: Heat labile exotoxin
Gamma-exotoxin
: Heat labile exotoxin
B.t. strains producing beta exotoxin is prohibited in USA by EPA due to its relatively high
mammalian toxicity.
Spore forming (Facultative)- Non-crystalliferous
Bacillus cereus a common spore former and soil inhabitant, effective against
Coleoptera, Hymenoptera and Lepidoptera.
47
Non-spore forming bacteria
The digestive tracts of most insect contain many non-spore forming bacteria, many
of which are potential pathogens when introduced into insect blood.
(eg.) Serratia entomophila on grass grub in New Zealand.
Disease symptoms : Reduced feeding and reduced activity, fluid discharge from anus and
mouth, body becomes dark or black and finaly septicemia. Exceetory system is affected,
body cells get disintegrated, resulting in nervous system non-coordination.
Bacillus thruringiensis
Bacillus thruringiensis var. kurstali, which kills the caterpillar stage of wide array
ofbutterflies and moths. Avances in biotechnology improved the prospects for placing Bt
toxins within crop plants in a variety of ways. Genes responsible for production of Bt toxins
is incorporated into soil dwelling bacteria. When these altered bacteria grow and multiply
with in the plant, the Bt toxin is expressed with in the plant. So current plans are in
progress to develop transgenics in other words genetically modified crops that show
resistance to different crop pests.
Bt srains are highly specific in their activity like for examples
- Bt Var. kurstaki – caterpillars
- Bt Var. aizwai – wax moth larvae
- Bt Var. israelensis – mosquito, black fly and fungus gnat larvae
- Bt Var. tenebrionis-beetles (Colorado beetle larvae and elm leaf beetle larvae)
Table of Crystal proteins
Crystal proteins
Orders effective
1. Cry-I
Lepidoptera
2. Cry-II
Lepidoptera and Diptera
3. Cry-III
Coleoptera
4. Cry-IV
Diptera
5. Cry-V
Lepidoptera and Coleoptera
- First commercial product of Bt is Sporeine from France in 1938
- Kurstaki isolated highly potent strain in France (1962)
- First field release of Bt cotton was in 1996
- In India, the Bt cotton was released for commercial cultivation in 2002
- The estimated area of global area of genetically modified crops is 67.7 M. ha (2003)
- The GM crop which is widely grown is soybean for herbicide tolerance against Glyphosate
(Roundup)
48
- The maize transgenic crop is cultivated for the character of insect tolerance against
European corn borer, Ostrinia nubilalis which is a major pest in USA and other European
countries but this pest is not found in India.
- The cotton transgenic crop is cultivated for the character of insect tolerance against
American bollworm, Helicoverpa armigera which is a major pest in cotton growing areas
- Bt gene, Cryl Ac is inserted into cotton crop which protects cotton crop from American
bollworm attack
- Agrobacterium was considered as the vector for plant DNA transfer which was successful
for cotton gene transformation
Table 1 : Widely grown transgenics crop in world
Crop
Area (Million
Percent
of
global
Rank of crop
heactares)
GM area
Soybean
41.4
61
1
Maize
15.5
23
2
Cotton
7.2
11
3
Canola
3.6
5
4
Potato
NA
~1
5
Source : Economic times, 14th January, 2004
Table 2 : Transgenics produced for different trait
Character
Percent of transgenics so for produced for
this character
Herbicide tolerance
70
Insect tolerance
28
Quality traits
1
Status of genetically modified plants in India

The first experiment on transgenic plants in the field was started in 1995 when
Brassica juncea plants containing Barnase, Barstar and Bar genes were planted at
Gurgaon in Haryana.

Mahyco introduced Monsanto’s Bollgard® Bt gene into the Indian cotton hybrids
by backcrossing with a transgenic line. This Bt cotton confers protection against
Indian cotton bollworm, Helicoverpa armigera.

The area under Bt cotton in India is 1,00,000 ha in 2003 (Economic times, 15th
January, 2004).

The “Refuge” strategy is followed to avoid resistance development in Helicoverpa
armigera where 20% of the area cultivated should be under non Bt cotton.
49

Mahyco in collaboration with Monsanto (USA) released Bt cotton varieties as MECH
(Mahyco Early Composite Hybrid) series for different regions of India like MECH-12,
MECH-162, and MECH-184 for south and central India whereas MECH-912 for
north and central India.

Genetic Engineering Approval Committee (GEAC) is responsible for monitoring of
genetically modified crops cultivated in India which comes under Ministry of
Environment and Forests.
Development of Transgenic plants against various insect pests
Crop
Gene
Target pest
Rice
crylAb
Chilo supressalis
Rice
crylAc
Yellow stem borer:
Scirpophaga incertulas
Potato
cry 13a
Colorado potato beetle:
Leptinotarsa decemilineata
Tomato
cry 1Ac
Fruit
borer
:
Helicoverpa
armigera
Brinjal
cry 1Ab
Shoot and fruit borer :
Leucinodes orbondalis
5. FUNGI
Facultative parasites, multiplied using artificial media. Fungal infection is called
mycosis. Fungi produces endotoxins which causes the insect death. Entomogenous fungi
belongs to four main classes of fungi, fiz.,
Ascomycetes
- Cordyceps
Basidiomycetes
- Septobasidium
Deuteromycetes
- Beauveria, Metarhizium, Verticillium, Asprgillus, Hirsutella
Phycomycetes
- Coelomomyces infecting mosquitoes Entomophthora
infecting aphids
Deuteromycetes and Phycomycetes are most important.
(a) Green muscarding fungus – Metarhizium anispliae
Used on coconut rhinoceros beetle, rice leaf folder and BPH. It infects all the stages
of insect. Infected body is mummified and shrunken from original ‘C’ shape, becomes dried
to hard structure, body covered with dark olive green powdery mass of spores. It is mass
multiplied in carrotbroth. Commercial products available are Metaquino and Bio-1020.
50
Mass production : Carrot broth method
Take 40 g of carrot bits in 250 ml conical flask
with 65 ml of water

Autoclave at 20 Psi for 30 min

Cool and inoculate with the fungus

Fungal can be applied to manure pit after a fortnight
(b) White muscardine fungus – Beauveria bassiana
Attacks, silkworm, Spodoptera and Castor semilooper Body become soft and
breakable, body get dried and giving a milky liquid Common product Boverin.
(c) White halo fungus – Verticillium lecanii
Infects coffee green scale, Coccus viridis and green house whitefly. Infected body is
mummified, becomes hard and covered with filamentous white hypae.
Mass production
Take 65 g of sorghum grain in 250 ml conical flask
with 25 – 30 ml of distilled water

Autoclave at 20 Psi for 30 min

Cool and inoculate with fungus

Fungal culture can be used after 3 weeks of growth
(d) Hirsutella thompsoni is used for the control of citrus rust mite. Product available is
Mycar.
Entomopathogenic fungi

Louis Pasteur, was the first to use fungus on grape vines in the vine yards to
control the tiny inhabiting insect.

E. Metschnikoff (1879) & J. Krassiltchik (1888) produced the fungus, Metarhizium
anisopliae to control the wheat cockchafer, Anisoplia austriacea and sugarbeet
weevil, Cleonus punctiventris.
Mode of action : In contrast to oral route of infection by which entomopathogenic bacteria
and viruses enter th host insect, entompoathogenic fungi are able to penetrat directly the
outer integument. Once its attaches to the host, fungus penetrates the insect body wall
with the help of hyphae produced from the spores. The invasion of the hyphae in the cuticle
51
is through wounds, joints between segments or through sense organs. The fungal enzymes
that mention the cuticle accelerate the physical process of penetration. Once the hypha
enters the body’s circulatory system, a number of options are available for the fungus. The
cause of the insect’s death is extensive fungal growth in the haemolymph and poisoning by
fungal toxin.

Fungal formulations are usually made up of growing hyphae and spores.

Activeingredients are mixed with bentonite, kaolin clay or a carrier base.

Formulations are supplied as wettable powder or as a dust for spraying.
Commercially used fungi for insect control
Fungi
Target pest
Trade name
Beauveria bassiana
Cotton bollworms
Boverin®
Coffee berry borer
Hirsutella thompsoni
Phytophagous Mites
Mycar®
Metarrhizium anisopliae
Sugarcane pyrilla,
Biomax®
Rhinoceros beetle
Verticillaium lecanii
Whiteflies,
Aphids
and
Vertilec(®
Scales
Nomouraea rileyi
H. armigera, Achaea janata,
S. litura
Aschersonia aleyroides
Whitefly
--
Pandora delphacis
BPH and GLH of rice
--
Protozoans
Protozoa : The insect larvae infected with protozoan shows symptoms of soft body and
the body is easily breakable.
E.g., Farinocystis tribolii infects Rust red flour beetle, Tribolium castaneum
6. PROTOZOANS
Protozoa kill the insects either directly or by reducing the fecundity of the adult.
Their effect on the host is Chronic. They prolong the larval life in the field, thus exposing
the insect longer to predators and parasitoids. There are called debililating infections. They
are always associated with other insect pathogens.
e.g. Neogregarines Mattesia grandis against cotton bollowrn Microsporidians Farinocystis
triboli against red flour Nosema heliothidis on cotton American bollowrm Noseme
melolonthae on June or Chaffer peaster.
Rickettsiae : These are the microorganisms that are intermediate between viruses and
bacteria. These are obligatory pathogen, which attacks midgut epithelium.
E.g., Rickettsiella melolanthae infects lamellicorn beetle.
52
7. RICKETTSIAE
Rickettsiae are intermediate microorganisms between bacteria and virus. It require
long time to kill the host and are not considered for rapid insect control. Host specificity of
rickettsia is low and are pathogenic to arthropods and also to warm blooded vertebrates.
(eg.) Rickettsiella melolonthae on the grub of lamellicorn beetle causing Lorsch disease.
8. NEMATODES
Most nematodes infect their insect host as juveniles, entering through cuticle or
midgut. In the insect body nematodes under goes a rapid growth, leaves the host, enter the
soil and moults to form an adult nematode. Important entomogenus nematodes belong to
three families of the Phylum Nematoda are
Merminthidae
- Agamermis decandata on grasshopper
Rhabditidae
- Neoplectana carpocapsae known as DD-136. It serves as a carrier
for
the
bacterium
Achromobactor
nematophilus
which
causes
septicemia to the host.
Tylenchidae
- Heterotylenchus.
53
Lecture No. 4 (Unit I)
Handling of Natural Enemies
Collection, Preservation and Shipment of Biotic Agents
Insects are found practically everywhere and occupy about 75% population in the
animal Kingdom. Certain groups of insects are found only in very special places like plant
galls, flowers, nodes of leaf roots, stem, mushrooms, decaying fruits, under loose bark of
trees, dry leaves, stones, trashpiles and rotting logs and are attacked by parasitiods and
predators. The untrained person can go through habitats literally swarming with insects
and be totally unaware for their presence unless he/she is bitten or stung by these
creatures. Several authros have highlighted various devices viz., net, aspirators, killing
bottles/ jars, traps, beating sheets used for collecting the insects in general, especially
order-wise, their preservation and shipment (Peter son, 1948; Oldroyd, 1970, Borror, De
Long and Triplehorn, 1981).
Collection :
There are few points that will sharpen one’s powers of observation and perception,
test one’s skill and patience and provide pleasure of collecting insects, as with hunting,
fishing or bird-watching. Similarly, parasitoids and predators can be searched along with
their hosts prevailing in their specific haultats.
Collection of host larvae of chewing insects and their natural enemies :
1)
Caterpillars feeding externally on leaves, buds or flowers can be collected by beating
on a white sheet or sweeping.
2)
Caterpillars inhabiting inside the galls, leaf miners, stem tunnelers, borers of twigs,
shoots, buds, flower-heads, fruits or roots, etc. can be taken out easily by carefully
opening the plant parts.
3)
Sometimes larvae can be obtained by way of manipulating decaying vegetables,
fruits, animals and fecal matter.
4)
Immature stages of aquatic insects can be collected by aquatic dip net hand sieves,
enamel trays, buckets and glass jars.
5)
Larvae of insects associated with stored products can be obtained by breaking the
seeds or careful manipulations of the stored commodities.
These larvae may be either reared in the insectary, if possible on artificial medium
or on natural food plants and observed for emergence of natural enemies. Immature stages
of parasitic or predatory forms can also be obtained by collecting the infested hosts from
the field and dissecting them under a stereoscopic binocular microsecope especially for
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
54
taxonomic studies and not for culture initiation. Thus several egg-larval, larval parasitoids
and larval predators can be collected.
Collection of adult hosts and adult natural enemies :
Certain lepidopterans, neuropterans, hemipterans, coleopterans, etc. are also
attracted to the light source / light trap and may be collected and used for developing the
insectary. They can easily be spotted in the field especially collinellid predators which are
predatory in both, larval and adult stages and are more active during bright sunny hours
10 am – 3 pm.
Collection of piercing and sucking insects and their natural enemies
The piercing and sucking insects like aphids, jassids, whiteflies, scale insects and
mealybugs remain outside and with their mouthparts pierce through the epidermis and
suck the sap. Movement of ants and mould formation indicates the presence of these pests.
They may be collected as follows :
Table 1: Survival and seasonal occurrence of true aphidophagous coccinellids at Delhi
Ladybird predator
Preferred
Season
Plant Host
Prey
Aphis craccivora
Cowpea
Kharif
Lipaphis erysmi
Mustard
Rabi
Non-aphid prey
Sorghum
Summer
Brumoides suturalis
Aphis gossypii
Gourds
Kharif
(Fabricius)
A. craccivara
Beans
Rabi
Non-aphid prey
Sorghum
Summer
Adonia varieagata (Goeze)
Aphid/non-aphid
Complex*
Coccinella septempunctata
Aphis gossypii
Cotton
Kharif
Linnacus
L. erysimi
Mustard
Rabi
Non-aphid prey
Sorghum
Summer
A. gossypii
Cotton
Kharif
Brevicoryne brassicae
Cabbage
Rabi
Non-aphid prey
Sorghum
Summer
Complex*
C. transverssalis Linnaeus
Complex*
Menochilus sexmaculatus
A. craccivaora
Cowpea
Kharif
(Fabricius)
L. erysimi
Mustard
Rabi
Non-aphid prey
Sorghum
Summer
Sorghum
Kharif
Complex*
Micraspis discolor
Rhopalosiphum maidis
55
(Fabricius)
R. maidis
Maize
Rabi
Non-aphid prey
Sorghum
Summer
Propylea dissecta
A. craccivaora
Cowpea
Kharif
(Mulsant)
R. maidis
Maize
Rabi
Non-aphid prey
Sorghum
Summer
R. maidis
Maize
Kharif
L. erysimi
Mustard
Rabi
Non-aphid prey
Sorghum
Summer
Complex*
Scymnus spp.
Complex.
Fauna of sorghum mainly Perigrinus maidis, Atherigona soccata, Chilo partellus, immature stages of the
above predator and other insects, mites of minor importance as well as pollen grains from associated
weeds.
1. Nymphs and adults of jassids, whiteflics may be collected through net or aspirator.
2. Colonies of aphids and coccids (scale and mealybugs) may be removed alongwith the
plant parts and caged on natural foods in the laboratory. Subsequently, these may
be kept under observation for the emergence of natural enemies. Several predators
viz., coccinellid, syrphids, neuropteran, etc. in varous Tages like egg, larval, pupal
along with secondary parasitoids hyperparasitoids on these predators especially on
grubs and pupae may be recovered. Likewise, different groups of parasitoids on
these pests may be obtained without disturbing the hosts as immature parasitoids
complete their life cycle within the host body. As and when coccinellid grubs and
aphid lions (larvae of neuropterans are noticed along with the field collected hosts,
should be removed and kept in individual vial as their larvae are cannibalistic in
nature.
Collection of insect eggs and their natural enemies
Collection of eggs of phytophagous insects viz. lepidopterans, hemipterans,
Coleopterans and dipterans is done mainly on the basis of visual observations. However,
location of the eggs laid by these insects may be explored based on the sound knowledge of
biology and bionomics of the pest.
The egg-masses/eggs collected from the plant / plant habitat are brought to
laboratory and kept under observation for the development of egg and egg-larval parasitoids
mainly and occasionally for the predators or hyperparasitoids. The egg collection of
entomophagous
ptermalids,
insects,
spalangids,
like
Chalcidoids
culophids,
(trichogrammatids,
aphelinids,
encyrtids,
mymarids,
cleonymids,
elasmids,
siniphorids,
euplemids, callimomids, curytomids, miscrogasterids, perilampids,l eucharids, chalcidids,
leucosphids) ichneumonids, braconids and dipterans is either cumbersome or difficult as
56
are mostly internal. However, when laid on the host body or apart from the host body, they
hatch with in a day or two, hence collection becomes practically impossible. Therefore, they
may be reared in order to get their progeny without disturbing them.
Collection of coccinellid eggs
Predatory ladybird beetles lay their eggs mainly in egg masses on the plants, weeds
and under the most field soil and may be collected through visual examination. The identity
of particular predator species is easily done on the basis of their laboratory rearing during
larval / adult sage.
Collection of neuropteran eggs
Often these eggs (individual or in groups) remain hanging through its stalk attached
to the plant hosts and may be searched with little efforts.
Collection of epipyropid eggs
Metallic coloured egg-masses of Epiricania melanoleuca an external parsitoid of
Pyrilla perpusilla Walker may easily be searched on plant host leaves (sugarcane, paddy,
sorghum, maize, etc.) and processed for conservation as laboratory rearing of the parasitoid
on alternate host is not so for explored.
Collection of mantid eggs
The mantid eggs (ootheca) are often encountred attached to the twigs, in orchards
and are well known. These may be brought to the laboratory for further studies along with
stick.
Collection of syrphid eggs
The scattered eggs are normally found in between the aphid colonies developing on
the plants and may be removed gently with the brush or picked up alongwith the leaf.
Collection of eggs of hemipteran predators
The pyrrhocoreid predators, Antilocus coqueberti lays eggs in the groups of 80-120
loose eggs in the crevices of field soils near the plant host and may be searched by digging
the soil near the plants like cotton, okra, hollyhock. These eggs look like the eggs of red
cotton bug hence are reared for determining their identity.
Predators of pentatomid, lygaeid, mirid, anthocorid and reduvid, normally lay their
eggs on plant parts in groups.
In general collection and determination of identity of eggs or larvae or pupae of
entomophagous insects remains in question as taxonomy of immature stages is less
developed. Often rearing helps in identification that too through the taxonomists. Hence
some of the natural enemies introduced in our country (Appendix-I) / being reared in
various laboratories (Table – 2) may be procured while initiating the projects on biological
pest suppression in order to get pure and identified culture.
57
Preservation
Natural enemies killed with a paper strip dipped in benzene, or KAAD mixture
(Kerosene – part, 95% ethyl Alcohol 7 – 10 parts, Glacial acetic acid – 2 parts, Diaxone 1
part) can be preserved in 75% ethyl alcohol, after washing in 70% alcohol. For permanent
storage of specimens, a mixture of 75% ethyl alcohol (95 parts) as well as glycerine (5 parts)
is suggested. If parasitic forms are dissected from the host body in saline water, such
immature stages should be passed through 25% and 50% alcohol before being transferred
into 75% this will avoid shrinkage and can be stored for a longer period. Constant
supervison is needed to avoid drying of the specimens due to evaporation of alcohol and
should be added as and when needed. Small gellatinous capsules normally used for
medicines can be used for storing the parasitoids. These capsules are perforated at 4 – 5
places with a fine needle before putting specimens inside. Finally capsules which need less
space as compared to vials are dipped into the glass bottle containing alcohol (75%) and
secured tightly. According to Peterson (1948) host larvae containing parasitic forms when
killed in KAAD mixture and preserved in 95% alcohol remain in good condition even after
considerable long periods and the stages of internal parasitoids can easily be determined.
Certain parasitoids, predators and their cocoons / puparia can be mounted on
paper strips (carding) as these are convenient to handle and preserve for further studies
(Novitzky, 1956; Borror, De Long and Triplehorn, 1981; Noyes, 1982) sometimes minule
stages may be preserved in the form of permanent slid also.
Predatory mites
1)
Keep a drop of mounting media like Hoyer’s chloral hydrate (Berlese’s fluid) which
contains gum arabic-30g, distilled water 50 cc, chloral hydrate – 200 g and glycerine
– 20 cc on the slide and place 3 – 4 specimens of smaller arthropods and cover it
with coverslip.
2)
Heat gently on flame unless bubbles start coming out. Observe under stereoscopic
binocular for air bubbles. Re-heat if bubbles remain till it is cleared.
3)
Dry in oven at 40 – 500 C for 24 – 48 hrs.
4)
Take out slides from oven and seal with cutex or some other water proofing material
viz., DPX, Gyptal etc.
Adult parasitoids
1)
Take an adult parasitoid preferably alive one and give gentle touch of 10% KOH
(Potassium hydroxide solution)
2)
Add fine droplet of glacial acetic acid and fuschin stain.
3)
Treat with clove oil a mount in Canada balsam.
58
Immature stages of natural enemies
It is difficult to prepare permanent slides of immature stages of insect parasitoids
and certain predators as these become transparent and unvisible on long term storage.
However, a skilled and devoted worker can attempt to prepare good slides based on
experiences, following under mentioned points.
1) Dissect the material into Bouhin’s solution and keep specimen in cavity slides.
2) Steps 1 and 2 as under adult parasitoids.
3) Treat with Carboxylol and go on adding Canada balsam simultaneously to avoid
shrinkage and mount it.
For studying purposes the temporary slides may be prepared by way of dissecting
the specimens in Bouhin’s solution and mounting into glycerine.
Composition of Bouhin’s solution
Picric acid (saturated aquous solution )
45 ml
Glacial acetic acid
5 ml
Ethyl alcohol
45 ml
Formaldehyde (40%)
5 ml
Packing and shipment
Packing and shipment of natural enemies and their host is a common practice
followed either for determining the identity of the dead specimens or for supplying the
nucleus culture of certain parasitoids, predators and their alternate laboratory hosts. Live
insects should be sent in plastic containers or card board or thermocol packing (Fig. 4) with
proper labelling and sent through air mail so as to reach the destination quickly and safely.
Fig 4.1 : Thermocal packing (Consigment).
Some of the precaution should be undertaken while mailing (Shipping) of live insects.
59
1)
When tin/plastic container is used for packing, the lid should be perforated with
numerous small holes.
2)
Never pack small insects like Trichogramma, Telenomus, inside the tin or plastic
containers without using tubes as are very minute and escape through the holes.
Prefer to send parasitised Trichocards, rather than adult parasitoids.
3)
Keep standard size of the container which is easily acceptable to the post office.
4)
Always put/smear a line of honey as adult food inside the tubes for chalcidoids
before packing.
5)
Always use a food media (honey + agar) for cocinellid predators while preparing the
parcel. These lady birds may continue to feed on this media while on travel and
survive for a longer period.
6)
In case of puparia of tachinid parasitoids packing should have either a layer of moss
grass or sponge in bottom followed by a layer of cotton placed on the top of the
container.
7)
For packing and shipping the larvae which have cannibalism in 2nd or 3rd instar,
every larva should be kept in individual vial alongwith sufficient and suitable food.
8)
The parcels / packets should always be labelled with labels like “Handle with
Care”, “Live Insects”, “Quick Delivery”, “Avoid Heat”, etc.
9)
For predcators like neuropterons’, shipment of its eggs alongwith the prey (fridged
eggs of rice meal moth) and strips of corrugated papers should be preferred. So that
even if there is hatching during transaction the predator larvae will get prey and
hiding place and avoid cannibalism.
10)
Avoid sending the host larvae and prefer pupae. If larval shipment is essential
provide sufficient food for them along with damp moss, tissue paper or muslin in
order to avoid decomposition due to succulent food material.
11)
Always pack newly laid eggs/ newly formed pupa / newly emerged adults so as to
reach destination safely even if it is delayed in transaction due to unavoidable
circumstances.
12)
Keep the details about insect viz., host, locality, name of collector, date of collection
for getting the identity while name of the insect, medium on which reared, number
of insects, date of hatching, emergence / food material / medium or host required
for rearing etc. for initiating the culture in new laboratory. It should follow a letter
separately as per the norms of the postal services where in further details alongwith
a counter slip for acknowledgement may be given.
13)
Always write / type address in bold words, clear and correct for quick disposal and
delivery.
60
Important points to remember
1) When using entomopathogens

Correctly identify your pest.

Choose the correct variety / strain of microbials for the pest to be controlled.

Older caterpillars are more difficult to control than young ones. Monitor plants
frequently and make applications when larvae are young.

Some of these are inactivated by sunlight and has a short activity period. Multiple
applications may be necessary for a continuing pest problem.

Carefully read the label and follow all directions regarding storage, mixing,
application and safety. Remember that the pesticide label reflects the laws; uses and
practices, not authorized on the label are illegal.
2) When ordering natural enemies

Know the specific pests you need to control.

Know the best natural enemies, either single or in combination, for the target pest
or pests.

Know the proper timing of release of the natural enemy, based on the life cycles of
both the pest and natural enemies.

Know the proper release rate for each natural enemy.

Calculate the amount of natural enemies needed, based on release rate, area to be
covered, and severity of the pest infestation.

Know the recommended frequency of release if multiple releases are necessary.

Provide a safe delivery address, where the shipment will be carried for as soon as it
arrives and where it will not be exposed to temperature extremes.

Understand proper release practices so that you will be prepared to make releases
when the shipment arrives.

Understand proper storage requirements, if releases are not to be made immediately
after arrival.
3) When you receive a shipment of natural enemies

Minimize exposure to hot or cold temperatures.

Carefully inspect the shipping container before opening, and note damage that likely
occurred during shipment.

Carefully inspect contents for damage.

Determine if you received the species and quantities you ordered.

Attempt to assess the quality of the natural enemies.

Contact your supplier immediately, if you are aware of any problems.

Make releases as soon as possible after receipt.
61

Store under proper conditions, if it is necessary to delay releases.

Make releases based on methods recommended by your supplier; releases thould
usually be made during a cooler part of the day and when rain is not imminent.

During release, check once again for quality characteristics.

Attempt to evaluate success of your release by monitoring natural enemy
populations and impacts on pest numbers.
4) When assessing the quality of natural enemies, evaluate the following
characteristics
1) Survival
2) Vigor
3) Quantity
4) Sex ratio
5) Proper species.
5) Consider the following factors when planning biological control programme
1) Effectiveness of the natural enemies
4) Cost
2) Sustainability
3) Safety
5) Environmental constraints to success.
Future Thrust
The use of biological control has become an accepted and established practice.
However, relatively few types of natural enemies are commercially available in India, and
these are targetted against small number of pests.
In the future, we expect to see more private companies, commercializing biological
control, from insectary production of biocontrol agents to providing pest management
consultant in the field. We also see a great potential for the development of grower
cooperatives for mass-producing and applying bioagents. Therefore, scientific research
must be combined with practical experience to determine efficacy of those agents how to
incorporate them into pest management programme and how to mass-produce them
qualitatively so that it will be cost effective. The number of commercially available natural
enemies is increasing and it will continue to do so as long as farmers see alternatives to
chemical control.
The Indian Council of Agricultural Research and the Department of Biotechnology of
the Government of India are extending ample support for research and development for
Biological Control of Insect Pests. The Government of India should extend further support
for indigenous commercial production of natural enemies / microbials by encouraging
private entrepreneurs within the constitutional framework. Educational programmes in
institutions particularly agricultural universities should give special emphasis on
development of biocontrol as one of the important aspect of IPM. Further, there is a need to
enhance inter-institutional collaborations in order to pool all the resources available to
develop biological control of insect-pest as front area, which would ultimately reduce the
pressure on chemical / insecticidal use.
62
Quarantine Handling of Entomophagous Insects
Introduction
The Purpose of handling intentionally imported entomophagous insects under
quarantine conditions is to prevent any concurrent introduction of undesirable species of
phytophagous insects, hyperparasites, weed pests, or plant diseases. In order to confirm
the habits of newly introduced species, it should be propagated for at least one generation
in quarantine.
In order to implement this objective in California, an agreement among authorities
of the United States Department of Agriculture, California State Department of Agriculture,
and the University of California was reached (Swain 1952) wherein the quarantine
personnel of the Department of Biological Control of the University of California is charged
with the safe handling of such material. Under the provisions of this agreement, shipments
from foreign collecting areas which display the proper federal permits (United States
Department of Agriculture, Agricultural Research Service, Plant Quarantine Branch, form
PQ-21) normally proceed undistrubed through customs and quarantine inspections at the
port of entry to the biological control quarantine laboratory. This results in receipt of
imported material with minimal delay. For example, average travelling time for shipments
sent air mail from Tokyo, Japan to California is three days; from New Delhi, India and
Capetown, South Africa six days.
Importance of the quarantine facility and procedures to biological control projects
can be appreciated when it is realized that proper handling of imported material is essential
in order to obtain the beneficial insects with which biological control entomologists must
work. In the chain of events which make up a biological control programme, quarantine
handling is actually the link between foreign collecting and the insectary production
programme which permits dispersal.
It is no understatement to say that the success or failure of importation
programmes depends in large measure on the degree of training, resourcefulness, and
dedication of quarantine personnel. Thus, it follows that personnel who will perform this
vital function should possess certain basic qualifications, namely. 1) the attitude that each
imported species or strain of primary parasite or predator may contribute significantly to
control of a pest species; 2) profound interest in insect biologies; 3) a general taxonomic
knowledge within major entomophagous groups; and 4) a knowledge of disease symptoms.
Paramount to the foregoing qualifications is complete acceptance of rigid discipline
regarding personal habits and handling procedures in order to eliminate any possibility of
dangerous organisms escaping to the local environment.
63
The quarantine laboratory
The basic function of a quarantine laboratory is to provide facilities which will
permit the handling of imported material in a manner that precludes escape of potentially
dangerous organisms. Ideally, it is a completely equipped self-contained, insect-tight,
highly functional laboratory (Doutt 1951a) – literally an insectary within in insectary. Some
of the finest importation programmes had very simple but highly efficient quarantine
facilities, the effectiveness of which was made possible only by the highest devotion to duty
of the personnel in charge. An excellent example is the quarantine insectary utilized during
the introduction of entomophagous insects into Fiji for control of the coconut moth (Tothill,
Taylor, and Paine 1930 p. 21, plate 14). Similar quarantine field insectaries are currently
used in Australia and Mauritius. Certain Commonwealth Institute of Biological Control
stations utilized a separate building for quarantine use.
64
Figure 4.2 : Floor plan of biological control quarantine facilities. Portions of existing buildings were
remodelled to provide these laboratories.
A) Moorestown, New Jersey (U.S.D.A.).
B) Albany, California (University of California).
A autoclave. AC air con ditioner. EC emergence chambers. L laboratory. LBT laboratorytable. lt light
trap. O office. Q quarantine. QA quarantine anteroom. R refrigerator. S sink. SRY stairway.
Equipment for the handling of insects will be much the same as used elsewhere in
the insectary with the proviso that it be restricted to the quarantine rooms. Because of the
independent nature of the quarantine facility, it is necessary to provide adequate internal
storage for glassware, cages, cloth, and miscellaneous supplies for the manufacture of
propagation equipment.
65
In the event that large quantities of hibernating parasitized host material must be
held in quarantine in order to synchronize the life cycles of the expected parasites with
their locally established hosts, it will be necessary to obtain adequate refrigeration within
the quarantine laboratory. In order to satisfy diapause requirements of European corn
borer larvae, large amounts of hibernating material were held in refrigerated rooms at 34 0
to 360 F for about three months (Baker, Bradley, and Clark 1949, p.48) (Figure 46, facing
p. 320). Household refrigerators will adequately store considerable amounts of hibernating
material but when refrigerated cooling is used it is very important to keep a close check on
the relative humidity as it may become dangerously low.
Desiccation will be minimized if the refrigerant in the cooling coils is approximately
50 below the desired temperature of the cold room or refrigerator.
A univoltine pest species held
at normal temperatures until issue of
its parasite complement may create a
space
problem
in
the
quarantine
laboratory. For the purpose of holding
numerous grubs of Popillia japonica
Newm. pending emergence of Prosena
siberita Fabr., King and Hallock (1925)
used a cage of 22 ft x 7 ft x 6 ft
dimensions. These are two situations
which required special techniques and
they
clearly
indicate
the
need of
strictest quarantine precautions when
considerable quantities of imported
living host species must be held for an
extended period.
(Figure 4.3 : During the introduction of
parasites of the European corn borer it was
necessary to hold in quarantine large
quantities of overwintering material in large
refrigerated rooms (Photo copied from Baker, Bradley, and Clark 1949))
In order to dispose safely of shipping containers and shipment residues, an
autochlave or incinerator should be located preferably within the quarantine laboratory. A
small and fairly inexpensive portable autocalve with a capacity of approximately 800 cubic
inches will suffice for sterilization of material normally received via air mail. Unwanted
shipping materials ultimately should be burned.
66
If it is physically impossible or impractical to build an incinerator into the
quarantine area, it should be located near by for use by insectary as well as quarantine
personnel. In this event, for added safety, material such as shipping containers or bulky
plant material should be transported from the quarantine room to the incinerator in sealed
multilayer paper bags. Conventional cyanide jars may be used to destroy hyperparasites or
other unwanted arthropods as they emerge from shipments.
Procedures for Handling material in quarantine
Examination and Processing of the Shipment
1)
To minimize the possible transport of organisms into or out of quarantine, it is good
practice for quarantine personnel to wear a laboratory coat, particularly during the
processing of new shipments. This coat should be white for easy visibility of insects
which may alight on it and it may hang in the quarantine room between periods of
use. It should never leave the quarantine area without first being fumigated in a
large cyanide jar, or being placed in a heavy paper bag which is then sealed and
promptly carried to the fumigation chamber. Personnel leaving the quarantine area,
i.e. anteroom, should first undergo a decontamination process which may consist of
inspection before a full-length mirror, blowing pressurized air over the garments
and, if necessary, a change of laboratory coat. This procedure should be carried out
where a light trap will attract escaped insects.
Once a shipment has been taken into the quarantine room, it is placed for
opening (size permitting) in a large sleeve-cage, which should be stocked
continuously with a number of vials, forceps, scissors, collection aspirator, etc., for
the purpose of handling the enclosed material.
2)
Opening packages while working through sleeves is important not only in preventing
the escape of insects but also for human comfort as was learned during the
importation of brown-tail moth parasites (Howard and Fiske 1911, p. 164) when it
became necessary to protect personnel from the irritating effects of spines that
occur on the host larvae.
3)
For ease of collecting, packages are opened before a screened ‘transfer cage’ which
traps nearly all the insects as they fly towards the window.
4)
If a small room is not available for the opening of large shipping containers, a cloth
enclosure (large enough to accommodate the package plus the head and shoulders
of the quarantine worker) may be suspended from a light framework within the
quarantine room. Such an arrangement has been used by Australian workers
particularly in the handling of large quantities of fruit fly parasites shipped from
foreign insectaries.
67
Since shipments destined for the University of California quarantine are usually airletter packages, the following comments will pertain chiefly to the processing of such
containers.
1)
After first gently squeezing or shaking in order to detect broken containers inside,
the shipping envelope or wrapping is carefully cut open. If the shipping container is
broken, it is possible that insects may be loose within the package. If collectors have
observed the precaution of enclosing all shipping containers in stout cloth sacks
before placing them in shipping envelopes or boxes, safety in handling at the
receiving end is greatly enhanced.
2)
If breakage has occurred, one end of the enclosing cloth sack is cut open and the
sack is everted, spilling the contents into a suitable container while working
through sleeves.
3)
With unbroken containers the collection of parasites which emerged en route is the
first concern. Usually the packages are opened within the previously mentioned
cage and the parasites or predators allowed to fly to the glass, where they may be
collected with an aspirator.
4)
It is particularly important not to use an anaesthetic at the initial opening of a
shipment if the natural enemies that emerged en route appear to be weak as
expressed by immobility, unsteady gait, or inability to cling to a vertical glass
surface, or if dead insects are in the containers. Certain coccinellids are rather
easily killed by CO2. Weakened insects are safely handled only be gentle aspiration
or by picking them up with a small brush. The vial into which they are placed
should then be provided with a few fine streaks of honey for food, care being taken
to avoid large droplets in which the insects could be trapped.
5)
No inflexible rules apply to the feeding of adult parasites. In practice, honey seems
to provide moisture and food sufficient to sustain most adults for somewhat long
periods. Taylor (1937, p. 234) reports that only enough water should be added to
allow the honey to flow freely. This rule of thumb is probably a good one to follow
since honeys from different sources vary widely in sugar content. At humidities
below 55 per cent most honeys become stiff and must be diluted with water or
glycerine in order to enable the parasites to ingest them.
6)
Occasionally, living parasites are observed mired in honey which may be provided in
the shipping containers. This probably occurs when the parcel is opened and the
contents are suddenly exposed to light, thus stimulating the insects to increased
activity. If this situation arises, a drop of distilled water will float the trapped
68
parasite free of the honey. Then it can be retrieved from the water with a fine needle
or brush and placed in a dry vial where usually it will preen itself dry.
Storing Adult Parasites
Tolerance of adults of different species of parasites to given temperatures is highly
variable and should be investigated for individual species if it becomes necessary to store
them.
Most adult parasites of homopterous pests can be held in vials for several weeks at
550 to 600 F in darkness if they are removed to approximately 800 F for 20 minutes for
feeding and exercise two or three times weekly; on the contrary, Meteorus sp. will tolerate
400 F for 90 days and Mormoniella vitripennis Wlk. has produced viable eggs after five
months at 4.40 C (DeBach 1943a). Care must be taken to replenish the food, usually honey
streaks, and occasionally vials should be changed. In this manner, when males were
lacking it has been possible to hold virgin arrhenotokous females after they have deposited
male eggs and subsequently to mate them with their own male progeny and thereby initiate
a culture. Then, too, subsequent shipments may contain males, thus permitting utilization
of the stored virgins. In general, the best storage temperature is one at which appreciable
visible activity ceases. This will vary with the species.
Incidental Inclusions – Detection of Disease
A side benefit of the importation of expressly sought exotic natural enemies is the
incidental inclusion in the shipments of associated entomophagous species which the
foreign collector sometimes has overlooked or about which he has not known if he has
lacked the time or facilities to determine just what species were attacking the host.
Quarantine personnel should be alert to such unexpected species and where definite host
relationships and preferences are learned (a bonus derived from the effort of initial
segregation) tests should be conducted comparable with those accorded the sought-after
species. The rarest species in a shipment may be an efficient natural enemy in the new
environment.
After emergence is complete and before the material has been fumigated, and as a
final step before sterilizing or incinerating shipment residues, the host material should be
re-examined in detail for evidence of disease. If the services of an insect pathologist are not
locally available, suspect material should be sent to a recognized laboratory of insect
pathology.
Termination of Emergence and Disposal of Shipping Materials and Residues
After parasites begin to emerge from foreign material, the question of how long to
hold it for complete emergence must be resolved, especially if only a few individual
parasites are being reared out. Experience has taught that it is usually time to dispose of
69
host material if emergence of hyperparasites significantly exceeds that of primary parasites
(except in species whose males develop as hyperparasites) or if dissection reveals obviously
dead hosts or dead immature parasites concurrent with a sharp drop in numbers of
emerging primaries. Proper disposal is extremely important, particularly if shipments are
arriving from areas known to harbour plant pathogens which are not present in the area of
the quarantine laboratory. Although federal quarantine regulations specifically prohibit
entry of the plant host or such diseases, no chances should be taken with the residues
Figure 4.4 : (Left). Autoclave – a safe means of disposing of shipment residues.
Already mentioned in the section on facilities and equipment were such items as
cyanide jars, incinerator, and autoclave. Incineration is a safe method of disposal for
shipping containers and wrappings. Glass and certain wood or metal containers can be reused if they are sterilized in an autoclave. If shipment residues are not to be retained for
the collector’s examination upon his return, they, too, should be destroyed by burning.
Recording of quarantine data :
Written records associated with the quarantine handling of imported material are of
utmost importance. The sender’s and receiver’s form has been mentioned as the original
record of importations, and the reader is again referred to figure 51 in order to note the
type of information required. Upon the receiver’s completing the form at the finish of
emergence, the data can e utilized to make a cross index to species of parasite, host insect,
70
host plant, and locality. Names used must be verified by taxonomists. This matter is
elaborated later under ‘Value of Taxonomy to Quarantine Work.”. The receiver’s final entry
on the S. and R. form is a summary of the daily emergence records (figures 48 and 50)
which are initially affixed to the segregated increments. A good practice is to compile a
semi-annual or annual summary of shipments received, along with pertinent biological
data developed in quarantine. Propagation records include daily observations on testing
procedures and become a part of the original data pertaining to importations.
Figure 4.5 : Processing a shipment within an isolation (sleeved) cage, as viewed looking down through
the glass top.
Beginning clockwise with the left hand, items include plastic packet containing parasite material on leaf
fragments, cloth sack which enclosed the packet, shipping envelope, S and R form, collection aspirator,
daily emergence form, the length of Plastic hose is used to jar insects from shipping vials for recollection, forceps, pencil (handy for reaching into vials and killing unwanted organisms), waxed pencil,
brush for handling very frangile or weak insects, scissors, and razor blande for opening wrappings, 8dram vials for segregation of material.
In the Department of Biological Control of the University of California, after a
species has been properly screened and its primary habit established, and after a culture
has been enlarged to the point where it can be consigned to project personnel, a form
entitled ‘Quarantine Release Authorization’ is filled in and countersigned by the chairman
of the department. Copies go to the chairman and the project leader for the purpose of
providing a written record of the transaction, as well as a summary of the biological
information developed in quarantine, such as notes on fecundity, longevity, host-feeding,
sex ratio, and host preference which will be used as a guide by project technicians in the
early phases of developing a mass production programme. As a safeguard, upon releasing a
species, a small insurance stock of the new species is retained in quarantine until project
technicians have their cultures will established.
71
Value of taxonomy to quarantine work
Although in the applied sense the taxonomic status of imported beneficial insects is
secondary in importance to their biological attributes, ultimately it is necessary for
purposes of communication to have names for them.
Accurate specific determination of imported entomophagous insects and their hosts
often involves considerable delay, but since generic identification is usually readily
available, the identity of the insects is retained by use of the S. and R. number and the
shipment item number, e.g., Metaphycus sp. S. and R. 1895-#2. If a large number of the
unknown species is derived from several shipments over a considerable span of time from
the same area, the species may be referred to by a code name such as Aphytis sp. ‘A’,
Prospaltella sp. ‘G’, or Metaphycus sp. ‘C’.
The practical value of rapid determinations is readily appreciated when several
shipments arrive during a brief period and it therefore becomes physically impossible to
propagate all the species represented. Therefore becomes physically impossible to
propagate all the species represented. Therefore, if certain importations can be assigned to
species which were rather extensively studied or tested in the past, they can be deemphasized in favour of the newly imported species in the shipments, they placing
maximum effort where it offers the most promise of applied use.
Since it is virtually impossible to identify moving insects accurately, taxonomic
determinations are made of dead specimens from shipments and from specimens which
died during testing. Specimens of species unknown to the quarantine worker are promptly
sent to a taxonomist, as well as specimens for verification of determinations made in
quarantine.
In order to maintain accurate records it has become a practice at Riverside to affix
vials containing dead insects to the identification memo shown in figure 58. Information
from the collector regarding locality, date, host plant, and information from quarantine
personnel such as S. and R. No., and host preference are placed on this memo which is
then sent to the appropriate taxonomist. The taxonomist returns this form with a
determination, and it is then attached to the original S. and R. report. This procedure
facilitates changing of names in the records occasioned by subsequent taxonomic
information and helps to identify the insect in question, particularly when several
shipments over an extended period are involved. Code names are then related to the
determination.
Relation of quarantine to other activities
The indispensable role of systematics in quarantine work has been emphasized. On
the other side of the coin it can be readily appreciated that the quarantine worker has
something to offer the systematist, for it is his privilege to observe biological traits which
72
may be the first indications that the species he is handling is not necessarily what the
taxonomist determined it to be (Flanders 1953b). Differences may be evident in
characteristics of immature stages when adults appear to be identical. For example,
differences in pupal pigmentation as well as responses to various temperatures led to
critical study of the adults which developed from these immature stages with the result
that the number of known species of Aphytis has been considerably increased (DeBach
1959)
In the event that new species are successfully propagated, the taxonomist receives
adequate series thereof and parallel series can be sent to other taxonomists or museums
for their reference collections.
One important bit of information which the quarantine biologist can contribute
concerns the correct association of males and females of the species with which he is
working, because taxonomic descriptions of parasitic Hymenoptera are primarily concerned
with females, the male often being incompletely described or omitted.
Importations are an obvious source of new host and distribution records.
Unfortunately, this type of information is rather slow in getting into entomological
literature.
The biologies of imported entomophagous insects may be considered of either
academic or practical interest, depending upon the host preference and relationships
involved. In actual practice, species which show promise of exerting some degree opf control
over an agricultural pest, as deduced from preliminary laboratory testing, are consigned as
soon as possible to the projects concerned in order to implement detailed testing, mass
production, and colonization, all of which are beyond the scope of the quarantine
laboratory.
MASS REARING OF HOST INSECT (Corcyra cephalonica Stain.)
Precautions
a. Use low heighted room or cloth cabin for rearing of Corcyra, as the . i escaped moths
resting on the walls and roof ceiling can be collected easily.
b. Use separate room for mass rearing of Corcyra to avoid the iadulteration of the culture.
Blotting paper should be used to collect the eggs to facilitate cleaning.
c. Each tray should be kept for 3 months and after use, clean them with 2% formalin,
d. Use only 200 Corcyra adults per oviposition jar or 1000 adults/drum to avoid
overcrowding during oviposition (cage / drum).
e. Moths should be fed with Vitamin E capsules to enhance fecundity.
f. Treat the Corcyra eggs with UV rays to prevent hatching during . parasitization by the
Trichogramma or to store for some period.
73
The Corcyra eggs are used in biological control as unnatural / factitious host for
mass rearing/ multiplication of certain parasitoids like Trichogramma, Apanteles spp^,
Bracon spp. and predators like Chrysoperla, etc.
Mass rearing of Trichogramma chilonis Ishii
Precautions

To avoid contamination of culture and host larvae feeding on parasitized eggs, the eggs
of the host should be frozen at 0-5°C for 2 hrs in refrigerator or expose to UV light
irradiation for 45 minutes to kill the host embryo as well as contaminating species.

Runts (underdeveloped individuals) can be avoided by keeping the parasite: host ratio
1:6.

Use only 0-24 hrs old eggs of host, Corcyra for assured breeding of the parasites.

In case of need of transfer of the parasitized egg cards, transfer can be done on third
day of parasitization which has given blackenings to the host eggs.

During shipment, Trichocards should be packed keeping the parasitized surface on
inner side.

Probable date of emergence of adult parasitoids should be specified on the cards for
guidance of the user.

Cut pieces of Trichocards should be stapled on the lower side of the leaf to provide
shade and to avoid direct exposure of sunlight.

Card, pieces should be stapled in morning hours and just before emergence to avoid
predation.

In the case of release of adult Trichogramma, open the bag / container after 8 days of
egg parasitization, move it along the rows in cropped areas and go on tapping the bag
till all the aduit parasitoids fly out.

Refrain from using the pesticides in the field where Trichogramma are released. If need
arises, use selective/safer pesticides. Ensure that pesticides are used 10 to 15 days
before or after the Trichogramma release.
74
Mass rearing of egg larval parasitoid Copidosoma koehleri Blanchard
Precautions for handling
Potato tuber moth is a serious pest causing damage to the crop in the field and
tubers in storage. Copidosoma koehleri is a potent egg- larval parasitoid of the pest. Mass
productioi technique of the parasitoid is given below.
Precaution for Copidosoma
a) Select potato tubers free from pesticides treatments.
b) Treat potato tubers before release of parsitized eggs sheet with formaldehyde.
c) Treat rearing jars, basket, glass vials and other equipment with 5% formalin solution to
maintain sanitory conditions.
d) Puncture potato tubers just before hatching of parasitized eggs.
e) Use sufficiently dry and sterilized soil for pupation.
f) Due care should be taken while releasing mummies so that they should not be destroyed
by predators.
Mass rearing of Predators Chrysoperla carnae Stephens
Precautions
• Rear the grub stage individually to avoid cannibalism.
• Release should be made in early morning hours to settle larvae on crop canopy.
• Avoid to release freshly chrysopid laid eggs as they may be parasitized / predated in the
field.
• Do not pesticides in the field where predators are released, other wise use selective / safer
pesticides after or before 10 – 15 days release.
Mass rearing of Predators Cryptolaemus montrouzieri mulsant
Precautions
• Do not feed adult predatory beetles only on honey-agar diet, but supplement with mealy
bugs eggs as it affects fecundity of the predator.
• Avoid insecticide application in the field where predatory beetles are released. If
insecticide spray is necessary, keep 7-10 days gap between release of predator and
insecticide spray, and /or follow staggered spray technique. For this, first spray alternate
tree and release the predator on remaining untreated (alternate) trees after 4 to 5 days.
Fifteen days thereafter, spray predator released trees and release predators after 4 to 5
days on pre-sprayed trees for faster control of the pest by chemical as well as biological
method and conservation of the released predators.
Predators
1. Definition : “A free-living organism throughout its life, it kills its prey, is usually larger
than its prey and requires more than one prey to complete its development.
75
2. Characteristics

A free living organism throughout its life cycle and usually with long life cycle

Requires several prey to complete its development

Larger than its prey

Kill their prey immediately

Usually both immature and adult stages are predatory, except some cases eg.

Blister beetle larvae feeds on eggs of grasshopper, whereas the adult feeds on
flowers.
3. Differences between predators and parasitoids
PREDATOR
PARASITOID
Very active
Sluggish
Stronger and larger
Smaller and not stronger
More intelligent than prey
Not more markedly intelligent than the host
Habitat is independent of the host
Habitat is made and determined during their
development by the host
Life cycle is longer
Life cycle is short
Prey is seized, killed and eaten immediately
Takes longer time to kill the host
Several insects are attacked in a short
Requires only one host
period
Attack the prey to obtain food for themselves
It is for providing food for the offspring
Locomotary organs and mouth parts well
Not so well developed
developed
Ovipositor and oviposition not specialized
Ovipositor well developed and oviposition
specialized
Not specialized in food habits
Exhibit food specialization
4. Adptations in insect predators
Vision : Compound eyes are large
eg. In dragonfly the compound eyes are holoptic with 20,000 ommatidia in each eye to
perceive even the slight movement in the field of vision.
Mobility of the head enhances the vision eg. Preying mantid. Protruberant compound eyes
eg. Robberfly.
Special organs
01]
Mask: Labium is modified as prehensile organ in naiads of dragonfly, which is
otherwise a face mask, to catch prey.
76
02]
Raptorial leg : The prey is cautght between the grooved femur and spiny tibia, where
the coxa is elongated to extend the length of prey capturing. eg. Fore legs of
preying mantids, water scorpion, giant water bug.
03]
Basket like leg : Due to the anterior shifting of the thoracic sternal plates, all the
three pairs of legs are found clustered as basket near the head to capture and
devour the prey while at flight. eg. Dragonfly.
04]
Mandibulosuctorial mouth parts for piercing and sucking the body fluid. eg. Antlion
grub, Chrysopid grub.
05]
Horny proboscis eg. Robberfly adult.
06]
Stout mandibles eg. Dragonfly
Special adoption
01]
Fast flying capacity is found in most of the flying predators for chasing and
capturing the prey.
02]
Rapid pounce upon the prey while at flight eg. Robberfly.
03]
Trapping : Antlion grubs and ground beetle grubs construct conical and vertical
pits, respectively for trapping the prey.
04]
Stinging predators : Few wasps sting and paralyse the caterpillars and store them
as food for the young ones without decay due to the antibiotic property of the venom
injected by the wasp while stinging.
5. Predators of agricultural importance
Predators which prey on insects belongs to different groups.
Group
Organism
Predatory Stage
Arachnids
Spiders, scorpion and mites
Nymph and adult
Insects
Dragon fly (Odonata)
Nymph and adult
Preyingmantis (Dictyoptera)
Nymph and adult
The larvae are taken in plastic containers with 1-2 cc of corcyra eggs and loose paper strips.
The paper strips along with larvae sticking on them are dropped in the field at random
while walking across the field.
Predators may be
Monophagous : Highly prey specific and feeds on one species of prey.
eg. Vedalia beetle Rodolia cardinalis feeding on cottony cushion scale, Icerya purchasi.
Oligophagous : Feeding on several prey species with broad host range.
eg. Green lacewing Chrysoperla camea on aphids, mealy bugs and scales.
Stenophagous : Feeding on few prey species with restricted host range.
eg. Aphid feeding Coccinellids and Syrphids.
77
Lecture No. 5 (Unit I)
Nutrition of Entomophagous Insects and Their Hosts
Nutritional Requirement of Insects
Introduction
The Culture of entomophagous insects is usually accomplished by rearing the
insects on living hosts, either natural or unnatural. Success of this type of programme is a
matter of logistics and synchronization of three life cycles: the plant, the phytophagous
insect, and the entromophagous insect.
Replacing or by-passing part of the food chain with artificial diets involves more
than just supplying nutritional requirements for growth or reproduction. The diet must first
be attractive to the insect. Beck (1956) proposed several definitions of nutritional
requirements in order to prevent confusion. Nutritional requirements should refer only to
the chemical factors essential to the adequacy of the ingested diet. Chemical feeding
requirements are chemical factors important to normal feeding behaviour, and physical
feeding requirements are the insect’s requirements as to dietary texture, position, light
intensities, and other physical factors influencing feeding behaviour. It is primarily because
of these latter two classes of requirements that no universal diet is likely to be found to
culture all insects; furthermore, even in one species the diet of the immature stages may be
quite different from that of the adult.
The qualitative nutritional requirements are quite similar for most animals.
Approximately 30 basic chemicals are required for growth and reproduction. Included are
essential amino acids, most of the vitamins of the B group, a sterol, and certain minerals.
Luckey (1954) titled an article A Single Diet for All Living Organisms and gave the
composition. Luckey realized that a diet with the proper formulation of all nutrients
required to rear all species may never be known, but because of the similarity in nutritional
requirements between animals he proposed ‘Universal Diet No. 1’. Undoubtedly, this diet
would support growth for some polyphagous insects, for they would probably eat it, but the
chemical feeding requirements and physical feeding requirements would not be satisfied for
most phytophagous, predatory, or parasitic insects.
The determination of the nutritional requirements of insects has unfolded rapidly in
recent years and can be followed in a series of reviews on insect nutrition. Since Uvarov’s
(1928) review when scarcely a single specific nutritional requirement had been determined,
only a few insects have been reared on purely artificial diets in the laboratory. One of the
first artificial diets was formulated by Loeb (1915) and Guyenot (1917) for rearing
Drosophila.
A few other workers reared cockroaches, flesh flies, mosquitoes, and some
stored-product pests in the laboratory on various artificial diets.
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
78
Some of the B vitamins known to be required by higher animals were shown to be
definite requirements for insect growth and metamorphosis. One amino acid was distinctly
implicated as a requirement. The nutritional importance of certain salts was shown
especially in some aquatic insects; as for lipid soluble factors, cholesterol was found to be
required by at least one species. The nutritional value of specific carbohydrates was also
tested for adult insect utilization.
B vitamins known to be required by higher animals were shown to be essential in
the diets of most insects tested in the absence of micro-organisms in the diet. The first
studies on replacing the total protein by use of amino acids were achieved, and it became
apparent that the 10 amino acids required for insect growth were quite similar to those
required by higher animals. The importance of some fatty acids was shown in a few insects,
and it appeared that a complete chemically defined diet for Drosophila had been achieved
for supporting growth of the larvae.
Artificial Diets
All degrees of artificiality in diets have been used for culturing insects from the pure
chemical one (defined diet) to media composed of a natural food but mechanically changed
(cooked, ground, and blended) which can be termed ‘prepared media’.
Aseptic culture involves sterile techniques such as autoclaving the diet and
introducing eggs or larvae that have been sterilized, usually with chemical disinfectants.
However, known specific micro-organisms can be introduced into the sterile system to serve
as food or to provide growth factors for insects to be cultured. Pure culture, known today as
axenic culture, is the rearing of one or more individuals of a single species on a non-living
medium. The other extreme is xenic culture in which the number of associated organisms
in the culture are unknown.
Host-specificity of phytophagous insects also is a stumbling block when attempting
to culture them on artificial media.
If Fraenkel’s (1953, 1959) concept is valid, that the basic nutritional requirements
are similar for most insects, that these requirements are present in most plants, and that it
is the odd chemicals in plants that either attract or repel the phytophagous insect, the use
of artificial diets should be simplified. For to a nutritionally complete medium (which could
be almost any plant that satisfied the physical feeding requirements), the addition of a
feeding attractant should satisfy the chemical feeding requirements. Removal or
‘neutralizing’ or repellent chemicals from an unnatural plant medium then should permit
the development of an insect. ‘Neutralizing’ host-plant resistance factors is perhaps
possible, for Beck has shown that one resistance factor in corn is not effective in stopping
the development of corn borer larvae if a relatively high sugar concentration is present in
the plant, even though sucrose is not required by the young larvae (Beck 1957). Fleschner
79
(1952) believes chemical or dust deposits on leaves lowers host plant resistance to mite
attack. Fraenkel’s basic idea that all plant cells are uniform in nutritional composition
requires further evaluation, for although cells may be qualitatively similar in nutrient
composition, nutritional imbalances may exist for certain insects ingesting these cells
because of quantitative differences in the proportions of nutritional elements. Recent
developments on the physilogical factors of resistance in a few plants are discussed by
Auclair (1958), with emphasis factors of resistance in a few plants are discussed by Auclair
(1958), with emphasis on osmotic (and turgor) pressure of different plant varieties
influencing aphid feeding.
In spite of complicating nutritional requirements, host selection factors, chemical
feeding factors, and physical feeding factors, some phytophagous insects have been
cultured on artificial diets. Furthermore, parasites and predators have, in turn, been reared
on insect hosts cultured on artificial diets, and even a few entomophagous species have
been cultured directly on a artificial diet.
Entomophagous Insects Cultured Directly on Artificial Diets
Agria affinis, a dipterous parasite of Choristoneura fumiferana (Clem.), is one of the
few parasites mass-cultured thus far on an artificial diet. House and Tract (1994) found
that three parts of pork liver and one part fish (salmon or catifish), each weighed
separately, then mixed and reduced to a smooth, paste-like consistency by a food grinder,
produced an effective medium for culturing this sarcophagid species. Arthur and Coppel
(1953) cultured Sarcophaga aldrichi Park.,
a parasite of C. fumiferana and Malacosoma
disstria Hbn., on the same type medium. R. Smith (1958)_ reared 40 generations of
Kellymyia kellyi (Ald.), a sarcophagid parasite of grasshoppers, on pork liver alone. He also
reared this species on a mixture of powdered milk, powdered eggs, and brewers’ yeast
moistened with water to the consistency of a thick paste. The flies larviposited freely on the
liver and dead grasshoppers but not on the milk-egg-yeast mixture.
Box (1952a) was able to feed Exeristes roborator Fab. on chopped meat mixed with
an extract of pink bollworm, Pectinophora gossypiella (Saund.). The parasite laid its eggs on
this material when placed in a piece of corn pith containing a groove. The material was
covered with a piece of white paper perforated with a pin. The eggs were then removed and
placed on pink bollworm larvae ‘partially’ killed with wet heat, and by this method he was
able to raise a large population of Pimpla at the time when there was a shortage of
Pectinophora.
Among the predators cultured directly on artificial diets are few coccinellids, but to
date they have not been mass cultured on any artificial diet. Hawkes (1920) found that
adults of Adalia bipunctata (Muls.) will cagerly cat pounded dates, upon which they can
liver for months. The newly hatched larvae could not use the food, but Hawkes managed to
80
bring one larva from the second instar to the adult stage, and most older larvae could be
kept for a long time on this diet, but ultimately died without growing. Azumkowski (1952b),
experimenting with Colemegilla sp. which feeds extensively on various lepidopterous eggs
(1952a), found that the adults fed well on fresh liver or raw meat and lived longer than
those fed aphids, but these beetles did not oviposit, and larvae reared on this medium died
before reaching maturity.
After suppliementing the liver diet with aphids, larval feeding increased from 30 to
70 per cent, and such larvae were able to reach the adult stage. Supplementing the liver
with a ‘Multivitamin Roche’ mixture of liver plus vitamin C increased the number of larvae
reaching maturity over liver plus aphids. Szumkowski also obtained greater fecundity and
fertility by feeding adults liver plus vitamin E, and liver plus vitamin E plus aphids, than
when feeding only liver or raw meat alone. Smirnoff (1958) cultured 19 coccinellid species
on a mixture of agar, cane sugar, honey, royal jelly, alfalfa flour yeast, and pulverized dry
insects which are natural prey of the species to be reared. For larvae this mixture should be
supplemented with three parts beef jelly to one part royal jelly. Hagen (unpublished reports)
obtained some egg deposition from Hippodamia convergens. Guer, adults, that had
overwintered, by exposing the beetles to droplets of the following fluid diet mixture : 6 g
fructose, 3 g enzymatic protein hydrolysate of yeast, to mg choline chloride, 0.1 ml of (50
mg cholesterol plus 25 drops ‘Tween 80’ plus 1 ml 95 per cent ethyl alcohol), and to ml
distilled water.
Entomophagous Insects Produced on Hosts Cultured on Artificial Diets
Host insects cultured on
artificial
media for use
in
the
propagation of
entomophagous insects have been few. But, sine new techniques are permitting more and
more insects to be cultured on aritificial diets, no doubt more parasites and predators will
be cultured on host insects so bred. Simmonds (1944b) attempted rearing some parasites
on Drosophila pupae, and later (1953) he was able to massculture Spalangia drosophilae
Ashm. (which was being released against the fruit fly, Oscinella frit (L.)) on Drosophila
melanogaster Mg. reared in a prepared medium Maybee (1955) also used D. melanogaster
as an unnatural host to culture continuously the proctotrupoid Loxotropa tritoma (Thoms.)
which naturally parasitized the carrot root fly, Psila rosae (Fabr.). P rosade itself has an
obligatory diapause, and hence is of limited use in rearing this parasite. Maybee used an
agar-base medium for culturing the Drosophila.
Theron (1947) cultured several ichneumonid species on the larvae and pupae of the
flase codling moth, Argyroploce leucotreta Meyr.,
and the codling moth, Carpocapsa
pomonella (L.) which in turn were reared aseptically on an artificial medium supporting
introduced moulds. Theron used Ripley’s et al. (1939) diet technique developed for rearing
81
A. leucotreta. The codling moth parasites reared by Theron included Trachysphyrus
sexannulatus (Grav.), Gryptus sp., and Coccygomimus heliophila Cam.
The pteromalids Nasonia (=Mormoniella) vitripennis (Wlkr.), Muscidifurax raptor Grlt.
and Sand. and Spalangia sp. were cultured by DeBach (1940, 1942) on housefly puparia.
The host material was cultured on Richardson’s (1932) medium. Schneiderman and
Horwitz (1958) produced N. vitripennis on the flesh fly, Sarcophaga bullata Park., puparia
which were raised from maggots cultured in raw hamburger on brewers’ yeast and
powdered milk.
Opius longicaudatus (Ashm.) was propagated on Dacus dorsalis Hend. cultured in
an agar-base drosophila-type medium by using 3 per cent agar and covering the medium
with cheesecloth (Marucci and Clancy 1950). These workers also propagated a cynipid,
Trybliographa sp. and Tetrastichus dacicida Silv., on both D. dorsalis and Ceratitis capitata
(Wied.) larave developing in agar base media. Several other species of Opius were
propagated on D. dorsalis which was cultured in artificial diets developed by Maeda, Hagen,
and Finney (1953) and Finney (1956). O. oophilus Full., which oviposits in eggs, and O.
vandenboschi Full., which oviposits in first-instar larvae of D. dorsalis, were cultured from
sections of either apple or guava which had first been exposed to fruit flies for heavy
infestation followed by exposure to the Opius spp. adults for parasitization. After parasite
oviposition, the fruit sections were placed on nonaseptic media to permit the fly larvae to
develop on ample food and parasites to emerge from the fly puparia (Finney and Hagen,
unpublished reports).
Several species of grasshoppers of Crytacanthacrinae and Oedipodinae were
cultured on an artificial diet which permitted propagation of some of their parasites (R.
Smith 1952).
A few predators have been produced by providing unnatural or factitious hosts
cultured in artificial media. Readio (1931) reared nymphas of Reduvius personatus (L.) on
house flies and Tribolium larvae grown on artificial media. Struble (1942) was partially
successful in rearing a bark beetle larval predator, Temnochila virescens (Fabr.), on
artificially cultured larvae of Lucilia sericata (Meig.). However, the first-instar larvae of this
ostomid needed to be fed scolytid larvae, for they could not break the integrument of the
flesh fly larva. Balduf (1941, 1948) made a series of biological studies in the laboratory of
Phymata pennsylvanica americana Melin by feeding the bugs on artificial-media-reared
adult Drosophila melanogaster and Musca domestica L. Sinea diadema (Fabr.) were fed the
same flies (Balduf 1947b). Comparing the biological performance of both predatory bugs
when fed the flies, Balduf (1947a) found that Phymata did better, for it ate both fly species
as long as they were moving. However, Sinea in the fourth and fifth instars and adult stage
could not cdapture Drosophila adults because of their small size, while Musca was a little
82
too large for easy capture. Thus Balduf concluded that an intermediate size prey would be
more suitable for Sinea. However, Sinea would feed on recently killed Drosophila if starved.
West and DeLong (1955) reared the reduviid Zelus exsanguis (Stal) in laboratory during the
summer by providing miscellaneous insects collected from general sweeping, but in the
autumn and winter, Zelus nymphs were fed cultivated Drosophila melanogaster adults until
the fifth instar was reached, and then fed red-pine sawfly larvae, collected from cocoons;
the adult bugs were fed cultured house-flies.
Artifical diets used in culturing immature host stages
The preceding two sections dealt with relatively few cases where entomophagous
species have been cultured on artificial diets or on hosts which have been cultured on
artificial diets. However, in as much as nearly any insect cultured on an artificial diet
potentially represents either a natural or an unnatural host for culture of entomophagous
species, it is important for our purposes to examine the use of artificial diets for culture of
various potential hosts.
The list of immature stages of phytophagous, mycetophagous, and saprophagous
insect species that have been cultured on artificial diets is constantly increasing. The
nutritional requirements of phytophagous insects have been recently reviewed by Friend
(1958a, b), and Grison (1948, 1951) and Friend (1955) discuss problems in culturing
phytophagous insects on artificial diets. The laboratory culture of a variety of insect
species, including some species that have been propagated on artificial diets, has been
treated by Galtsoff et al. (1937), Campbell and Moulton (1943), Peterson (1953), and Fisk
(1958).
There are a few ‘standard’ artificial diets that have been used to culture some
common test insects. Haydak (1936, 1943) was able to culture Galleria mellonella, Achroia
grisella
(Fabr.),
Plodia interpunctella
(Hbn.), Oryzaephilus
surinamenisis
(L.),
and
Trogoderma spp., as well as other Dermestidae and grasshoppers on the following diet :
four parts fine corn meal, two parts whole wheat flour, two parts skim-milk powder, two
parts standard wheat middlings, and one part dried yeast. Equal parts of this dry diet (in
grammes) and liquid mixture containing one peart any and one part glycerine (by volume)
are mixed. This is a particularly good diet for wax moths. According to Haydak the dry diet
can be mixed in different proportions with the liquid phase depending upon the feeding
habits of other insects.
A more purified diet was used by Fraenkel and Blewett (1943) for culturing Ephestia
moths and five common species of Coleoptera infesting grains and flour. This diet is
composed of 50 parts casein, 50 parts carbohydrates (starch or glucose), one part
MeCollums’ salt mixture No. 185, one to three parts sterol (usually cholesterol), five parts
dried brewer’s yeast, and 15 parts water.
83
Since
these
earlier
diets,
which
were
employed
mainly
for
propagating
cerealinfesting insects, some progress has been made in culturing phytophagous insects on
artificial diets. However, such investigations have been along lines of determining
nutritional requirements, mostly under aseptic conditions. These research diets do not lend
themselves immediately to mass-culture techniques, but provide us with a knowledge of the
nutritional, chemical, and physical feeding requirements necessary to permit growth and
development. Therefore, substitution of the pure chemicals with cheaper, more whole
foodustuffs that would provide similar chemical and physical feeding requirements could be
used. Even aseptic conditions employed in culturing insects can probably be circumvented
by adding chemical micro-organism inhibitors and altering the pH. The technique of using
homogenates and extracts of natural host plants and storing these produces in deep freezes
has been used successfully in propagating some phytophagous insects attacking host
plants that are only seasonally available.
It is appropriate to include a short review of some of the different types of diets used
and especially to refer to the different techniques used in the culture of these nonentomophagous forms, for they may be cultured as hosts, or prey for parasites or
predators. Instead of treating the insects by their type of food habit, the species will be
considered under their taxonomic order.
Acarina
Some predatory mites will evidently feed on other substances besides their prey. In
the field and laboratory, certain species of typhlodromids that naturally prey upon
tarsonemid mites have been observed to feed on aphid and white-fly honeydew which
permits these mites to survive when host populations are at low densities (Huffaker and
Kennett 1953, 1956). These workers (1953) also found that these typhlodromids would eat
sugar solutions, egg yolk, and other liquid foods when they were hungry. The typhlodromid
mites that Chant (1958, 1959) studied in England usually fed upon tetranychid mites, but
he found that they could live for a considerable length of time on apple leaves without other
food, and could develop and ovrposit in an apparently normal fashion when provided only
with fungal spores or plant pollen. Since the predatory mites will feed on liquid and solid
foods there exists the possibility of culturing typhlodromids on artificial diets.
Artificial diets for adult insects
Many insect species that have been studied need only to ingest water and a
carbohydrate in the adult stage for survival and reproduction. The nutrients essential for
reproduction in this type of insect are carried over from the immature stages to the adult.
Evidently, there are all degrees of transfer, for some adult insects may not have to feed at
all while in others a carbohydrate, protein, minerals, several B vitamins, water, and
84
possibly cholesterol are required for fecundity and fertility. Adult insects requiring such
complex diets have received little food reserves from their immature stages.
A clue to those species that require a complex diet in the adult stage for
reporduction duction is that they usually exhibit a distinct preoviposition period. There are
some species which will deposite a few eggs after a relatively short preoviposition period,
but if fed only carbohydrates fecundity drops rapidly. The initial egg production resulted
from a limited amount of stored nutrients; therefore, to have sustained egg production, the
essential nutrients must come from extrinsic sources. Some insects, when provided with a
simple carbohydrate diet, will never produce eggs. To determine if it really is the deficient
diet that is responsible for the failure of egg deposition and not the absence of some
extrinsic ovipositional stimuli, an examination of the ovaries will show the extent of
ovigenesis.
Diptera
It was apparently in the Diptera that adult nutrition was first discovered to
influence reproduction and longevity of insects. Guyenot (1917), experimenting with a
different Drosophila medium, found that when the adults were fed egg lecithin normal egg
deposition occurred. Drosophila adults are usually exposed to, and feed on, the same
medium in which the larvae are cultured. Special handling of the Drosophila adults is
covered by Kerr (1954), who fed adults equal parts of honey and compressed bakers’ yeast
formed in a thin syrup. Gerolt (1957) apparently exposed the adults to the larval medium,
but gives detail on handling and sexing adult D. melanogaster.
Some agromyzids have been cultured in the laboratory and although the larvae were
reared in various plant leaves the adults had to be provided with at least a carbohydrate
solution (20 per cent sugar water) to prolong longevity and obtain good egg deposition
(Freeman and Guyton 1957).
85
Lecture No 6 (Unit I)
Dynamics of BC Agents vis-a-vis Target Pest Population
Population dynamics and role of biotic and abiotic factors
The oscillation or fluctuation in the insect population observed over period of time,
due to different nature of biotic factors acting on them is known as population dynamics.
Here the insect population level fluctuates around the general equilibrium position (GEP).
Biotic factors
Biotic factors of the environment tend to modify the activities of insects. Individuals
within a population enter into varied interactions with each other besides interacting with
the adjacent population. These interactions may be positive or negative according to
whether it produces beneficial or harmful effects on the interacting individual or
population. In the positive interaction the individuals live adjusting each other which is
known as either mutualism or commensalism.
1. Competition
The active demand by two or more individuals of the same species of population
(Intraspecific competition) or members of two or more species at the same trophic level
(Interspecific competition) for a common resource or requirement, which is limiting is
known as competition. Both the types of competition lead to density and diversity of a
population. Example for these type of competition are given below.
Interspecific competition
Accidental introduction of oriental fruit fly, Bactrocera dorsalis replaced
mediterranian fruit fly, Ceratitis capitata in Hawai (1940). This is called Ganse’s principle
or Competitive exclusion principle. Red flour beetle Tribolium eliminates saw toothed
beetle Oryzaephilus when both are grown in the same flour.

Crowding in aphids results in dispersal of winged form.

Cannibalism is noticed in American bollworm larvae and chrysopid grub.
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
86

Increased crowding (population pressure) in rice weevil Sitophilus oryzae leads to
reduction in fecundity.
2. Parasites and Predators
Parasite : An organism which is dependent for some essential metabolic factor on another
through out it all life stages, which is always larger than itself.
Parasitoid : An insect parasite of an arthropod which is parasitic in its immature stage
killing its host in the process of development and adults are free living.
Predator : An organism that capture and feeds on other insects.
Interactions between predator and prey are different from the parasite-host
relationship in that the predator and prey maintain an equilibrium more dynamic than the
parasite and its hosts. The parasites in general when the rate of parasitism is high, cause
death and result in elimination of hosts. But the predator never eliminate the prey
completely.
Abiotic factors
1. Physical factors
Temperature : It has got a profound influence on the life cycle of insects by affecting the
number of generations. Normal life activities go on smoothyl at a specific temperature or at
a specific range of temperature called optimum range of temperature. The rate of
chemical reactions and metabilic processes are influenced by temperature. Insect enter into
diapause, if the temperature is below optimum called hibernation or above optimum called
aestivation. Life in this universe exists within a range of 200 to 1000C.
Maggots of Chironomids and other dipterans thrive upto 55 0C. Snowfleas
(Collembola) survives near polar region of earth at high mountain ranges.
Examples illustrating the influence of temperature on insects are given below.
 Bed bug completes 12 generations in tropics and only two generations in cold
climate.
Influence on fecundity
 Diamond back moth (DBM) lays more eggs at a larval temperature of 180C than
220C.
 Grasshopper lays 20 to 30 times more eggs at 320C than 220C.
 Ovipsition of bed bug is inhibited at 80C to 100C.
 Pediculus spp. (Head and body louses) does not oviposit below 250C.
 High temperature induces more early shoot borer attack in sugarcane.
 Larval period of sugarcane internode borer is shortened 16 – 24 days in summer
and prolonged 141 – 171 days during winter.
 Swarm migration of desert locust. Schistocerca gregaria occurs at 170C to 200C
87
2. Light : Regularly occurring daily cycles of light and darkness have been known to exert
influence on the behaviour and metabolism of organisms. The response of organisms to
environmental rhythms of light and darkness is termed photoperiodism. Each daily cycle
inclusive of a period of illumination followed by a period of darkness is called photoperiod.
Photoperiod is an important factor in diapause induction, because the intensity of diapause
is directly related to day length. Influence of light on some insects are given below.

In silk worm moth Bombyx mori longday late embryonic development induces the
adult to lay diapausing egg.

In aphids short day length produces sexual forms and long day length produces
asexual forms like parthenogenetic viviparous reproduction. This is known as
seasonal dimorphism.

Many insect species are not equally active throughout the 24 hours of the day.
Some are primarily nocturnal dark active, diurnal day active and others referred as
crepuscular active at dawn and dusk.

Fruit flies Tephritids lay eggs only in light.

Codling moth, cotton bollowrm Earias spp, Red hairy caterpillar oviposit in dark.
3. Humidity
Entomogenous
fungi,
fungus
attacking
insect
requires
high
humidity
for
multiplication and spread. Coffee green bug Coccus viridis is controlled by white
halofungus Verticillium lecani, which multiplies faster at high relative humidity. High
humidity induces the incidence of BPH in rice and aphids on other crops. For termites
(Isoptera) humidity is an important ecological factor. They move towards zone of high
humidity. Worker termites have a humidity preference of 90 to 95 per cent and live longer
at 100 percent relative humidity under starve condition. Groundnut leaf miner incidence is
less in irrigated crop than rainfed crop due to RH factor. Influence of humidity on fecundity
is given below.

Migratory locust Locusta migratoria failed to produce eggs at 40 per cent RH, but
at 70 per cent RH only it sexually matures.

Rice weevil Sitophilous oryzae fecundity is less at 34 per cent RH and maximum
at 70 per cent RH.
4. Rainfall : Some of the effects of rain fall on the biology of insects are given below.

In Red hairy caterpillar (RHC) and cutworms rain fall is essential for adult
emergence and pupation.

Excess rainfall brings the whitegrubs to soil surface, so that it get predated by
birds.

Excess rainfall control aphids, diamond backmoth (DBM).
88

Intermittent low rainfall increases BPH and thrips incidence in rice.
5. Wind :

It helps in the dispersal of insect species besides interfering with their normal
feeding, mating and multiplication.

With the help of the wind current Helicoverpa adult fly up to 90 km.

It also help in the dispersal of aphids over large area. Indirectly it helps the insects
by altering the temperature and RH.

Mites also get dispersed by wind.
6. Topographic factors : Besides mountain, large areas under water, viz., lake, sea, etc.
also act as physical barrier for the spread.
7. Soil type : It plays an important role in the multiplication of insect species.

Wire worms multiply in heavy clay soil with poor drainage and white grubs
multiply in loose sandy soil with better drainage.
8. Water :

Standing water helps in the multiplication of insects like mosquitoes and certain
beetles.

Running stream is preferred by odonates, caddis flies etc.
Manipulation
Conservation biological control has received considerable attention in the recent
past (Barbosa, 1998; Pickett and Bugg, 1998). The successes in classical biological control
have provided the background and encouragement for efforts in the manipulation of
natural enemies. Such manipulations include conservation, augmentation, habitat
management and genetic manipulation (Elzen and King, 1999). During the 1980’s.
Manipulation refers to those procedures that help the establishment and activity of natural
enemies. Manipulation of a natural enemy or its environment may be justified if a definite
need exists and a reasonable assurance of success is possible. Certain factors associated
with the habitat, the host, or the natural enemy itself may render an entomophagous
organism ineffective as a biological control agent, but still be subject to manipulation. The
habitat may have certain adverse climatic factors, such as heat, cold, low humidity or wind.
Unattractive or otherwise unsuitable host plants may be present, or there may be a scarcity
of food or water for adult natural enemies and inter-specific competition among natural
enemies.
Habitat management
Habitat management, a subset of conservation biological control, is an emerging
technology to enhance biological control in an agro-ecosystem by preserving or enhancing
89
its plant diversity or providing adequate refugia for pest’s natural enemies (Landis et al.
2000). The focus is to manipulate the environment to enhance the survival and
physiological and / or behavioral performance of natural enemies to increase their
effectiveness. Generally, the area manipulated may be the crop field itself, the land at field
margins, or the surrounding agricultural landscape. However, it is important that the
sources should be provided at a correct time and in a spatial array that allows the natural
enemies to take full advantages of them.
1) Vegetation manipulation
Vegetation manipulation in agro-ecosystems and their surroundings is an important
practice used to enhance beneficial arthropods in agricultural crops (van Emden and
Dabrowski, 1994; Shenk and Bajwa, 2001; Jayraj and Saminathan, 2001). Providing “right”
vegetation diversity may improve availability of pollen, nector, honeydew as food for adult
natural enemies, shelter or a moderated microclimate and alternate hosts or prey.
2) Provision of alternate food sources
Recently, several studies have demonstrated the potential for establishing flowering
plants in or around farm fields to attract natural enemies and enhance biological control in
the adjacent field (Luna and Staben, 2000). Food sources for adult natural enemies can be
improved by planting suitable plants (Table 1). Intecropping chickepea with corriander was
found to increase the activity of Campoletis chloridae and decrease in population of H.
armingera (Turkar et al., 2000), Similarly strip-cropping in cotton enhanced the population
of predators on cotton (Parajulee and Slosser, 1999). Kavitha (2002), observed that the
predator population was maximum in cotton plots stripcropped with sorghum (16.74),
followed by maize (14.68) and cowpea (12.77). Inter-planting maize and cowpea in cotton
acts as a source of the predators to cotton crop. The function of the border maize and
cowpea may be attributed to the abundance of floral nectar and alternate prey (aphids),
shelter, mating and oviposition sites harbored in the border crop compared with
monoculture cotton having lesser biodiversity.
Table 1 : Increased parasitism due to the presence of adult food sources
(Modified from Powell, 1986).
Parasitoid
Pest
Crop
Food sources
Campoletis chloridae
Helicoverpa armigera
Chickpea
Coriander
Tiphia popilliavora
Phyllophaga spp.
Various
Nectar from weeds;
Rohwer (Tiphiidae)
Lachnosterna spp.
honeydew from scale
insects
Aphelinus mali
Haldeman
Aphids
Apple
Nectar from the
honey plants
90
(Aphelinidae)
Phacelia and
Eryngium
Apanteles
Colias philodice
medicaginis
Godart
Alfalfa
Nectar from weeds;
honeydew from
Muesebeck
aphids
(Braconidae)
Aphytis proclia
Quadraspidiotus
Orchards
Nectar from the
Walker
perniciosus
honey plant Phacelia
(Aphelilnidae)
Comstock
tanacetifolia
Bentham
Various species
Malacosoma
Apple
Nectar from weeds
Sugar-cane
Nectar from weeds
americanum F. Cydia
pomonella Busck
Lixophaga
Rhabdoscelus
sphenophori
obscurus Boisduval
(Euphorbia species)
Villeneuve
(Tachinidae)
2) The use of elemental marker rubium also showed that syrphid flies, parasitic wasps and
lacewings fed on flowering cover crops in orchards and that some moved 6 ft. high in the
tree canopy and 100 fleet away from the treated area. Thus, planting flowering plants and
perennial grasses around farms may lead to better biological control of pests in nearby
crops (Long et al. 1998).
3) The long-practiced method of clean cultivation for weed control may be undesirable from
the standpoint of removing wild plants infested with honeydew-producing insects or
containing nectaries. The adult females of many natural enemies need a source of
carbohydrates, such as nectar, to mature their eggs. Some weeds have been shown to fulfill
a role as providers of carbohydrate. Absolutely clean field, devoid of all weeds, may not be
the ultimate goal in sustainable agriculture (van Emden, 1990).
Provision of alternate hosts
1) Colonization of alternative insect hosts may improve synchronization between a pest and
its natural enemies. Alternate hosts of natural enemies can also be made available through
vegetation diversity where these can multiply in large numbers before attacking the target
host (Table 2). Higher parasitism of Acherontia styx eggs on sesame by Trichogramma
chilonis in cotton-sesame intercropping results in the increased parasitism of Helicoverpa
armigera eggs on cotton.
91
2) Collection and rearing of H. armigera eggs from cotton plants in intercropping revealed
31.5 per cent parasitisation by T. chilonis whereas no egg parasitism was observed in pure
cotton crop (Ram et al., 2002). Similarly Trichogramma Spp. heavily parasitized eggs of
Catopsilla pyrenthe on wasteland weed Cassia spp. (Patel and Yadav, 1999). Interspersing
C. occidentalis with hybrid cotton 10 resulted into very high rate of egg parasitism by
Trichogramma spp. in bollworms, Earias vittella and H. armigera (Yadav et al., 2001).
Table 2 : Examples of increased parasitism due to the presence of alternate hosts
(modified from Powell, 1986)
Parasitoids
Pests
Crops
Alternative hosts
Macrocentrus
Cydia molesta Busck
Peaches
Lepidoptera
species (Braconidae)
Archytas
species
on
weeds
Heliothis virenscens
(Tachinidae)
F.
Scelionids
Eurygaster
Cotton
Cutworms on flax
Cereals
Pentatomidae
integriceps Puton
nearby
in
natural
habitats
Lydella
grisescens
Ostrinia
Robineau – Desvoidy
Hubner
nubilalis
Maize
Papaipema
Guenee
(Tachinidae)
nebris
on
giant
ragweed
Inagrus epos Girault
Erythroneura
(Mymaridae)
elegantula Osborn
Vines
Dikrella
cruentata
Gillette
on
blackberry
Lysiphlebus
Schizaphis
testaceipes Cresson
graminum Rondani
Sorghum
helianthi
Monell
(Aphidiidae)
on
sunflowers
Emersonella
niveipes
Aphis
Chelymorpha
Girault
Sweet potato
cassidea F.
Stolas
sp.
on
morning glory
(Eulophidae)
Braconids
Rhagoletis
Apples
pomonella Walsh
Trichogramma
Helicoverpa armigera
chilonis Ishii
Hubner
Trichogramma spp.
Helicoverpa armigera
Hb. Earias spp.
Tephritidae
on
weeds
Cotton
Acherontia
styx
Westw on sesame
Cotton
Catopsilia
pyranthe
on Cassia weed
The egg parasitoids, Anagyrus spp. overwinter on alternate hosts outside the grape
vineyard because the grape leafhopper does not overwinter in the egg stage. Thus, planting
92
these wild plants of genes Rubus in the viscinity of grape vineyard helped in increasing the
parasitism by Anagyrus spp. to control of grape leafhopper (Corbett and Rosenheim, 1996).
4) Provision of shelter
Among the earliest examples of habitat management were attempts to provide a
refuge / shelter for natural enemies of alfalfa pests displaced after cutting. Construction of
nesting shelters encouraged high local populations of Polistes wasps in cotton fields in the
West Indies and in tobacco fields in North Carolina, increasing the total predation of
injurious lepidopterous larave. Nesting boxes provided for insectivorous birds in some
intensively managed European forests also resulted in increased predator densities and
protection from defoliating insects.
Field margins and windbraks are a source of alternate hosts and provide shelter for
many natural enemies. In fact, some natural enemies can survive only if these ancillary
environments are present. In Europe, windbreaks and hedgerows have been used to
encourage the buildup of natural enemies. Also, modifying the native / wild vegetation
surrounding crop fields and orchards can favor a natural balance between pest arthropods
and their enemies (Rieux et al., 1999).
5) Effect of plant attributes on natural enemies
Plant characteristics are known to affect natural enemies. Some plants attract
natural enemies while others repel them. Egg parasitoids belonging to genus Trichogramma
did not parasitise eggs of Helicoverpa armigera (Hubner) on okra, pigeonpea, chickpea and
bottlegord but heavily parasitised on potato, lucerne, cotton, tomato, sorghum and maize
(Yadav et al., 1985; Pawar et al., 1989; Ram et al., 1998). The ichneumonid, Hyposoter
didymator showed preference for Spodoptera litura on castor and beetroot plants (Ballal et
al., 1987) Jalali et al., (1988) . Jalali et al., (1988) reported higher activity Cotesia kazak on
cotton, lkra and tomato as compared to other plants. Planting of marigold flowers attracts
not only H. armigera for egg laying but also higher parasitisatiion by T. chilonis (Patel and
Yadav, 1992).
6) Push-Pull strategy
Planting Sudan grass around maize field reduced stemborer infestation on maize
and also increased the efficiency of natural enemies (Khan et al., 1997b). Molasses grass
(Melinis minutiflora), when intercropped with maize, not only reduced infestation of crops by
stemborers, but also increased parasitism particularly by the native larval parasitoid,
Cotesia sesamae (Khan et al., 1997a). The plant releases volatiles that repel stem borers,
but attracts parasitoids without being damaged. Silverleaf (Desmodium uncinatum), a highvalue, commercial fodder legume, when intercropped with maize, repelled ovipositing gravid
stemborer females, and suppressed striga by more than 40 times.
93
Manipulation of cultural practices
Appropriate cultural methods can conserve and enhance natural enemies. The
timing and method of harvesting of some crops, such as forages, can sometimes be altered
to reduce the destruction of natural enemies. Minimum tillage not only conserves crop
residues and reduces wind erosion but also provides a habitat more favorable for many
natural enemies. Studies have shown that population of predacious ground beetles are
significantly higher in no-tillage fields than in conventionally tilled fields.
Properly timed irrigation may promote epidemics of fungal pathogens of insect pests
by providing the proper conditions of humidity in the microenvironment. Improperly timed
irrigation, on the other hand, may drive away beneficial insects.
Pre-harvest burning of sugarcane trash was found to have deleterious effects on
important parasites of Diatraea saccharalis. Post-harvest burning trash also kills large
number of overwintering natural enemies which as taken shelter in the refuge. Thus, the
post-harvest trash should be burnt after the month of February when natural enemies
leave the trash.
Minimizing pesticide use
To be effective, both imported and native natural enemies must be conserved within
the agricultural environment. The intelligent use of pesticides can alleviate the detrimental
effect of these chemicals on natural enemies. Some herbicides and fungicides also have an
adverse effect on natural enemies by repelling them from their hosts or reducing their
fecundity.
Habitat management technology is still in its early stages of development. Research
is in progress to find the role of local plant species and promising new plant introductions
in and around agricultural fields, taking into account both their benefits i.e. enhancing
beneficial arthropods the risks i.e. building insect-pests’ population. In case, we know how
a particular element of plant diversity could help in reducing the pest problem, that
element should be incorporated into the farm management plant. While implementing
habitat management, consideration should be given to the cost of developing and
maintenance of the beneficial habitat as well as the cost of any land that might be taken
out for production. A systematic and research oriented approaches to habitat management
may help the farmer in developing desirable habitats that match the needs of the beneficial
organisms as well as the pest management needs of the farm.
94
Lecture No. 7 (Unit II)
Mass Culturing Technique
Mass culturing of parasitoids
Rice moth, Corcyra cephalonica is a potential host/prey insect for rearing number of
parasitoids and predators.
Rearing of C. cephalonica
The larvae of C. cephalonica can be reared on cumbu grain. Heat sterilised broken
cumbu grain @2.5 kg along with 100 g of groundnut powder and 5 g of powdered yeast
tablet are taken in a wooden or plastic trays (45 cm x 30 cm x 10 cm). Streptomycin
sulphate 0.05 per cent spray is given @ 10 to 20 ml per tray to prevent bacterial infection.
Sulphur WP is added @ 5 g per tray to prevent storage mite infection. Corcyra eggs @ 0.5cc
(8000 – 9000 eggs)/ tray are uniformly mixed in cumbu medium and the trays are covered
with kada cloth, secured by rubber band. The hatching larvae feed on the grain by webbing
and larval period lasts for 30 – 35 days. The pupation take place inside the web itself. Pupal
period lasts for 5 – 7 days and adult moths emerge after 30 – 45 days from the date of egg
inoculation.
The emerging Corcyra adults are collected every morning and transferred to a
specially designed mating drum made of G.I. with wire mesh bottom where they are
provided with honey solution as food. The eggs are collected at the bottom on a blotting
paper kept in a tray. The eggs are cleaned with sieves or egg separator. One cc of egg will
contain approximately 16,000 to 18,000 eggs. About 100 pairs of Corcyra moth will
produce 1.5 cc of eggs during the four days of egg laying period. From each culture tray a
maximum of 2500 moths can be obtained. A tray can be kept for about 90 days for
collection of adult moths due to staggered development.
Mass culturing of egg parasitoid, Trichogramma
The eggs of Corcyra are sterilized by exposing to UV light (15 W for half an hour) to
kill embryo and are sprinkled uniformly on large egg cards (30 cm x 20 cm) divided into 30
rectangles (7 cm x 2 cm) by drawing lines containing a thin layer of gum @6 cc/card. These
cards are taken in large polythene bags (45 cm x 30 cm) containing nucleus egg card at a
ratio of 1:6 to fresh eggs and exposed for 2 days. They are kpet for another 2 days at room
temperature and on fourth day parasitized eggs turn black in colour. At this stage, the
parasitized egg can be used for field release or stored at 100C for a fortnight.
Field release : The parasitoids emerge 7 days after parasitisation under room temperature.
When cold stored, the cards are taken out and kept in room temperature for a day before
field release. The egg card’s cut into smaller cards along the lines and stapled on the plant.
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
95
Pest
Dose
Sugarcane internode borer
1 cc of parasitized eggs/release/ac; six releases at 15 days
interval from fourth month onwards
Cotton bollworm
6 cc of parasitized eggs/release /ac, 3 to 4 releases based
on pest intensity.
H. armigera on tomato
3 cc of parasitized eggs/release/ac, coinciding
with
flowering based on ETL or when 6 moths/six pheromone
traps are trapped.
Rice stem borer
T. japonicum -2 cc/release/ac; 3 – 4 release after planting
Rice leaf folder
T. chilonis -7 cc/release/ac; 5 releases at weekly intervals
from 30 days after transplanting.
Mass culturing of egg-larval parasitoid, Chelonus blackburni
C. blackburni parasitizes the egg stage but life cycle is completed in larval stage.
Corcyra eggs are sparsely sprinkle on white cards on a thin layer of diluted gum. After
drying, the adult parasitoids are allowed at one per 100 eggs into a plastic container and
covered with muslin cloth. After exposing for 24 hr, the cards are transferred to another
plastic container containing 250 g of broken cumbu grain. The parasitoids develop inside
Corcyra larvae and spin small white cocoons. The adults emerge in 15 – 20 days.
Field release : The emerged parasitoids are collected daily and taken to cotton fields for
release @ 1/plant (or) 8000 parasitoids/ac.
Mass culturing of Bracon sp. (Sandwitch method)
Bracon can be mass reared on Corcyra larva. The broader end of chimney is covered
with muslin cloth. Corcyra larvae are placed on the muslin cloth and tightly covered with
another muslin cloth by using a rubber band. Two mated female Bracon adults are released
to each Corcyra larvae through the narrow end of the chimeny which is closed with another
muslin cloth. After 3 – 4 hours the parasitized caterpillar are transfereed to containers
having folded papers. The female Bracon lays about 8 – 12 eggs on the ventral side of the
caterpillar and egg hatches in about 28 – 30 hours. The larval period lasts for 3 – 4 days
the pupal period 2 – 3 days. Life cycle is completed in 7 – 9 days.
Field release : B. hebetor is released @8000 adults/ac for cotton bollworm and B.
brevicornis is released @ 10 adults/tree for coconut black headed caterpillar.
Mass culturing of larval parasitoid, Eriborus trochanteratus
E. trochanteratus can be reared on Corcyra larvae under laboratory condition. The
adult parasitoids (1:1 male : female) are released into mating cage (30 x 30 x 30 cm) with
adult food kept in a sponge. Next morning the females are spearated and transferred to
glass or plastic containers. Corcyra larvae @10/female parasitoid are allowed for
96
parasitisation. The container is kept upside down on a sheet of paper for 3 hr. The
parasitoids inject their eggs into host larval body. After 3 hr, the parasitized larvae are
transferred into a container with broken cumbu grains for further development. The
parsitized cocoons are collected after 10 days from the rearing containers and kept
separately for adult emergence.
Field release : Release 800 adults/acre.
Mass culturing of larval parasitoid, Gonozus nephantidis
This can be mass cultured on the natural host, coconut black headed caterpillar.
Adult parasitoids are released in specimen tube for 1-2 days for mating with 30% sugar
solution as food. One female parasitoid is taken in a 7.5 cm specimen tube with a medium
sized black headed caterpillar for parasitisation. The parasitoid lays 10 – 15 eggs on host
larval body surface. The grubs feed on larval contents from outside and kill them. The
grubs are collected and transferred to setting paper strips in which they construct silken
cocoons.
Field release : The emerging adults are released in coconut garden @ 10 adults/tree.
Mass rearing of pupal parasitoids, Trichospilus pupivora and Tetrastichus israeli
T. pupivora and T. israeli are parasitic on the pupae of coconut black headed
caterpillar, H. armigera, Spodoptera and castor spiny caterpillar, Ergolis etc. They can be
mass cultured on the pupae of the above host insects. Fresh pupae of the host insect are
transferred @ 5/tube of 15 x 2.5 cm size and 30 mated female parasitoids are released into
each tube with 50 per cent honey as adult food. After 2 days, the parasitised pupae are
transferred in test tubes for parasitoid emergence. It is a gregarious parasitoid which
completes the life cycle inside the pupae of the host. Larval and pupal period lasts for 6
days and 8 – 10 days, respectively.
Field release : Release @ 20 adults/tree for coconut black headed caterpillar.
Mass rearing of potato tuber moth
Laboratory host : Potato tuber moth, Phthorimaea operculella Z.
Target pest : Potato tuber moth in field and storage.
Mass culture of potato tuber moth as host

Obtain adults or eggs sheets of the potato tuber moths. Adults of PTM can be
obtained from potato stores or from field at harvesting. Whereas, egg-sheets can be
obtained by caging adults in glass breeding jars containing tissue papers or muslin
cloth cover. Provide 20% honey solution in cotton wicks to adults for feeding.

Collect paired male-female moths or fertilized sexed female in breeding glass jar and
keep the tissue paper in it for egg laying. Otherwise provide muslin cloth strips for
egg laying. During pre-oviposition and ovipositon periods, adults may be provided
with honey solution. Cover the breeding jar with muslin cloth using rubber bands.
97

Female moths oviposit on tissue paper / muslin cloth strips. Many times, eggs can
be deposited underside of the muslin cloth on top. Potato tuber slices of 0.5 cm
thickness can be placed on muslin cloth cover, which attract the females for egg
deposition on it.

Take medium sized potato tubers and clean them with duster cloths. The cleaned
potato tuber can be nailed by rolling them on puncturing brush fitted on wooden
platform. It is necessary for making easy entry of PTM larvae into tubers, besides
through eye buds.

The punctured potato tubers are then placed on a layer of coarsely sieved sterilized
soil in the plastic basket. A layer of soil (1.5 cm thickness) maintains dryness in the
rearing basket and also provide ideal site for pupation of the PTM larvae.

Place the egg-sheet (500 – 700 eggs) of PTM on the top of freshly punctured tubers
with egg surface of the sheet down side. These eggs hatch within 4 – 5 days and the
larvae find site of eye buds / punctures for entry into tubers.

Each basket may be held for larval feeding up to 15 days from the time of egg
hatching by keeping them in BOD incubator at 27+1oC temperature.

After 15 days, the expended tubers may be discarded. Collect the pupae of PTM
embedded with soil particles by sieving the soil. The pupae attached to the bottom of
the basket are delicately scraped with a flat piece of metal sheet. Place such pupae
in incubator at 27+1oC temperature for moth emergence, in the wide mouth plastic
jar covered with muslin cloth held in position by rubber band.

After 4 – 7 days, moths emerged from pupae may be collected, sexed / paired and
then keep them in breeding jars to obtain eggs on tissue paper / muslin cloth
sheets.
Mass culturing of C. koehleri (Exotic parasitoid of PTM)
C. koehleri is a monophagous, egg-larval parasitoid and hence it could only be
reared on PTM. In addition to this, it bears a characteristic of polyembryony. Following
steps are involved in mass rearing of the parasitoid.

Collect the egg sheets of PTM and keep them in a glass jar.

Collect the paired male-females of C. koehleri adults with the help of aspirator and
release them in the jar containing PTM egg sheets. This jar may be closed with
muslin cloth and rubber band.

An adult female parasitoid can able to parasitize about 50 – 70 eggs of the host.

After exposing the host eggs for parasitization up to 24 hrs, transfer the parasitized
PTM eggs on the punctured tubers layered over a sterilized coarsely sieved soil,
already kept in plastic basket as described. If required, potato tubers may be treated
98
/ sprayed with 0.1% formaldehyde solution, before exposure to parasitized PTM egg
and dried in shade for preventing the development of mould on tubers during larval
developmental period.

A parasitoid develops within the egg and larval stages of PTM and completes its life
cycle within 20 days. After 15 days of larval development, fully parasitized
(moribund) larvae come out of tubers and form a pupal case attached with soil
particles. Such pupal cases of mummified larvae (mummies) can be separated from
soil particle by sieving.
These mummies are kept in wide mouth plastic jar and cover with muslin cloth and
rubber band, and keep in incubator at 27+1oC temperature.
As per requirement, the mummies can be separated from pupal cases with the help
of forceps. These mummies could be stored up to one month period at 100C temperature.
Field Utilization of C. Koehleri
Each female parasitoid lays eggs in the PTM eggs and each parasitized egg can give
50 – 70 adult parasitoids due to polyembryonic condition of reproduction. This parasitoid is
used for the control of potato tuber moth in the field as well as in storage conditions. For
field use, keep the C. koehleri mummies in perforated plastic vials and hung these vials
with thread and bamboo sticks at 5 m distances in potato field. Otherwise, adult
parasitoids emerged from mummies in plastic jars can be released in potato field by moving
jars considering the wind direction. Whereas in storage, mummies are placed in the heaps
(Arnies) of potato tubers or adults could also be released at site.
Dose of field release and in storage
Release of 2,00,000 adults or 5,000 mummies / ha in potato field and follow 4 releases at
weekly interval (i.e. release of 50,000 adults or 1,250 mummies / release / week for an
hectare area and perform such 4 releases commencing 45 days after planting of tubers).
Release one mummy / 4 kg stored tubers under storage condition.
Precautions

Select potato tubers free from pesticide treatments.

Treat potato tubers before released of parasitized egg sheet with formaldehyde.

Treat rearing jars, basket, glass vials and other equipments with 5% formalin
solution to maintain sanitary conditions.

Puncture potato tubers just before hatching of parasitized eggs.

Use sufficiently dry and sterilized soil for pupation.

Due care should be taken while releasing mummies to avoid destruction by
predators.
99
Lecture No. 8 (Unit II)
Insectary Facilities and Equipment
Fundamentally, the word ‘insectary’ means a place wherein insects are housed or
propagated. Therefore, technically the insectary concept would embrace the entire range
from caged individual limbs or trees, as the vedalia beetle, Rodolia cardinalis (Muls.), was
first propagated in California, or Cryptognatha nodiceps Mshill. in Fiji, to the oppsite
extreme of modern climate-controlled installations such as the insectaries of the Canada
Department of Agriculture, Entomology Research Institute for Biological Control, Belleville,
Ontrario, Canada, one of the Commonwealth Institute of Biological Control laboratories at
Bangalore, India, and at Rawalpindi, Pakistan, and the insectaries of the University of
California, Department of Biological Control at Albany and Riverside.
If justification for the existence of insectaries is necessary, it is clearly given by
Beckley (1956) who stated...... the primary reason for the existence of the Associates
Insectary is to promote biological control.
This statement takes on added significance
because Mr. Beckley is responsible for pest control on 10,000 acres of oranges and lemons
in or near Santa Paula, California and therefore views biological control strictly as an
effective and economic means of controlling mealy bugs and soft scales, partilicularly
Saisetia oleae (Bern). Another growers co-operative insectary exists in Filimore, California
and here Metaphycus helvolus (Comp.) is propagated for release against S. oleae on 70,000
acres of citrus.
There are only two examples of proved commercial insectaries and they clearly
represent the ultimate goal of insectaries, namely, to provide safe, economical pest-control
service for the grower.
Therefore, careful selection of insectary workers is particularly important since the
propagation
of
insects
demands
highly
specialized
and
extremely
varied
work.
Consequently, no amount of formal training can prepare a person completely for the work.
A high degree of interest, curiosity, and enjoyment in working with living animals usually
will indicate capability in this direction. Of course, formal traning in entomology is highly
desirable and usually necessary at the supervisory level. True, practical training is of great
benefit, but a somewhat broader background of insect behaviour, physiology, taxonomic
affiliations, and biologies will better equip personnel to cope intelligently with insectary
problems as they arise.
Further, it is very important that all persons working in the insectary realize the
need for the faithful performance of their particular jobs in the over-all propagation
programme, and in so far as it is practical, all personnel should develop a basic
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
100
understanding of all operations within the insectary. Large programmes require a higher
degree of specialization among personnel than do programmes where perhaps two or three
persons perform all the duties associated with insect propagation-insect handling as well as
mechanical knowledge and ability adequate to keep insectary equipment functioning
properly.
If the insectary serves several projects under different project leaders or is of
commercial scope, it seems desirable to have a superintendent who is responsible for the
physical facilities as well as for co-ordination of the various aspects of the propagation
programmes, such as making decisions regarding assignment of space in order to minimize
intercontamination of cultures.
Location of Insectary
From the standpoint of climate control within the insectary, an area of temperature
climate offers the best location. In tropical and subtropical regions, with the exception of
certain insular areas, the climatic area chosen should be cool, as in the higher elevations,
for it is far easier and cheaper to control heating requirements than to provide facilities for
adequate cooling. Experience has show that inadequate temperature control during
climatic extremes usually results in loss of cultures and can neutralize the work of several
months.
The ground area occupied by an insectary is determined by the projected purpose
and the value placed on such a development by persons interested in it. One or two rooms
in an existing laboratory may be considered adequate for certain programmes.
Easy access to the insectary is a necessity. This includes roads, approaches, ramps,
and loadig areas, which are necessary for convenient handling of host-plant material,
lumber, refuse removal, and miscellaneous items of large bulk.
Where possible, consideration should be given to locating the insectary away from
the immediate vicinity of agricultural areas. A minimum buffer zone would be perhaps a
quarter mile for these reasons : (1) reduces hazard of insecticidal drift from a crop area
entering the insectary; (2) reduces chance of contaminant species of parasites or hosts
entering the insectry; and (3) reduces chance of hosts (pest insects) moving from the
insectary to the crop area. The few preliminary tests which have been concluded to date in
order to determine the effect of air pollutants on insects indicate deleterious results to
certain aphelinids and coccinellids. Therefore, unless adequate filter systems are
contemplated, it would be well to avoid locating an insectary where urban or industrial
atmospheric contamination (smog) is a problem.
The terrain of the building site need not be perfectly level. Because of the prime
importance of climate control within the insectary, the insulation gained by going below
ground may be worth considering. Where contour, water table, and subsoil conditions
101
permit, a basement or cut into a hillside may offer tremendous savings in future airconditioning costs, as well as permit more accurate climate control.
If the above ground portions of an insectary can be oriented with the greatest length
arranged in a true east-west direction, especially in areas with hot summers, natural
lighting gains uniformity, the wall on the sunny side can be shaded by an overhanging roof
or louvres, and only the short west wall is exposed to the greatest heat source and can
therefore be shaded by awnings, louvres, or trees.
Landscaping in the immediate vicinity of the insectary should exclude plant species
which serve as hosts of phytophagous insects scheduled for propagation within the
insectary. It is well to keep all foliage away from walks and entrances in order to reduce the
possibility of carrying contaminant organisms into the insectary on clothing.
Building specifications
A permanent insectary is one whose projected use will extend over several years.
Temporary insectaries are those with a projected short-term use such as for a few
seasons.
Exterior Design : The primary insectary problem is climate control, and many aspects of
design revolve around this on necessity regardless of the permanent or temporary status of
the insectary. A field station of the Commonwealth Institute of Biological Control at
Fontana, California, is an example of a highly functional insectary in keeping with its
residential surroundings (fig 8.1).
Figure 8.1 : Commonwealth
Institute of
Biological
located
Control
insectary
at
Fontana, California, is an example of a highly
functional insectary designed in the motif of
its
surroundings
–
in
this
instance,
a
residential area.
A) Exterior – south aspect.
B)
Floor
plan.
Cooling
is
provided
by
refrigerated room air conditioner placed in
window openings. The larger storage area is
cooled by an evaporative cooler.
Interior Design
The floor plan will be determined
by the proposed use of the insectary. If
the intended use is for research and
limited mass culture, a quarantine area should be provided and the main insect-handling
area may consist of one or more large rooms which may be partitioned into smaller rooms
as needed. The insectary at Albany is an example of this type
102
Figure 8.2 : Insectary of the Department of Biological Control (University of California) located at Albany,
California – an example of open planning.
A. Exterior : south aspect.
B. Floor plan. Most of the partitions are temporary and can be moved as propagation need dictate. The
quarantine laboratory is in a separate building. Shelter areas are roofed over only and serve as storage
for cages, melons etc.
Legend :
CS
Cold storage
R
Rearing
ST
Storage
L
Laboratory
S
Sink
V
Vestibule
103
LBT
Laboratory table
O
Office
SR
Sterile room
WH
Water heater
It the intended use is for large-scale mass-culture programmes, a different design will be
called for. The commercial insectaries at Fillmore and Santa Paula illustrate this type
Figure 8.3 : Commercial insectaries.
The
buildings
shown
plus other
similar structures at each location
serve the acres indicated.
A. Fillmore Citrus Protective District,
Fillmore, California, serves 7,000
acres of citrus.
B.
Associates
Insectary,
Santa
Paula, California, serves 10,000
acres of citrus.
In order to provide ample
space for manceuvring bulky items
such as carts and racks, corridors
are at least 4 feet wide and
doorways at least 3 feet wide.
Ceiling height is recommended at 7
feet. This height can be reached
easily by a person 5 feet 10 inches
tall, thus facilitating collection form
the ceiling in open-room cultures. Cost of air conditioning is reduced considerably by eliminating
excessive head space.
Because fine dust is harmful to beneficial insects, dust control in the insectary is
very important, particularly in open-room culture. Since a concrete floor is a major source
of dust from the scuffing of its surface, it usually requires covering, at least in rearing
rooms. Painting and waxing are adequate only if they are maintained properly. A more
satisfactory solution is to cover concrete floors with linoleum or asphalt-vinyl tile. Wooden
floors do not present a critical dust problem, but they are far more expensive to maintain
and are easily damaged by water. Another source of dust is the water-atomizing humidifier,
which provides a fine, rapidly evaportating spray which effectively increases the humidity,
but distributes minerals from the water on near-by objects which, in time, constitute a
definite dust problem.
104
Facilities for adequate storage of equipment and supplies are considered when the
floor plan is designed. Interior finish of rooms is related to the size of the insects to be
handled. Ledges around windows, fixtures, or ducts, can be eliminated by flush design. A
smooth plaster finish painted with a glossy moisture-resistant paint coloured off-white
(blue or green tint) greatly facilitates the collection of insects and enhances illumination
within the room. Paints containing fungicides may be desirable, but paints having
insecticidal properties should not be used. All plaints used in culture rooms should be
thoroughly tested for toxicity to beneficial insects. A convenient screening procedure
involves placing in pint-size glass fruit jars a piece of woood or pressed board which has
been painted with a sample of the paint to be used and allowed to air-cure for 30 days.
Honey streaks are provided on the glass and parasites are added. Control insects from the
same culture are placed in an empty glass jar with honey provided. Mortality counts at 24
hour intervals will reveal the needed information. In order to reduce variables, it is
necessary to subject both test and control jars to equal conditions of light, temperature,
and humidity during the test period.
Window lighting alone is of insufficient intensity for suitable plant growth. Artificial
lighting usually with be required for this purpose. An even light gradient, i.e., north light in
the northern hemisphere, may be helpful in certain handling procedures such as
phototropic collection of hosts or emerging adult beneficial species which exhibit positive
phototaxis. On sunny sides of the buidling’s exterior, louvres may be used to achieve a
similar result. In order to reduce thermal conductivity and to assure insect-tightness,
windows should consists of a rigidly fixed inner pane which is of large dimension and free
of cross bars (an aid in collection of insects by aspiration) and flush with interior wall
surface, and a removable (for cleaning) but weather-tight outer pane. Approximately 2
inches of air space should separate the panes. If a quarantine area is a part of the
insectary, an added degree of safety may be achieved when the outer panes consist of wirereinforced glass. Improvements in artificial lighting, coupled with the known lack of control
over, and inadequacy of, window light to produce healthy plants, have stimulated interest
in windowless insectaries. Such a commercial insectary is advocated by DeBach and White
(1960). Advantages of below-ground or windowless construction include increased
insultation and controllable lighting. Of course, a dependable source of electrical power is a
necessity if this type of structure is to be used.
Utilities
The number of ceiling light fixtures will depend on the proposed use. In general,
natural lighting should be a secondary consideration mainly because of its unpredictability.
Adequate light for growing plants can be achieved by portable racks containing ‘cool white’
105
fluorescent tubes, or by providing ceiling hooks from which lights can be suspended
directly over growing plants.
It is very important to provide sufficient numbers of electrical outlets. Usually such
receptacles will be located just above table or work-bench heights and in the ceiling. A
general rule is one double outlet per 8 linear feet of wall.
Where a relatively large installation is contemplated, it is desirable to have a
separate power substation provided by the utilities organization which serves the area. By
so doing, adequate electricity for the insectry is assured, and the increased line load will
not affect other buildings in the immediate vicinity. Where electricity is a primary source of
power, power failure can devastate the insectary effort, not only by disrupting air
conditioning, but also by freeing several hundred thousand lepidopterous larvae which
were confined by hot-wire barriers. Portable gasoline-powered generators can be utilized to
maintain minimum operational requiremnts when such emergencies arise. This also
emphasizes the importance of location, design, and insulation, for, if done properly, a
building can withstand interruptions in utilities of several hours without suffering damage
from fluctuations of temperature.
Since the hazard of contamination is ever present, it is imperative to reduce this
free-living population to the lowest possible level. Chief requisites of construction for light
traps are a light source and a baffle system that directs insects into the trap and prevents
their leaving it. Light traps ordinarily are placed against or in walls, and should be so
located that they do not present a collision hazard to personnel or equipment passing near
them. Since light traps operate constantly, they should be located away from doors or open
windows to which insects out-of-doors may be attracted after dark and thereby gain entry
to the insectary.
Domestic (portable) water at 40 to 60 pounds per square inch pressure should be
available at all sinks and in all rooms. If possible hot water should be available to personnel
at cell sinks.
Distilled water is a necessity for preparing nutrient media for plants. Further, as a
last rinse in washing glassware it greatly reduces hard-water film. During warm weather,
distilled water can be colleted from the cooling coils of refrigerative-type are conditioners.
Such water is satisfactory for rinsing glassware but due mainly to a relatively high copper
content it may not be used in nutrient media or in hydroponic solutions.
Natural or bottled gas may serve as fuel for the heating system and for conventional
burners at laboratory tables. For reasons of safety, gas appliances should be vented to the
outside of the building.
Air pressure at approximately 60 pounds per square inch in all propagation rooms
and at sinks will prove useful for drying washed fruit, for blowing debris out of corners of
106
cages, for operation of certain types of temperature and humidity controls, and in water
spray humidifiers.
Air vacuum at 10 inch minimum mercury for collecting insects may be more
necessary than air pressure facilities, if these must be choice between two because
collection is one of the most time consuming and necessary functions of insectary
operation. An alternative method is to make use of available portable electric machines,
which provide both positive and negative air pressure.
Floor drains appropriately located in propagation rooms and perhaps main
halloways will facilitate sanitation. Such drains should have dirt-tight covers; otherwise,
their traps will become clogged and become an additional item of maintenance. Toilets and
lavatory facilities should be located away from the propagation rooms.
Air conditioning and enviromental control
Basically, there are two concepts of design involved, namely, central system which
consists of positive and negative pressure ducts radiating to and from all rooms from one or
more heating and cooling units, and individual air conditioning for each room. The chief
disadvantages of the conventional central system are : (1) lack of adequate control in
individual rooms; (2) all the rooms are affected during periods of routine maintenance or
breakdown; add (3) high initial cost. The main disadvantage of air conditioning rooms
individually is the possibility of higher cost of maintenance per room, which may be
compounded in time by unavailability of parts.
Since temperature fluctuations of 20F and relative humidity fluctuations of 4 per
cent are adequate for normal insectary work, moderately priced controls will satisfactorily
operate either system. Also to be considered is the possible need of providing for fluctuating
temperatures. In this regard, Stein (1960) indicated that Trichogramma cacoeciae Marchal
propagated at fluctuating temperatures of 160 and 260C was approximately ten times as
effective against codling moth eggs when compared with parasites propagated at a constant
temperature of 270C.
Ventilation, or recirculation of air within insectary rooms, is a vital part of the airconditioning problem. Munger (1955) demonstrated the need for fresh (filtered) air in the
culture of citus red mite, and the principle appears to have a bearing on the degree of
success of culturing certain insects as well.
Heat pumps which automatically control both heating and cooling are increasingly
available on the American market. The 1960 cost (installed) is approximately 8600 per ton
cooling capacity for household units. In a temperate climate, a 3-ton unit of this type could
be used to air-condition an insectary of 1,000 to 1,600 square feet, providing insulation
and ceiling height were proper. Since this is a forced-air system, a certain amount of duct
107
work is required. A return duct to the machine insures a degree of recirculation. Air filters
and humidifiers can be supplied for ‘heat pumps.’ Only time will tell the story on
maintenance costs and efficiency, but it appears that ‘heat pumps’ have much to offer in
the area of packaged climate control.
Temporary Facilities
Temporary facilities should conform to the general planning of a permanent
installation and in practice should give several years’ dependable service. For example,
many existing laboratories have ceilings 8 feet or more in height. The construction of a false
ceiling at the suggested 7-foot height will make air conditioning easier and more
satisfactory. The false ceiling may be made of light gauge gypsum board, 1/4 - inch
plywood, or 1/8 inch masonite nailed to the lower edges of properly spaced joists and
furring strips. The seams can be covered with tape and the entire surface then painted.
Additional mechanical features, such as wiring, plumbing and duct work can occupy the
space above the false ceiling.
Figure 8.4 : Wiring diagram for simple laboratory heaters. If assembled as indicated, the fan will
operate continuously and the heating element will be energized only when the contact points of the
thermostat close.
Heating may be achieved by household-type electric or gas heaters. If the latter are
used, it i important to have them vented to the out-of-doors. At Riverside, one
thermostatically controlled 1,200-watt electric heater with a built-in fan adequately heats a
well-insulated 14 ft. x 30 ft. room with a 7-foot ceiling. Smaller rooms (12 ft. x 12 ft. x 7 ft.)
can be heated with similar but smaller heater fans or by bare wire heat cones with a fan
behind them.
108
Figure 8.5 : Diagram of evaporative cooler used indoors to increase relative humidity. If wired in this
manner, the fan runs continuously. Water recirculation pump is activated by humidistat.
When considering cooling for small areas, the prevailing relative humidity out-ofdoors must be known. Evaporative coolers are useful in arid climates, but are much less
effective where the humidity is in excess of 50 per cent during hot weather. Air conditioners
of the refrigerator type, although more costly, are effective regardless of existing outdoor
humidities.
Relative humidity may be regulated by either reduction or increase to a desired
level. The former maybe accomplished by either a refrigerative cooler which locks free
moisture as ice on its cooling coils, or an exhaust fan in the attic which can reduce relative
humidity to the level of outside conditions by pulling air through the building.
In arid climates it is relatively simple to increase humidity by using evaporative
coolers housed completely within the building. Figure shows a wiring diagram of such
apparatus. Minor mechanical changes of the evaporative cooler allow the fan to operate
constantly, and the recirculating water pump operates only when an increase in humidity
is called for by a humidistat. Additionally, desirable features of both systems are increased
air circulation and filtration of the air. A 2,000 CFM evaporative unit is considered
adequate to humidify approximately 4,000 cubic feet, providing insulation and vapour
sealing are adequate.
Recirculation may include or exclude outside air. If outside air is to be forced into
the insectary, it is necessary to provide adequate filtration in order to prevent the entry of
unwanted organisms or particulate matter. In arid areas, especially during periods of high
temperature and low relative humidity, it may be necessary to exclude outside air and
recirculate the air within the building in order to maintain the desired relative humidity
109
unless air enters through an evaporative cooler. If evaporative coolers are used, it is
necessary to provide filtered openings, such as partially opened windows or doors, in order
to assure a flow of cooled air through the building. Otherwise, excessive humidities as well
as heating will result.
Permanent Facilities
Larger permanent insectary installations may be provided either with cntral systems
of climate control or with individual room air conditioners. If the insectary consists of more
than one wing, or group of rooms, a ‘heat pump’ for each wing may be desirable. In the
original insectary at Riverside a system of dampers in the supply and return ducts permits
the recirculation of air solely within one wing or through the central humidifying and
heating units.
At Riverside the recently completed insectary addition is equipped with a central
steam-heating system and a central chilled-brine cooling system with individual rooms
controllable +20 between 600 and 900 F and +4 per cent between 40 and 80 percent R.H.
Heated or cooled air and steam for humidity enter rooms through a common duct as
thermostats or humidistats dictate. The central cooling system actually consists of two
operating units each of which has two compressors. The designed capacity of the
compressor is such that three of them will provide adequate cooling. The fourth compressor
is a reserve or safety feature which guarantees continuous operation of the air-conditioning
equipment if one unit becomes inoperative. Because of excessive summer heat, additional
features include insulation of ducts and ceiling as well as a provision for cooled air to be
continually blown through the attic space from a large evaporative cooler. The roof is
covered with light-coloured crushed rock embedded in tar over four layers of asphaltimpregnated paper. In the older portion of the insectary the central evaporative cooling
system failed to give adequate temperature control. Therefore, it was necessary to install
household refrigerated room air conditioners in several of the existing rooms, and they have
been quite satisfactory during the past five years.
Maintenance and Safety
Preventive maintenance needs to be planned and practised throughout the year,
and it is highly desirable to have a person of varied mechanical interests on the insectary
staff. Replacement of items of equipment and anticipated maintenance costs must be
considered in projected budgets. Because of the mechanical features pertinent to insectary
operation, safety to personal becomes an important consideration. All moving machinery
should be well shielded. Indeed, the initial design should include all possible safety
features, such as fire prevention, low electric-shock hazard, well-lighted stairwells and
entrances, and non-opposing doors. Miscellaneous safety facilities include a well-stocked
first-aid kit (all personnel should be well trained in emergency first-aid techniques),
110
adequate fire extinguishers at appropriate locations, fire blankets, and mild alkaline
solutions for neutralizing the effects of acid burns.
Furnishings and Equipment
Insectary furnishings include standard items such as work tables, storage cabinets,
chairs, and basic office equipment. Mobility is a desirable feature for bulky items such as
laboratory tables, large ranks, and plant-growth lighting devices. Retractable wheels
present added versatility for such equipment. For most efficient use of space, cages, racks,
or tables should be designed in equal multiples of dimension.
Cages or housing insect cultures are perhaps the items most used in the insectary
and range in complexity from those which can be made easily be laboratory personnel from
such articles as glass jars, lamp chimneys, cellulose acetate, and a great miscellany of
small cages for limited propagation, to those requiring the services, of an experienced
carpenter or cabinet maker.
Principles of Cage Design
In order to have assurance that an insect cage is escape-proof, particular care must
be given to such matters as mortised and glued joints, glazing, properly attached cloth
areas, and well-scaled doors. Door gaskets may consists of felt cloth or of light sponge
rubber. Cloth and gasketing should be glued and stapled, or tacked to the cages. Ease of
cleaning is important and is possible if the bottom of the door is flush with the floor of the
cage.
Figure 8.6 : Apparatus used
for
ventilation
of
sleeved
cages. The fan creates a
negative
pressure
in
the
common duct with the result
that air is pulled through the
cages which are placed with
their cloth backs against the
ports. It is doubtful if aphids
or mites can be continuously
propagated
numbers
indoors
sufficient
in
for
economical insectary procedure
without
providing
adequate ventilation (Finney
1960).
An isolation or sleeved cage was developed for handling parasitic insects by workers
in the U.S. Department of Agriculture in 1907 (Howard and Fiske 1911, S.E. Flanders, in
1934, redesigned this type of cage, and since then several modifications of it have been
111
made, but this is the type3 most often used in insectary rearing programmes at the
University of California.
When insects are propagated indoors, proper ventilation and filtration of air can be
prime factors of success. This is true in open cult in open culture where an entire room
serves as a cage (previously discussed under recirculation), as well as with conventional
rearing cages, each with its own particular micro-environment. The size mesh of cloth or
sereening to be used is determined by the size of the smallest insect to be contained in or
excluded from the cage. If the inclusion of growing plants causes excessive humidities or
unfavourable odour concentrations, exhaust apparatus similar to that shown in figure 8.6
should provide the needed ventilation. In addition, it may be necessary to pass the air
through activated carbon filters in order to get rid of chemical pollutants.
Figure 8.7 : Basic optical
aids necessary for efficient
insectary work.
A.
Low-power
magnifiers
used in routine mass culture.
Left
to
right:
Illuminated
magnifier (fixed), hand lens
portable
self-illuminating
magnifier, and head loupe.
B. Higher power magnifiers
used in research insectaries.
Left to right : Stereoscopic
dissecting microscope gives 9
x to approximately 100 x
magnification; the supporting
mount permits adjustment in
three
planes;
illuminator
flood
with
lamp
variable
transformer control. At the
right
is
a
compound
microscope with its substage
illuminator. Shown attached
are a card for converting ocular micrometer reading to microns and camera lucida. The variable
transformer can be used for adjusting light intensity which is of particular value when working with the
latter.
Because many adult and immature beneficial insects are quite small, certain optical
aids, together with adequate illuminator, are necessary for efficient insectary procedure.
Magnifications of 9x to 54x, as provided by conventional stereoscopic dissecting
112
microscopes, are sufficient for most routine needs. Adequate extension supports provided
with a rack and pinion focusing mechanism, and ball and socket swivel mounted for
vertical and horizontal swinging give added usefulness to the instrument. Other optical aids
that are useful are hand lens of 14 x or 20x, head lens of 3x or 4x, a reading glass, and
illuminated magnifiers. The research laboratory will, of course, have need of instruments of
greater power.
Collection, packaging, shipping, and storage equipment
Certain specialized techniques and items of equipment have been developed
regarding procedures for collection, packaging, and storage of beneticial insects pending
shipment.
Figure 8.8 :
Phototaxic
collection of
insects.
A) Left : Shadow
collection of black
scale
crawlers
(Fillmore
Citrus
Protective
District
Insectary,
Fillmore).
Crawlers leave the
drying twigs and
collect below the
light
at
the
shadow line. Right
:
Shadow
box
used for collection
of
smaller
numbers
of
lecaniine crawlers.
When the doublewalled cover is in
place ventilation is provided through cloth-covered holes in the interior wall. Crawlers move towards
light which enters through the glass or celluloid front and collect at the V-shaped shadow. The interior is
painted with non-reflective black paint.
B) Amphitheatre – oleander scale crawlers (University of California). Crawlers are attracted towards the
vertical fluorescent tube and collect in windrows at the shadow line case by broad V’s of cardboard.
C) Leptomastix (Associates Insectary, Santa Paula). The demountable box is at floor level. When the
slide is removed, the parasites which are positively geotaxic (and therefore on the floor) and positively
113
phototaxic move into it. When a predetermined number of parasites have entered the box, the metal
sliding covers are replaced. The box is then removed and taken to mealybug-infested citrus groves
where the parasites are released.
Collection in the insectary may be accomplished by three general methods used
singly or in combination, namely : (1) utilization of insects’ inherent behavioural taxes; (2)
anaesthetization; and (3) aspiration.
Perhaps the most commonly used behavioural taxis utilized for insect collection is
phototaxis. The first-instar, or crawler, stage of many scale insects as well as adult
entomophagous insects exhibit strong positive phototaxis. The light source may be an even
light gradient from a window or an artificial light. Figure 8.9 shows equipment and methods
designed to collect insects by light attraction.
Figure : 8.9 : Apparatus for blending CO2 and ether for anaesthetizing parasites. From right to left : CO 2
cylinder with pressure regulator, warm air source to prevent pressure regulator from freezing up during
prolonged use, flask of ether in water bath at room temperature (80 degrees), tubing in the rubber
stopper of the ether flask. CO2 does not bubble through the liquid ether, but passes in and out of the
flask, picking up sufficient ether to produce the desired effect.
Because most adult beneficial insects are attracted to light, care must be taken in
open culture to shield hot light bulbs with cloth or panes of glass to prevent parasites from
contacting them.
114
Figure 8.10 : Modified hair dryer
used to collect parasites directly
into a carton for storage prior to
shipment (U.S.D.A., Moorestown)
Strong
anemotaxis
may be taken advantage of for
concentrating large numbers
of
insects.
Use
of
this
response was utilized in the
collection of Aphytis. In a
different
and
application,
currents
novel
artificial
were
useful
air
in
obtaining large numbers of
eggs of the moth, Sitotroga
(Flanders 1934). Moving air
pressed
the
abdomens
screen
against
and
the
females’
a
wire
resulting
pressure stimulated egg deposition. Although currently not used, a collection technique
which utilizes the chemo and phototaxic responses of adult Cryptolaemus montrouzieri
Muls. was developed by W.C. Beckley, manager of Associates Insectary at Santa Paula,
California. The chemo-stmulant consisted of smoke from smouldering rags in a bee smoker
which stimulated the beetles to fly to the nearest light source, in this case a cloth-covered
window. A large, flat funnel is utilized to scoop them into plastic tubes.
Anaesthetization by combining CO2 and ether was developed by Finney, Fanders,
and Smith (1947) in connection with the mass production of Macrocentrus ancylivorous
Roh. and its host, Gnorimoschema operculella (Zell.), the potato tuber moth. CO2 used alone
will anaesthetize insects for only short periods and is consequently of limited value in
mass-production work. However, it is adequate and safe for handling small numbers of
insects rapidly, for instance in making quick sex-ratio counts or for removing contaminant
organisms Ether used alone may weaken or kill insects and when used repeatedly or in
quantity in a poorly ventilated room constitutes a definite explosion and fire hazard. Mixing
the two gases by means shown in figure 8.10 greatly extends the anaesthetization period as
compared with CO2 used alone and minimizes the danger o explosion.
Collection by aspiration may be by simple mouth aspiration or by utilizing
mechanical suction such as that provided by household vacuum sweepers, portable devices
115
designed for laboratory use, or piped-in vacuum. Collection by use of a modified hair dryer
is illustrated in figure 8.10. Mouth-operated aspirtors should be equipped with filters in
order to protect the operator from inhaling fine debris. Several types of aspirator collectors
are shown in figure 8.11.
In mass-culture work, large numbers of a beneficial species can be collected by
anaestheticzation in a closed unit and placed in storage containers during one continuous
operation. Volumetric counting of parasites or predators is convenient and accurate but
can be used only when size and sex ratio are uniform. In a technique development by
Bedford (1956) for the collecting, counting, and packaging of Chelonus texanus Cress, the
parasites were attracted by a double light source directly into the shipping container, and
were easily counted as they passed through slots in cork on the way.
After insects are counted they are placed in containers for transportation to
colonization sites. Choice of container depends on the mode of transport. Insulation against
lethal temperatures extremes and an available food supply for the insects while in transit
are prime considerations. Convenient storage containers are small, heavy paper cartons of
one-half-pint or one-pint size. Glass or plastic tubes and vials can be used for small
numbers of beneficial species. Containers must be supplied with streaks of honey or honeyagar as food for the insects as well as bits of shredded wood or paper for resting sites. Paper
or shedded wood which have been impregnated with honey may be used satisfactorily to
provide both food and resting surface. Such containers may be packed in sturdy boxes or
cartons for shipment.
Most adult entomophagous insects may be satisfactorily stored at 500 to 600 F and
at approximately 75 per cent relative humidity. At this temperature they are relatively
inactive and consume little food. Conventional household refrigerators equipped with
modified thermostats are usually adequate for this purpose. If it is necessary to accumulate
collections for several days or, in some cases, weeks before shipping the insects or taking
them to the field for colonization, they will remain in good condition if the containers are
removed two or three times weekly from 600F and warmed to approximately 800F for 20 or
30 minutes. During this time the insects become active, feed, and defecate. With this
procedure it is always important to provide a supply of food and moisture. Otherwise, loss
of vigour and increased mortality may follow. Honey seems to provide both requirements.
Typically, shipments from insectaries will be for direct release at the destination.
Because of the hazards inherent in such procedure, the matter of proper quarantine
handling enters the picture. Although immature parasites may survive adverse conditions
better than adults, detection of potentially harmful species is most accurately performed by
screeining the latter. Shipments of parasitized hosts can be safe under closely controlled
116
conditions as in mailing cards to which are attached eggs of Sitotroga parasitized by
Trichogramma. It is presumed that collections of beneficial species will be thoroughly
checked by the shipper and only pure cultures of the beneficial form included. As an added
precaution, such material, prior to field release, should be re-examined at the destinuation
for the presence and removal of unwanted species.
Figure 8.11 : A) Collecting devices utilizing air flow.
At the top is a glass tube with the end narrowed. The
hose holds a bit of fine mesh cloth over the opposite
end of the tube. Just below is the collecting device
developed by A.J. Nicholson (C.S.R.R.O., Div. of Ent.,
Canberra, Australia) in conjunction with population
studies utilizing blow flies as test animals. Air
pressure only is needed as direction of air flow can be
controlled by the two-way valve arrangement beneath
the major tube either to such insects in or blow them
out. The other devices are made from copper tubing of
various sizes and they fit into standard straight-sided
shell vials. The three at the right were designed for
collecting predaceous mites. The small one in the
centre fits into a 1½ - dram shell vial.
B) Diagram of aspirator. A construction diagram of the
uppermost aspirator in the group on the left in figure
A.
Legned :
A
Intake tube
B
External shell of suction line
C
Stopper
D
Bead of solder around tube
E
Suction line
F
Cloth or roo-mesh screen
G
Receptacle
C) Portable collector. The portable collector is made
from a clothing vacuum cleaner. Two size – D
flashlight batteries provide power to run the tiny
rotary fan. This collector is very useful in the
insectary as well as in the field.
The problems of shipping are concerned
with local transportation which may involve
insectary,
vehicles,
institutional,
and
or
utilization
local
of
growers’
public
or
governmental transportation facilities.
117
Prevailing weather has a profound influence on the chosen mode of shipment, particularly
when extremes of temperature prevail.
Since containers in which parasites or predators are colleted and stored in the
insectary will usually be too fragile to serve as shipping containers, local shipment of
beneficial species requiring not over 8 hours in transit may be satisfactorily accomplished
by placing the containers of insects in a small, insulated, prechilled chest. The addition of a
frozen gel packet will prolong the effectivencess. In order to avoid subjecting the insects to
lethal or weakeningly low temperatures, some sort of insulation between the insect
containers and the frozen gels or ice should be provided. This technique was developed by
Brennan and Mail (1954), who made shipments of adult Culex tarsalis Coq. with low
mortality in up to 50 hours in transit. Figure 8.11 shows an insulated chest and gel packet
as used at the University of California.
Instead of using an ice chest for storage of the insects, wrapping the insect
containers in set cloth will adequately cool them by evaporation for a few hours in arid
areas if temperatures do not exceed 1100F. This is effective if such preparations are
transported in wire baskets within the shaded vehicle, for its movement causes air flow
around the package of insects, thereby increasing the rate of evaporation. A conventient
carrying case, employing the evaporative cooling principle, for local transport has been
developed at Riverside and is shown in figure 8.11. An automobile which was provided with
mechanical refrigeration was used for redistributing large numbers of parasites of the
spotted alfalfa aphid throughout the hot interior valleys of California. Holloway (Allen,
Holloway, and Haeussler 1940) reported a high degree of success in shipping parasites of
oriental fruit moth by surrounding the insect containers with a thick layer of moist cotton
and corrugated paper which was held in place by light canvas.
Shipments requiring commercial or governmental freight or mail services, as with
interstate transport, must necessarily be handled in a somewhat more formal manner and
must display proper shipping permits in order to expedite prompt delivery. For long
distances, air mail and airfreight offer the most rapid means of transportation. Adult
parasites often suffer a rather high mortality from starvation or temperature extremes while
en route. Unless these problems can be satisfactorily solved it will be necessary to ship
immatur stages. When quarantine regulations permit, parasitized hosts on portions of their
host plant may be shipped. Such material may be mailed in small flat packages contained
in large cloth-reinfored paper envelopes and shipped via air mail – a technique utilized by
foreign explorers. It is sometimes more satisfactory to place bulky material in small porous
boxes and send them air freight. The latter method is well adapted to shipping of adult
predators and large adult parasites which seem to require more room for relatively
unrestricted movement than do small parasites.
118
Sanitation and safety equipment
In order to prevent contamination of cultures of entomophagous species or their
hosts, one of the most important principles to observe rigidly is never to bring field material
or host-plant material into the insectary unless it has been fumigated or is securely caged.
Continuous successful insectary operation can be achieved only if one is constantly
aware of the interrupting factors which may be encountered and may need appropriate
preventive measures. Disease can quickly neutralize a propagation programme by either :
(1) assuming devastating epizootic proportions with immediate effect, as virus diseases of
lepidopterous larvae; or (2) acting as an insidious devitalizing agent, as microsporidian
diseases of eggs or larvae. Entomophagous mites can have an equally devitalizing effect on
their insect hosts. Fine duet from poor-quality concrete floors or from free-spraying
humidifiers can settle on host material and greatly reduce activity of parasites.
Figure 8.12 : Methods for counteracting
lethal
high
temperature
during local
shipment of parasites.
A) Prechilled chest and frozen gel packet
utilized in shipments requiring added
protection
from excessive
heat.
Note
buffer between frozen gel and containers
of insects.
B)
Carrying
case
which
cools
by
evaporation. The plastic hose is used for
jarring the lnscts out of the tubes at the
release site.
Facilities and equipment which will
improve sanitation for the building
are
vacuum
brooms,
cleaners,
sweeping
brushes,
compounds,
mops, buckets, antiseptics, such as
aqueous solutions of formalin or
chlorine, and also adequate storage
space for these items. The best
sterilization method for glassware is
autoclaving. Next
best
are
deep
sinks or trays wherein glassware
and plastic cages can be immersed in antiseptic solutions.
Waste disposal presents a special problem. A safe procedure to prevent living
insects leaving the environs of the insectary is burning or fumigating host-plant material
119
used in the culture programme. Arrangements for regular removal of trash should be made
with local agencies.
Subsidiary facilities
Greenhouse : Greenhouse facilities may be necessary for culturing host plants. In general,
however, a plant propagation greenhouse does not provide an ideal environment for the
mass culture of entomophagous insects because of factors which are largely beyond the
control of the insectary operator, namely: (1) light and humidity conditions cannot be
economically standardized; (2) contaminant species of phytophagous insects will invade the
plants; and (3) predators or secondary parasites cannot be excluded. Specially designed
greenhouses for the quarantine handling of imported weed-feeding insects would overcome
the disadvantages just mentioned, but the cost probably would prevent their being used for
commercial production of entomophagous insets.
Storage
Storage of Equipment : Storage of equipment has been mentioned previously and is
here re-emphasized for it is a most necessary facility both within the insetary in the form of
reasonably dust-proof cabinets or drawers for storage of cloth and glassware, and outside
as weather-proof sheds or buildings for unused cages, certain host material, and tools.
Storage of host material : Storage of host material may require considerable space
and is usually provided independently of the insectary building. A commercial insectary
may require several hundred square feet for such purposes. Because of the necessity for
maintaining several insect species simultaneously, a research insectary also may require
relatively spacious facilities for storage of a variety of host-plant materials such as banana
squash, melons, potatoes, citrus fruits, and grains, and, in addition, lath house or
greenhouse space for leafy plants.
Seasonally available host materials such as melons and other cucurbits require dry
storage. Ordinarily, an open shed with a water-tight roof provides adequate protection.
Material of this sort is safest when placed in layers one or two deep on sturdy, airy racks.
Thorough screening will minimize rodent damage.
Grains and cereal products should be fumigate upon receipt and then placed in
insect proof containers for storage.
In warm regions certain host materials such as potatoes and citrus fruits do not
keep well without refrigeration. Refrgerated rooms will be required to maintain the
necessary 380 to 420F.
Fumigation
Fumigation facilities are necessary if proper sanitation and prevention of
contamination by insects are to be achieved. Nicotine and HCN fumigation chambers can
be easily and economically constructed. For example, a large box or framework enclosed by
120
a plastic tarpaulin will suffice. Injury to potted plants from methyl bromide usually can be
averted if relative humidity in the fumigation chamber does not fall below 80 per cent.
Persons who conduct fumigation must be thoroughly conversant with the dangers of
the materials used and the necessary antidotes, and must understand what consititutes
adequate fumigation for the particular material being tested. Except for the occasional
necessary fumigation of insectary rooms, fumigation should be performed well outside of
the insectary. If a permanent fumigator is contemplated, it should be located downwind of
the main building and equipped with an exhaust stack high enough so that discharged
fumes will not endanger personnel or cultures in the insectary or near-by installations.
Workshop
If research is to be a major insectary function, facilities for construction and repair
of cages and other paraphernalia are necessary. Power tools such as table saw, grinders
and sanders, jointer, bandsaw, and drill press require the services of someone trained in
their uses. A collection of conventional hand tools such as hammers, saws, chisels, planes,
screwdrivers, wrenches, brace, bits, and clamps of various sizes will suffice for most
repairs. Electrical and plumbing facilities also will need occasional attention. In additon,
painting supplies will be needed, and safe storage is required for flammable materials. In
short, the insectary programme will greatly benefit from the services of a skilled handyman.
Specialized Research equipment for Controlled Environment Studies
BIOCLIMATIC CABINET. Of proved value for determining biological potentials of
insects is the bioclimatic cabinet. ‘Each chamber is similar to a walk-in refrigerator, being
fitted with two doors separated by a 4-foot vestibule, and having an inner work space
providing floor area 6 feet by 6 feet for conducting experiments. Attached to the chamber
are various air-conditioning controls and devices that permit the air circulating within the
chamber to be heated, humidified, cooled, or dried, as desired. A major feature of these
chambers lies in their capability of controlling temperatures and humidities in smoothly
varying patterns such as occur naturally. Temperatures may be controlled to within plus or
minus one degree Fahrenheit over the range from -50 to +1250. Humidities, within this
same temperature range, may be controlled to within plus or minus
per cent relative
humidity over the range 20 per cent to 98 percent. At temperatures above freezing,
humidities may be controlled to as low as 10 per cent. Lights within the chambers are
automatically turned off and on by means of time clocks, and the settings of these clocks
are periodically varied in order to duplicate the variations in photoperiod as these occur
naturally.
Biotrone (Bio = life; trone = balance). In order to study the plant-insect complex
under a wide range of combinations of sunlight, temperature, and humidity, this highly
specialized greenhouse was conceived by S.E. Flanders an C.A. Fleschner.
121
‘The biotorone is a 12 ft. x 12 ft. glasshouse covered by a cubical lath-house
consisting of 165 interlocking aluminium louvres. The main purpose of the louvres is to
prevent excessive direct solar radiation, and they may be opened and closed automatically
in three ways, namely, by direct solar radiation, by a combination of direct solar radiation
and temperature within the glasshouse, or by a time clock. As a means of heat conservation
all louvres are closed at sunset and opened at sunrise the year around by the time clock
which is self synchronizing for seasonal change in length of day. In additon, the clock
energizes a remote bulb thermostat circuit which contains three separately located
thermostats. These thermostats are located on the tops of the lath house and shielded to
face east, south, and west. When a thermostat is closed by solar radiation in excess of a
predtermined intensity, only the louvres controlled by that thermosatat are closed. The east
thermostat controls the east side and the easterly half of the top. he south thermostat
control the south side and the entire top. The west thermostat controls the west side and
the westerly half of the top. The north, or entry, side is opened and closed by the clock and
stays open all day.
‘Heating or cooling within the glasshouse is accomplished by a thermostatically
controlled electric heater and a refrigerative cooler. A humidistat adds humidification by
means of fog-nozzles.
‘Fluorescent and incandescent lights within the glasshouse provide simulated
sunlight should the need arise.
The biotrone is located at the University of California, Department of Biological
Control, in Riverside.
Insectaries of the world
Table : A partial list of biological control insectaries
Country
Agrentina
Sponsoring organization
Date
Full-time
funded
personnel
Tecnologica
1935
22
Queensland Dept. of Lands, Brishbane, Qld.
1924
7
Canberra, * A.C.T.
1927
7
Samford, *Qld.
1958
3
Sydney, * N.S.W.
1953
3
Canada Dept. of Agr. Res. Branch, Belleville, *
1928
79
1937
20
Instituto
National
de
Agropecuaria, Buenos Aires
Australia
CSIRO.
Canada
Ont.
Chile
Ministerio de Agricultura, La Cruz
122
France
USDA, ARS Nanterre, Scine
1919
7
Minister dde P Agriculture, La Miniere, S. et O.
1957
5
Institut
National
de
la
Recherche,
1917
7
Biologische
Schidlings
1953
18
Landesanstalt fur Pflanzenschutz, Stuttgart*
1951
2
CIBC, Bangalore
1956
2
Indian Agricultural Reseach Institute, New
1938
11
Agronomique Antibes (A. – M.)
Germany
Institute
fur
bekimpfung, Darmstadt
India
Delhi
Israel
Citrus Marketing Board, Rehovot
1960
2
Japan
Faculty of Agriculture, Kyushu University,
1920
-
Fukuoka
Kenya
Dept. of Agr., Nairobi
1941
--
Mauritius
Dept. of Agr., Reduit
1924
--
New Zealand
Dept. of Scientific and Industrial Research,
1956
29
Ent. Div., Nelson*
Pakistan (West)
CIBC, Rawalpindi
1957
8
Peru
Estacion Experimental Agricola de la Molina,
1935
3
Lima
Switzerland
Swiss Federal Agr. Exp. Sta., Nyon, Changins
1960
4
Trinidad
CIBC, St. Augustine
1945
5
Turkey
Zirsi Mucadele Enstitudu Parazit, Laboraturai
1956
4
Dept. of Agr. Tech. Serv., Div., of Ent., Pretoria
1957
6
Union of Soviet
Ministry of Agriculture,
1947
8
Socialist
Tashkent*
Pyatigorsk*
1958
6
Batumi*
1947
5
Albany*
1943
33
Riverside*
1929
27
Diyarbakir
Union
of
South
Africa
Republics
United States
Univ. of California,
Associates Insectary,
Santa Paula, California
123
Fillmore Citrus Protective District, Fillmore,
1928
3
1924
3
USDA, Moorestown*, New Jersey
1949
2
CIBC, Fontana, CaliforniaBd. of Agr. and
1913
7
Bd. of Agr. and Forestry, Honolulu*, Hawaii
1913
7
Inst. for Plant Protection, Beograd (Zeman)
1954
6
California
Santa Paula, California
Fillmore Citrus Protective District Fillmore,
California
Forestryh, Honoulu*, Hawaii
Yugoslavia
Insectary Facilities and Equipment
Country
India
Sponsoring organization
Date
Full-time
founded
personnel
CIBC, Bangalore
1956
2
Indian Agricultural Research Institute,
1938
11
New Delhi
Factors Responsible for Trouble-Free Insectary Operation
Constant awareness and eternal vigilance should be watchwords for good insectary
operation. Here, the
ounce of prevention’
is indeed much more
to be desired than the
‘pound of cure.’ The
operator
should
always assume that
trouble is imminent
and a daily inspection
should be
routinely
made to detect it in
the incipient stage.
Figure 8.13 : Hot wire
barrier rearing tray.
A) General aspect : size
of this one is 20” x 40”.
The sides are 2 1/2”
high. Designed for use
124
with 115 V a.c., this device is extermely useful for open culture of lepidopterous larvae or wingless,
nonjumping insects. During operation the barrier is hot to the touch, not glowing red. Its gauge
(diameter) is determined by its length.
B) Details of construction : insulated lead-in wires are securely fastened between lock-nuts and
arranged as a crude maze in order to prevent escape of insects at that point. Bolts clinched tightly to
wall with washers on both siedes to prevent any looseness that would lead to arcing of the current.
Interior angles rounded at corners and bottom. Small tough wire is used to bind hot wire to the asbestos
sheeting on the sides. The binding wire looped over monel wire with ends extending through a small
drilled hole and out the other side. The wires are spread apart and a square-shouldered double-pointed
tack or staple is driven between them and across the drilled hole. The binding wires are brought
together over the staple and twisted together so that the hot wire is held snugly against the sheet rock.
The twisted wire is cut off to about ¼” long, turned down and the end driven into the wood so it will not
snag clothes or the person wroking with it.
The elimination of possible sources of pest organisms in the environs of the
insectary is one means of keeping the laboratory free of contaminants. This may be done by
eliminating all host plants, culturing only those that are virtually pest free by nature, or
routinely treating the host vegetation if the above means are not feasible. Because of the
possibility of insecticides drifting into the insectary, the treating of shrubs in the near
vicinity of the building is a definite hazard to the cultures in the insectary and should be
done only as a last resort and with great care. Elimination of plant hosts from the
immediate vicinity of the insectary is a better approach.
The utilization of small units greatly facilitates the detection of incipient pest
invasions in certain cages without threatening the remainder of the cultures.
The rapid turnover of cultures and the immediate destruction of used culture
material preclude the building up of possible pest organisms such as secondary parasites,
mites, psocids, and disease.
The use of effective barriers, such as the hot wire type, (figure 8.13) will prevent the
escape of larvae and crawling insects (Flanders 1945). Infestation of Pediculoides mites, an
organism of great concern to insectary operators because of its irritating effect on human
skin, can build up into traublesome populations on escaped lepidopterous larvae cocooned
in cracks or hidden nooks about the building.
The sterilization of equipment and facilities is essential in preventing the invasion
and spread of certain diseases infecting insect cultures.
Insects that have been subjected during their developmental stages to a stress of
some sort such as periods of famine or subnutritional diet, incompatible environment,
crowding, or injury may be more susceptible to the invasion of certain disease organisms or
latent viruses than healthy insects.
125
Biological Control of Contaminants
In certain circumstances a biotic agent may be used to control or even eliminate a
pest organism in a culture. For instance, the predatory thrips, Scolothrips sexmaculatus
Perg., was useful against the Pacific mite, Tetranychus pacificus McG., in the culture of the
red scale parasite Comperiella bifasciata How. (Flanders 1943b).
The bethylid Cephalonomia waterstoni Gah. completely eradicated an infestation of
the beetle Larmophloeus pusilus (Schonh.), which was very destructive to the eggs of
Sitotroga (Schread and Garman 1933).
Use of Hot Water to Control Contaminants
A hot water bath has on occasion been used to control pest organisms in the
insectary cultures. Wheat, the host medium of Sitotroga, was dipped in a hot water bath to
kill possible pests as well as to add humidity to the medium. Also, in the mass culture of
the potato tuber moth newly laid moth eggs were immersed in a hot water bath (118 0 F for
20 minutes) to prevent the further development of microsporidia in the cultures (Finney,
Flanders, and Smith 1947).
Chemical Control of Contaminants
Insecticidal applications for the prevention of pest organisms have been employed
with success in several instances. Sitotroga eggs were immersed momentarily in carbon
disulphide to prevent infestations of mites (Spencer, Brown, and Phillips 1935). Mortality
from virus infection can be markedly reduced by immersing eggs of Sabulodes caberata
(Guen.) in to per cent formaldehyde for 90 minutes.
Pediculoides ventricosus (Newp.) may be controlled by dusting the infested area with
a light film of flowers of sulphur which kills the mobile young attempting to travel through
the dust particles. Any field infestations brought into the insectary, especially infestation of
lecanine scale, or lepidopterous cocoon material that includes exhausted material, should
be eyed with suspicion and a circle of sulphur dust or of specific insecticides should be
placed around the area. Use of such insecticides in the insectary must be conducted with
extreme caution.
Rapid turnover and immediate destruction of used material tend to suppress
incipient infestations and prevent a flare-up of certain pests.
The recent development of silica gels has been a great help in controlling such pests
as cockroaches in the laboratory.
Because of its brief residual action, TEPP has been very useful in controlling
greenhouse pests on plants soon to be used with insect cultures in the insectary. Specific
acaricides have been used to control mite infestation on plants infested with insect cultures
such as spotted alfalfa aphid, Therioaphis maculata (Buck.)
126
Pyrethrum bombs, nicotine fumes, and TEPP smoke generators are used effectively
for general fumigation purposes in the greenhouse.
Methyl bromide fumigation is used routinely to defaunate growing plants before
bringing them into the insectary as well as to eradicate mealybugs on potatoes and tuber
moths in potato tubers destined for rearing scale insects.
Movement of Personnel Throughout the Insectary
Regardless of how insect proof, or how well arranged for isolating competitive insect
cultures the insectary rooms are, contamination may still occur if traffic of personnel and
visitors is not carefully regulated.
The work for the day should be scheduled to begin with the handling of uninfested
host media, and then proceed successively from the least to the most competitive host
species, thence to parasite cultures least likely to cause contamination, and then to
cultures more hazardous. Obviously, few species under one roof present less likelihood of
contamination occurring than when several are being cultured simultaneously.
If personnel is limited, it might be well to work with highly competitive insect
cultures such as mealybugs and potato tuber moth on alternate days. Pure cultures should
not be jeopardized for the pleasure or convenience of casual visitors. A visiting official with
a limited schedule might visit certain cultures before and after lunch, thus lessening the
contamination hazard with an interval of time.
Nicotine Hazards
Many entomophagous insect species are very sensitive to the effects of nicotine.
Smokers should always wash their hands thoroughly before handling equipment, such as
vials, feeding dishes, or cages, which the parasites will contact, and cages as well as
materials from which cages will be constructed preferably should be stored in a tobacco
smoke-free atmosphere.
Hazards of Dust
As a generalization, parasites are quite sensitive to dust parbicles. Tubes use for
collecting purposes should be well cleaned and stored in dust-free areas. Cages should be
wiped free of dust before they are used. Plastic tubes sometimes used in place of glasscollectin tubes are particularly difficult to maintain in a dust-free condition on account of a
static electrical charge usually associated with them, and they are not recommended for
use in hadling dust-sensitive species. Dust clinging to the bodies and wings of certain
insects cause death in a matter of a few hours; others are weakened and engage in
continuous cleaning activity to the exclusion of normal mating and ovipositor. Use of sealed
plastic bags for storing freshly washed and dried glassware is strongly recommended.
127
Sterilization of Equipment
Equipment used in the culture of host material subject to bacteria or virus diseases
should be either at autoclaved or treated with a good bactericide or virus inactivaor before
being re-used or placed in storage. Equipment not amenable to autoclaving should be
washed clean with a good detergent and immersed in an antiseptic solution such as dilute
formaldehyde.
Such equipment as metal cages or screen trays may be dipped in a scalding bath for
a few minutes in order to kill all insect material such as cocooned larvae, parasites, or
mites. This material, along with dried grass and debris, is then sufficiently loosened to be
easily removed with a strong spray of water.
Insectary Sanitation
Sanitation regulations strictly enforced will prevent losses from such pests as mites
and secondary parasites. The facilities and equipment of the insectary should be
maintained as immaculately as possible. The vacuum cleaner, brooms, brushes, soap and
water, and antiseptic solutions should be used unsparingly and routinely.
Hazards of Cage Materials
Before new cages or containers are used they should first be tried out
experimentally to determine whether any of the materials used in their construction are
toxic to the insects.
The first boxes used in the culture of Macrocentrus were constructed of redwood
and had to be discarded when experiments proved that they were poisonous to the
parasites (Finney, Flanders, and Smith 1947). Copper screen is toxic to some species of
insects when used as cage covering. The fumes from acetate sheets made into containers
may be toxic to insects. The sheets are usually taken from stocks where volatile gases have
been unable to escape. These sheets before they are used must be well aired and cured,
preferably in the sun, until all traces of the fumes have been eliminated.
128
Lecture No. 9 (Unit II)
Basic standards of insectary
Attributes of an Ideal Insectary
Estabilishment of the Isectaries
An ideal insectary should have the following considerations :
1]
It should be located in the Temperate zones. The general experience is that creating
warmer conditions (higher temperature) is easier as well as cheaper than cooler
(lower temperatures).
2]
It should at least be 0.20 0.25 kms (200 – 250 mtr.) away from the farm area in
order to avoid pesticidal drift, minimize chances of entering the undesired natural
enemies like Bracon spp. on Corcyra cephalonica Staiton and contaminate the host
cultures, avoid risk of laboratory host to escape and attack field crops.
3]
It should be away from the industrial area especially while the coccinellid predators
and certain aphelinids are cultured as these are prone to the deleterious effects of
air pollutants.
4]
Exclude plantation with in the immediate vicinity of the insectary while planning
landscaping as plants (Flora) attract undesired fauna, so as to avoid field
contamination.
5]
Orient the length of the insectary-building in east west direction, especially in areas
with hot summers, natural lighting gains uniformity. The wall on the sunny side
can be shaded by an overhanging / climbing plants using selected plants. Plants
having fauna which may affect the insectary cultures must be avoided.
6]
It should possess an area of 288 sq.m. (24 x 12 M) in order to house equipments,
generator and working personnels.
7]
It should have uninterrupted water supply.
8]
It should have dust/ant/rat proofing.
9]
It should have easy access for convenient handling of material, disposal of refuse
material, etc., hence, road should be permanent.
10]
It should be well equipped according to the requirements of a particular insect
species being mass produced in the insectary.
11]
Ceiling height should not be more than 2.5 M as it is not only convenient for insect
catching but also reduce load on air conditioners.
12]
The principles of constructing a good and low-cost building using indigenous
resources should also be the integral part of an ideal insectary besides security
measures.
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
129
Layout plans for insectary
The layout plans for need based insectaries should be planned separately for (i)
research insectary (Fig. 2) having reliable environmental control and (ii) Commercial
insectary (space shown for experimental laboratory in Fig. 2 may be conveseted into
commercial deals and public relations) with adequate facilities to have optimum
environmental control viz. 27+20C temperature, 60 + 5% relative humidity, light and good
hygienic conditions. The basic requirements of equipments and power source for research
insectary (research laboratory) and commercial insectary (commercial laboratory) are given
in Table.
EP = Egg parasitoids
PH = Poly/Glass house
LP = Larval parasitoids
PP = Pupal parasitoids
Fig. 2 Layout of an ideal research lab.
Basic requirements of glasswares and other items for an Insectary
Glass wares like glass tubes, petridishes, test tubes, vials, desiccators, cover slips,
slides, distilling set, hot plate, muslin cloth, marking cloth, jars, stapler, stapler pins, filter
paper, scissors, forceps, rubber bands, plastic trays, foam sheets (sponge), filter paper,
absorbent cotton, non-absorbent cotton, gum, reference cards, tricho-cards, lamps, cages,
pots, host insects, chemicals etc. are required for an insectary. These may be quantified
based on the requirement in the light of natural enemies multiplied / studied and type of
the insectary.
130
Table : Basic requirements of equipments and power source for an insectary
S.N.
Equipment
Purpose
1
Diesel
Generator
For neutralizing the effect of
40KV
(power
source)
Quantity required
(number) for
Research Commercial
Insectary
Insectary
One
One
electricity failures and thus
maintaining the desired
enviromental conditions of the
insectary.
2
Air
Conditioners
(1.5
3
ton)
with
organism handled there.
B.O.D. Incubators
For maintaining the parasitoids,
with stabilizer and
predators or their hosts and
humidity
studying their ecological
control
Six
Five
Two
One
-
Three
Two
One
One
Six
Four
Two
Four
One
One
requirements.
Advance
research
microscope
image
system
Ten
the rooms as per the need of the
stabilizer
device
4
For regulating the temperature of
For advance studies in biocontrol
with
which need high magnification and
analysis
their documentation. For general
Binocular
microscope having
studies
including
dissections,
measurements, etc.
provision for fitting
the occular micrometer.
5
IIot Air Oven
For sterilizing the glass wares and
food material like sorghum for rice
meal moth
6
Huidifiers
humidistat
with
For maintaining and observing the
relative humidity of the particular
room(s)
where
cultures
are
maintained
7
Desert Coolers
For reducing the temperature during
extreme summers.
8
Refrigerator
For storing the natural enemies/
certain media.
131
9
Deep Freezer
For storing the chemicals and cell
One
-
and
Two
-
For centralise stabilization of the
One
-
One
One
One
One
One
One
One
-
fractions.
10
Environmental
For
advance
ecological
Simulation
semiochemicals studies.
Chambers
Inline stabilizer
voltage for running
11
PC computer with
For recording, storing and analysing
printer
the data
Electric Typewriter
For typing the reports and other
documents.
12
Autoclave
For
sterilizing
the
media
and
plasticwares
13
Analytical
single
pan balance
14
Refrigerated
speed
For weighing the ingredients for
different media / artificial diet.
high
centrifuge
For isolation of semiochemicals and
cell fractions.
fuge
15
Homogenizer
For developing the media for insets
One
-
Gas Liquid
For analysing the constituents of
One
-
Chromatography
insect
(GLC)
required for developing the diets and
(amino
aids
lipids,
etc.)
isolation of semiochemicals
132
Lecture No. 10 and 11 (Unit III)
Colonzation, Techniques of Release of Natural enemies,
Recovery Evaluation
Colonization
It is a kind of manipulation of an organism to establish it in a new locality.
In India, Colonization efforts have also been made using indigenous, Epiricania
rnelanoleuca a parasitoid of Pyrilla perpusilla from North to other parts of the country
specially south. Aulhor is of opinion that term colonization may not be restricted only exotic
natural enemies. Howevcr, multiplication aspect is not always essential to be with
colonization as some of the potential natural enemies can be very effective and colonized
wliile their laboratory techniques for.multiplication are not developed. The other steps
(procedure) involved in colonization are more or less applicable also in qase of domestic
introduction (isolation of an indigenous organism and its introduction with in the country,
where it does not occur. Major components Colonization are :
1.
Introduction of natural enemy.
2.
Transport of natural enemy.
3.
Release of natural enemy.
4.
Recovery and establishment of natural enemy.
5.
Evaluation of natural enemy.
1. Introduction of natural enemies
The prime objective of natural enemy introduction (foreign exploration) is to
identify, select and import such organism which have promise to establish in the country of
introduction. These organisms should be imported in sufficient quantity at suitable stages
in healthy condition Dc Bach ( 1964 ) suggested undermentioned ecological points to be
considered for successful natural enemy introduction.
(i)
Climate : Is useful to import any natural ememy from a place having similar
climate of the country where imports are to be operated. The spectacular successes in our
Country due to introduction of ladybird Rodolia cardinalis (Muls.) may be attributed to the
similar climatic factors prevailing in these countries.
(ii)
Alternate Hosts : Many failures in the colonization of newly imported
natural enemies have been correlated with the absence of alternative host species to tide
over the scarcity of the principal host.
(iii)
Biological competitors for the host and Hyperarasitoids : During the
scarcity of sufficient host or its suitable stage which may occur due 'to attack of same host
by other species, the introduced natural enemy will have less chances of establishement.
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
133
Similarly, if an introduced organism is attacked by hyperparasitoids the chances of
colonization will be drastically reduced.
(iv)
Suitability of the host plant : Due to undesirability of a plant as a source
for shelter or food for the adult natural enemy, or a degree of physiological immunity
imposed upon the natural enemy by the host plant, colonization gets dilute. For instance if
a Trichogramma wasp is released against the borer pest in the ecosystem of sorghum and
chickpea, it will avoid chickpea plants and prefer to operate in the sorghum habitat ?
(v)
Physiological suitability of the Natural Enemy to its host : Physiological
unsuitability of natural enemy to their hosts sometimes is the another cause of failures in
colonization of imported parasitoids and predators.
(vi)
Dispersal of Natural Enemie : If an imported natural enemy has slow
dispersal, chances of successful mating will be more which will lead to successful
colonization.
2. Transport of natural enemies from laboratory to release site
Most natural enemies require a relative humidity of 50-80%, which can be provided
by placing moist material such as sterilized moss or synthetic sponges or cotton pads in the
container in such a way that natural ememies do not come into direct contact of wet
surface during transiction period. In order to avoid direct heat, release timing should be
decided in the early morning or late evening hours. In case of adults to be released, initial
feeding of honey or sugars or raisin should be provided to the natural enemies. If release
site is at a longer distance, direct heat to the natural enemies which often affects them due
to negligence is to be avoided.
3. Release of natural enemies against target pest
Clausen (1941 ) suggested that release in the evening around dusk restricts
dispersal while still providing a period of orientation to the new surroundings and might be
more desirable procedure during the hotter part of the year. Types of release material in
most of the parasitoids and predators is considcred to be the adult stage as compared to
the immature stages because adults can easily coup up with the new environment and are
low affected by biotic and abiotic factors. Colonization under cages (confined release) is
preferred over open field releases, i.e. natural enemies are released in the field in the
vicinity of suitable host.
Inoculative, inundative and host + natural enemy (accretive ) releases should be
decided based on the intensity of pest attack, availability of sufficient population of natural
enemies, type of habitat and other factors affecting release. Dosages of certain natural
enemies may be decided for proper management of the pest, Repeated releases as and when
necessary like chemical pesticides are to be made, as often natural enemies in India suffer
due to extremes of fluctuations in climatic conditions.
134
4. Recovery and establishment of natural enemies
Any progress towards establishment of an introduced species into the new
environment indicates the recovery. The main objective of recovery is to obtain a simple
qualitative assessment of the presence or absence of the natural enemy with little or no
reference to its probable efficiency. For sampling close examination of immature stages in
field or other possible methods of natural enemy collection may be deployed, Three species
of Trichogramma viz., T. exiguum (Barbados strain), T. perkinsii (Columbia strain) and T
japonicum (Philippine, strain) were released periodically against Chilo partellus in sorghum
field at Delhi. Inundative releases of Trichogramma exiguum during 1975 and onwards
indicated that the percentage stem tunnelling of sorghum at harvest due to C. partellus at
Delhi during 1976 was considerably reduced to 7.5% is against 33.4-53.6% during prereleased period. The egg mortality due to parasitization was upto 98.65%. These species
quickly established and were recovered from Nagpur and Delhi (Anonymous, 1982). In
India, several species of natural enemies have been recovered from the rclease sites or its
vicinity and found either temporary or permanentally establishad.
The exotic beetle, Sticholotis madagassa Weise released on 30.4.77 and 4.10.77 at
Harakhpur, Pipraich (Gorakhpur) against scale Melanaspis glomerata infesting sugarcane
were recovered after 6-7 months of releases from these sites (Misra et al., 1981). Similarly
Chilocorus cacti released in Deoria (Uttar Pradcsh) was recovered (Misra et al., 1984) while
only few immature stages were recovered when released, earlier at Bangalore during 197273 (Sankaran and Mahadeva, 1974). Weekly inundative releases of Trichogramma cacoeciae
pallidum and and T. ebryophagum against codling moth in Ladakh (Jammu and Kashmir)
were found effectively reducing the infestation by 17.5 to 37.5% (Pawar et. al., 1981 ) A total
of 17 releases consisting of 2018 grubs of coccinellid predator, Curinus coeruleus (Mulsant)
against subabul psyllid, Heteropsylla cubana Crawford between November 16 and
December, 1988 in an area of 100 m2, were successful in controlling the psyllid pest and
were subsequently recovered in March, 1989 for the first time in India (Jalali and Singh,
1989. The lady bird Cnpjolaemus montrouzieri (Fig. 5) which feeds on a variety of coccid
fauna is established in India and is very effective in controlling the mealy bugs (Gaulam el.
al. 1988, Mani, 1988). Similarly, due to domestic introduction of parasitoid, Epiricania
melanoleuca from Ullar Pradesh, the pyrilla is now kept under control in the states of West
Bengal, Bihar, Orissa, Rajasthan, Madhya Pradesh, Gujarat, Maharashtra, Tamil Nadu.
Kerala and Karnataka and is well established (Misraand Pawar, 1982a, 1982b, 1983, Pawar
et. al.1982).
5. Evaluation of natural enemies
To evaluate the effectiveness of newly introduced natural enemy, qualitative
evidence based on frequent, detailed and widespread field observation of a dedicated worker
135
is needed. It depends mainly on the wisdom, training, experience and sincere efforts of the
individual investigator. Sampling method and statistics play a great role in quantitative
methods of evaluation. Attempts are made to show the effect of the natural enemy or
enemies on the host population by analysing the host mortality at different stages due to
released organism. An effective natural enemy might be expected to show evidence of
control at the point of release within 3 host generations or 3 years (Clausen, 1951).
There are two Important systems followed normally for evaluating the effectiveness
of a natural enemy.
1. Experimental procedure (Natural enemy exclusion methods).
2. Analytical procedure (Use of life table technique).
Experimental procedure
Under this system barriers are created
between pest population and the natural
enemy.. The difference in density of pest
population is then ascribed to the excluded
natural enemies (De bach and Bartlett,
1964). It may be of two types.
(i)
Mechanical barrier method :
A
cage, cloth sleave, wire screen or any other
similar device
is
used to enclose~an
uninfested plant, branch section of bark
or field plot and the pest is introduced into
the protected zone. An adjacent infested
plot/cage/or
any
other
device
either
enclosed or enclosed similarly but With
opening enabling free access of natural
enemies, is used as a comparison.
Grubs sharing the prey
Fig. 5 Cryptolaemus montrouzieri preying on mealybug.
(ii) Chemical exclusion method : In this method, a selective pesticide is used to eliminate
or inhibit the population of natural enemies leaving behind unaffected (untreated) area.
The growth of host and infestation in an untreated plot (check or control) as compared
to treated plot provide evidence of natural enemy effectiveness. This practice is not
environmenlly friendly hence may be avoided.
136
Biological check method : This method has very limited
application in certain situations based on symbiotic
association between few ant species and homopterous
pests. These ants in exchange for the honey dew
excreted
by
homopterans
(membracids,
aphids,
fulgorids, coccids, etc.) protect them from the attack of
parasitoids and pmdators. By eliminating the ants from
the infested tree, the pest population is exposed to
natural enemies and may be compared with the check.
Fig 6 : Adults and nymphs of sugaracane leafhopper
parasistied by the parasitoid. Epiricania melanolecua.
Predatory spider after the prey.
Gram pod borer catepillar infected with
nuclear polyhedrosis virus
Fig 7 : Conserve the natural enemies
Analytical procedure
In this method a tabular accounting of mortality and its cause or causes (life table)
are determined in the light of various environmental factors. For life table construction a
series of density samples of the successive life stages (eggs, larvae, pupae and adults) of the
pest are collected and causes of mortality or decline in numbers during the life cycle are
measured over a sequence of 8-15 generations. The data so collected for each mortaty are
analysed statistically or represented graphically, for correlation between pest density and
changes in density and the amount of mortality resulting from each measured mortality
factor.
Lecture No. 12 (Unit III)
Conservation of Natural Enemies
137
Conservation of natural enemies
"Insects and Diseases, Weeds and Pests have their own controls -let these
operate and assist them"
—Fukuoka, 1978
"Conservation is a kind of manipulation of the habitat (environmental modifications)
which favour an organism for its survival and effective suppression of injurious fauna, in
order to keep natural balance in the eco-systcm."
Conservation of natural enemies (Fig. 5, 6 7) should be first consideration in
biological pest suppression ( biocontrol programmes ) as it avoids measures that destroy
natural enemies. Environmental factors viz., weather, food, sources other than food (place
to live), other organisms of the same species and other organisms of different species
including man, influence survival and activity of an individual natural enemy (Solomon
1949). Sugarcanc pyrilla epidemics during 1973-74, 1976 1978, 1984 and 1985 in the
states of Bihar, Uttar Pradesh, Punjab and Haryana were avoided by conserving prasitoid,
Epiricania melanoleuca Fleteher. Besides, several 'farmer's friends" viz. parasitoid,
rTetrastichus
pyrillae, on sugarcanc pyrilla, Platygaster oryza cameron on rice gall midge,
Orseolia oryzae (Woodmason): Diaeretiella rapae Curtis on mustard aphid, Lipaphis erysimi
Kalt. etc. and predators like syrphids on aphids, spiders, preying mantis, carabids, etc.,
preying on a variety of insect-pest fauna in different agro-ecosystems are either difficult to
mass produce or their multiplication techniques are yet to be developed, hence
conservation (measures that destroy natural enemies), and enhancement (use of measures
that increase longevity, fecundity, hatching and subsequent reproduction) arc indispcnsible
under Indian conditions.
Conservation and enhancement as discussed by Stehr (1982) earlier workers De
Bach ( 1964 ), Huffaker and Messenger (1976) and are summeriscd below:
(i)
Protection of natural enemies from pesticides.
(ii)
Developing resistant or tolerant natural enemies towards pesticides.
(iii)
Preservation of inactive stages of natural enemies outside the cropped area.
(iv)
Avoidance of harmful cultural practices for the natural enemies.
(v)
Maintenance of diversity and necessary hosts to tide over the difficult days of
natural enemies.
(vi)
Manipulating the alternate hosts for natural enemies.
(vii) Facilitating the availability of natural foods like nector, pollen grains, and honey
dew.
(viii) Spray or supply of food supplements to natural enemies, perpetuating in the field.
(ix)
Erection of artificial nesting shelters near field
for predacious
wasps
and
birds.
Compiled
by : Dr. U.P. Barkhade
& Dr.
A.Y. Thakare
138
(x)
Control (reduction) of undesirable predators which preferably feed on parasitised
hosts, sparing unparasitiscd host.
(xi)
Control of honcydcw feeding ants which interface with the natural enemies by way
of disturbing the process of parasitization.
(xii) Manipulation of favourable temperatures suited for the natural enemy, especially
under protected cultivation (glass/poly/ green house).
(xiii) Avoidance of dust (road dust or other dusts) which affect the natural enemies. Dry
conditions coupled with cultivation and elimination of ground cover result in dust
formation and accumulation.
It has been observed by the author that juicy sweet sorghum cultivars attract more
predacious lady bird beetles (Coccinella septempunctata and Menochiens sexmaculatus)
than traditional varieties. There are several practices including use of safer (less toxic)
pesticides viz. phosalone and endosulfan (Navarajan Paul et. at., 1979, Singh, 1986, 1989
a) which help conservation and enhancement of natural enemies noticed in India and
elsewhere. Some of the important practices which help natural enemy conservation and
enhancement arc represented in Table 11, and need to be undertaken / demonstrated at
farmer's field.
Table 11:Promising conservation & enhancement practices for some natural enemies.
Conservation
Country / place
Natural enemy
Pest spcies
References
Polistes
Leafworm
Ballou, 1915
Estabilishment
annularis
Albama
of artificial
(Linnaeus)
argillaceae
and
Crop
–
habitat
enhancement
practice
*
St. Vincent
Cotton
structures for
(Hlm.)
nesting
* Avoidance of
Germany,
Forest
Insectivorous
Forest pest
Bruns, 1959
weedicide, 2,
states of USA
Sugarcane
birds
Sugarcane
Mathes et. al.,
Parasitoids
pests
1953
Pteromalid
Bothynoderes
Van den
parasitoid,
punctiventris
Bosch and
Caenocripis
Germ
Telford, 1964
Egg
Leafhopper,
Doutt and
parasitoid,
Erythroneura
Nakata, 1965
4-D
* Shallow
USSR
Beet
ploughing
bothynoderes
Grom.
* Presence of
alternate crop
California
Grapewine
139
(wild black
Anagrus epos
elegantula
berries) for
Girault
Osborn.
Aphelinus mali
Eriosoma
(Haldeman)
lanigerum
hosting the
alternate
insect Dikrella
curentata
Gillett.
Wherein
parasitoid
overwinters.
Pruning of
Australia lation
Apple
infested twigs,
their storage
Wilson, 1966
(Hausm.)
and reinocu in
the same
orchard
Strip-
California
Alfalfa
harvesting
Aphidius
Acyrthosiphon
Van den
smithii
pisum (Harris)
Bosch et. al.,
Sharma and
Manduca
1967
Rao
sexta
Johannson
Avoidance of
North Carolina
Tobacco
tobacco
Trichogramma
Manduca
Rabb and
minutum Riley,
sexta
Bradley, 1968
varieties
Johannson
having more
sticky
Telenomus
exudates from
sphingia
the leaves.
(Ashmead)
Retaining
Huntingdonshre
Brussel
Predator,
Pieries rapae
Dempster,
weeds under
California
sprouts
Harpalus
L.
1969
special
rufipes
cricumstances
Avoidance of
San Joaquin
Citrus, garpa
Parasitoids
Scale insects
De Bach,
dust
Valeey
vine
and predators,
pacific mite,
1958 Flakerty
Metaseiulus
Willamette
and Huffaker,
occidentalis
mile pacific
1970
mite
Spray of food
supplements
California
Cotton, alfalfa
Chrysopa
carnea St.
Cotton pests
Hagen et. al.,
1970
140
(Mixture of
protein
hydrolyzate +
sugar + water)
Erection of
Texas
Citrus
wind breaks
Parasitoid,
Scale, Coccus
Reed et. al.,
Aphycus
hesperidum L.
1970
stanleyi,
(Compere)
Avoidance of
England
temperature
below
240C
in
Cucurbits,
Parasitoid,
Whitefly,
Hussey and
tomato, sweet
Encarsia
Trialeurodes
Bravenbore,
peppers etc.
formasa
uaporariorum
1971
Ghaen
Westwood
Trichogramma
Pieris rapae
the glass
house
Addition of
United States
Cole crops
laboratory
evanescens
reared
Westwood
Parker and
Pinnell, 1972
alternate host
Sitotroga
cerealella
(Olevier)
Galleria
mellonella (L.)
Avoidance of
India
Sugarcane
trash burning
Natural
Melanaspis
Shukla and
enemies of
glomerata
Tripathi, 1981
scale insect
India
Sugarcane
Epiricania
Leafhopper,
Joshi and
(ratoon crop)
melanoleuca
Pyrilla
Sharma, 1989
Fletcher
perpusilla
Walker
Avoidance of
India
Sugarcane
detrashing
Natural
Melanaspis
Jayanthi,
enemies of
glomerata
1991
White grubs
Jayanthi,
scale insect
Deep
ploughing
India
Sugarcane
Insectivorous
birds
1991
Lecture No. 13 (Unit III)
Augmentation of Natural Enemies
141
Augmentation
It is kind of manipulation within the organism, to increase its population and
efficiency through mass production technology and genetic improvement. The organism
used for the purpose may be of indigenous or exotic nature. The major components of
augmentation are
1. Multiplication of natural enemies .
2. Genetic improvement of natural enemies
Multiplication of natural enemy
(i) Host-range of natural enemy : scelionid egg parasitoid, Telenomus remus Nixon was
introduced into India in 1963 from Papua New Guinea and could be reared on Achaea
janata Linn. only for few generations. However, when reared on hosts viz., Spodoptera litura,
S. exigua and Agrotis spinifera (Hubner) (Ram Dass and Parshad, 1984), the parasitoid
could be reared successfully for several generations. Similarly, it became feasible to develop
mass, multiplication technique of coccinellid predator, Brumoides suturalis on mealybugs
after studying it host - range (Gautam., 1990 a)
(ii) Host effect on Natural Enemies : It is observed that if a host is comparatively bigger in
size, the progeny of the parasitoid is normally possesses good biological attributes viz,
longevity, fecundity and size of the parasitoids Gautam (1986a) studied the effect of
different noctuid hosts , on the parasitization by T. remus and found the parasitoid progeny
much superior when reared on eggs of Trichogrammatids Similar observations were made by
Dysart (1972) and Navaraja Paul et. al., (1981). Such natural enemies are consid-ered
superior for field relcases.
The parasitoid T. remus even though parasitises S. litura (unfertile eggs) where
certain essential aminoacids viz., cystine, citrulline, threonine, asparagine and phenyl
alenine were absent but failed to develop on the host and thus, lead to the collapse of
parasitoid progeny. However, it developed well on fertile eggs where all the
aminoacids
except phenyl alaninc were present (Gautam 1986 b ). Studies on the influence of
diapaused larvae of Chilo partellus (Swinhoe) indicated that the larval parasitoid, Apanteles
flavipes (Cameron) developed well, maintaining the biological attributes of the parasitoids at
par with non diapause larvae (Gautam, 1986 c). Further studies on host- parasitoid
relationship with regard to host age reveals that 0 to 72 hr old eggs of S. litura are accepted
by T remus with least detrimental effect on their progeny. Host eggs older than 72 hrs were
not parasitised by the parasitoid (Ram Dass and Parshad 1983)
Adults of Menochilus sexmaculatus were fed on several hosts and their effect on the
longevity, fecundity and egg size of the predator was studied. Longevity of adult M.
sexmaculatus fed on mealy bugs ranged from 22.7Compiled
to 40.14
days
in males
and
from 26.14
by : Dr.
U.P. Barkhade
& Dr.
A.Y. Thakare
to 42.71 days in femles. In case of those fed on Spodoptera litura and Corcyra cephalonica,
142
the period ranged from 2.57 to 3.86 days in both sexes. On an average 85.71, 22.85, 9.85
and 725.14 eggs were laid by a female when they were fed on Ferrisia virgata, F. virgata +
Planococcus pacificus, P. pacificus and aphids respectively. Little influence was found on egg
size and no effect on hatching due to feeding on different hosts (Gautam, 1989).
(iii) Influence of substrata and age of natural enemy on multiplication:
The ladybird predator, Coccinclla seplempunctata preferred to lay eggs on potato
infested with Ferrisia virgata, where 51.10% eggs were obtained as compared with 37.89%
laid on tissue papers which were at par among them selves. Very few (scanty) eggs (0.50%)
were laid on markin cloth compared with 7.95% on glass surface. On the contrary, female
preyed on aphids laid maximum eggs (61.64%) on multifoldcd tissue paper and the
minimum (0.50%) on Indian mustard twig, The eggs laid on other substrata, viz., jar
surface, cotton twig, and mark in cloth were 21.89, 14.35 and 2.13%, respectively (Gautam,
1990 b). In case of Brumoides suturalis, the ladybird preferred to insert eggs as many as
97.67% inside the cotton used for plugging the tubes and no eggs were laid on tissue paper
( Gautam 1990 a ). Similar trend was seen in case of other coccincllid predators viz.,
Scymuus nubilus, S. pyrocheilus, S.curtisii, Cryptolaemus montrozieri and Nephus regularis.
Adults of Menochilus sexmaculatus reared on F. virgata showed no particular preference to
different substrata viz. jar surface, tissue paper, on potato as indicated by the percentage of
egg laying viz., 36.51, 36.23 and 29.10, respectively. However, when preyed on Lipaphis
erysirni (Kalt.) and Aphis gosspii Glover it laid maximum eggs (71.75%) on cotton twigs
(Gautam 1990 c ).
Rearing of M. sexmaculatus either on mealy bugs using tissue paper or aphids with
cotton twigs upto 4th week (productive age) may be adopted as 73.44 to .81.47% eggs with
91.35 to 100% hatching are obtained in this period. Thereafter, adults may be discarded in
order to save laboratory host (Gautam, 1990c). Adults of C. scptcm punctata must be
released in nature after 10th week if reared on F. virgata and after the 14th week if preyed
on aphids, to save precious prey in the laboratory ( Gautam, 1990b ) as further rearing of
the predator becomes unproductive and uneconomical.
iv) Influence of adult food on natural ememies :
The sugar viz, fructose, glucose, lactose, maltose, galactose, sucrose and mannitol
have beneficial effect on the longevity of the adult parasitoids. Sometimes even a small
droplet of water is essential for the survival and increased longetivity of certain newly
emerged parasitoids like Campoletis chlorideae. Therefore, considering the impact of adult
nutrition on the longevity and fecundity of Chelonus blackburni, glucose 20% or fructosc
20% with 1% protinex can be used profitably in mass-rearing of these parasitoids (
Navarajan Paul et. al, 1986). A piece of raisin helps several natural enemies viz. Ananteles
143
spp., Bracon spp., Campoletis spp. to increase their biological attributes while sugar cubes
or crystal for tachinid parasitoids.
Influence of food supplements on the reproductive behaviour of the predator,
Chrysoperla carnea (Stephens) (earlier considered to be Chrysopa scelestes Banks in India)
revealed high degree of dependence on the type of food provided to the adults. The preoviposition was reduced lo 3.60 days in the, case of adults fed on flowers of ganja
(Cannabis saliva Linnaeus) followed by 6.80 days in castor (Ricinus comniuns Linnaeus) as
against 10.40 days obtained on 50% honey solution. There was many fold increase in
fecundity when females fed on composite food comprising flowers of cotton, okra, ganja,
maize and castor (81.60 eggs/female) followed by pollen grains of castor (269.60
eggs/female) as against honey (25.40 eggs/female) (Gautam and Navarajan Paul, 1988). In
general, addition of 10% sugar solulion as feed supplement to coccinellid predators
besides their prey is beneifcial and may be followed in multiplication programmes.
(v) Influencc of cold storage of host eggs on Natural Enemies :
In order to synchronise the availability of host eggs with the natural enemy
emergence as well as to sterilize and kill the host embryo, some times host stages are
preserved at lower temperatures, ranging from - 4°C to 50 C. Ram Dass and Atma Ram
(1983) studied that storage of Corcyra cephalonica eggs at -6 + 10 C beyond 8 days adversely
affected percentage parasitization and mean fecundity of Trichogramma exiguum whereas
no effect of freezing was observed on per cent emergence, per cent female and adult
longevity. Eggs of S. litura stored at various temperatures, namely, -6, 5, 1O, 15 and 20°C
revealed that 10°C temperature was suitable for parasitization by Telenomus remus
without affecting the biological altributes of the prasitoid. There was sudden decline in the
parasitization on 9th and 10th day beyond which host eggs were not accepted by the
parasitoid for parasitization. Cold storage of eggs in other temperatures showed very little
parasitization beyond 48-96 hrs (Gautam, 1987 a).
(vi) Influence of cold storage of natural enemies on its multiplication :
With a view to regulating the emergence of parasitoids viz., Trihogramma spp.,
Bracon spp. and Telenomus spp., a temperature range of 5-10°C is considered to be useful
for prolonging the immature stages of these natural enemies for 2-3 weeks while less than a
week when stored during adult stages that too after feeding them properly. Influence of cold
storage on the adult parasitoid and the parasitized eggs of S. litura by T. remus was
investigated by Gautam (1986d). The male parasitoid did not survive beyond 3 and 4 days
of storage of 5°C and 10°C, respectively. The females survived upto 7 days at both the
temperatures and parasitized 27.33 per. cent of host eggs at 5°C and 24.17 per cent at
10°C on the 7th day. Both the sexes could be safely stored upto 3 days at two temperature
without any detrimcntal effects on their biological attributes. Beyond this period, the
144
fecundity of parasitoid was reduced. The optimum age for cold storage of parasitized eggs of
S. litura at 100 C when reared at 27 ± 1.5°C was 6 and 7 days after parasitization, the
percentage emergence being 43.24 and 72.65 on 6th and 7th day, respectively. The other two
temperatures viz. 50C and 15°C did not prove to be favourable for the cold storage of
parasitized eggs. The 7 days old parasitized eggs could be safely stored for a week at 100C
without affecting the efficacy of parasitoids. There was a little effect upto 10 days.
Thereafter, a sudden decline in parasitoid emergence was observed upto 15 days, resulting
in no emergence on the 16th day.
(vii) Influence of different temperatures and relative humidities on natural enemy
multiplication :
The performance of T. rernus was studied at different combination of temperatures (
23 ± 1°C, 27 ± 1°C, 30 ± 1°C and 34 ± 1°C ) and relative humidities ( 25 ± 5, 50 ± 5, 75 ± 5
and 90 ± 5 per cent ). Among the various combinations, 27 + 1°C and 75 ± 5% RH was most
suitable for the mass multiplication of this parasitoid. It's life cycle was reduced to 7.00
days at 34 ± 10 C as against nearly two-fold increase to 13.70 days at, 23 ± 1°C. Different
levels of relative humidity, however, had no significant effect on the developmental period of
T. remus reared on S. litura (Gautam, 1986c). It was also studied in case of Trichogramma
brasiliensis where in most of the adults emerged were males when reared at temperature
exceeding 300C and was very detrimental in the mass production. However, when reared
between 25-270C, most of the adults produced were females, and had potential to breed
further without mating even for several generations. In general, temperature range of 2628°C and 60-75% RH is conducive for the multiplicalion of most of the insect species.
(viii) Effect of axenic rearing of natural enemies on mass production:
It has been a wide experience of insect rearing that when an insect is reared on
alternate host continuously for successive generations it normally looses its vigour.
Therefore, after certain generations either host is to be altered for few generations or
introduction of field population of natural enemy is suggested. Rearing of a larval
parasitoid, Campolctis chlorideae on its alternative host, S. litura serves purpose for 3-4
generations. Thereafter, change of its natural host, Helicoverpa ( Heliolhis ) armigera or any
other suitable host becomcs necessary in order to sustain the mass-production of
parasitoid. Rearing of aphidophagous coccincllids viz., Adonia variegata, Brumoidcs
suiuralis, Coccinella septempunctata, C. transversalis, Menochilus sexmaculatus, Micraspis
discolor, propylea dissecta, Scymnus pyrocheilus and S. curtisii on aphid ( Aphis, craccivora )
in the laboratory at 27 ± 1.5°C and 60 ± 5 % RH is successful only upto 5, 7, 5, 5, 20, 17,
16, 13 and 15 generations, respectively. Rearing beyond these generations is not possible
due to heavy larval mortality of the predator and reduction in hatching unless fresh field
145
population of ladybirds is inducted into laboratory stock. However, pre–oviposition period is
not affected due to axenic rearing of these lady birds.
Genetic improvement of natural enemies
The potential rate of incrcase or ability to outnumber host, recog-nised as a very
imporiant_attribite of an effective natural enemy. In India, Atma Ram and Sharma (1977)
attempted to improve the fecundity and sex-ratio of Trichogramma exiguum (earlier
considered as T. fasciatum) through selective breeding. A strain with improved fecundity
could be successfully selected through 16 generations. Sib mating resulted in reducing
considerably the heterogeneity present in the population undergoing selection. A similar
selection programme for improving sex-ratio, failed to yield significant results.
Studies conducted by the present author revealed that selective breeding could
increase mean fecundity of T. brasiliensis (Ashmcad) and did not produce homogenous
population even towards the end of 15th generation, suggesting use of other genetical
means to improve biological attributes of this egg-parasitoid.
Lecture No. 14 and 15 (Unit III)
146
Survivorship Analysis and Ecological Manipulation
Lecture No. 16 (Unit III)
Bankable Project Preparation
No. 20-38/2004-PP-I
Govemmeent of India
Ministry of Agriculture
Department of Agriculture & Cooperation
Krishi Bhavan, New Delhi
Dated the 26th July, 2005
To
The Secretary,
Department of Agriculture,
Govermment of All States/UTs
Subject : Guidelines for setting up of Bio-Control Laboratories.
Sir,
I am directed to enclose herewith a copy of guidelines formulated
for release of grants-in-aid/Central Assistance to NGOs/SAUs/KVKs for
setting up of Bio-Control Laboratories under the scheme “Strengthening
and Modemization of Pest Management Approach in India” for submission
of applications from different NGOs of your State.
Yours faithfully.
Lakhi Prasad
(Lakhi Prasad)
Under Secretary to the Government of India
Guideline for release of Grants-in-aid to NGOs for setting Bio-Control Laboratories under
the
Central
Sector
Plant
Scheme
"Strengthening
and
Modernization
of
Pest
Management Approach in India".
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
147
Objective of the Scheme
With a view to reduce the injudicious and indiscriminate use of chemical pesticides,
the Government of India has adopted Integrated Pest Management (IPM), encompassing
cultural, mechanical and biological methods and need based use of chemical pesticides, as
the cardinal principle and main plank of plant protection in the country. To achieve this
objective, establishment of adequate number of bio-control laboratories in the country is
essential. The Government of India has decided to involve non-governmental organization
etc. in setting up of these laboratories and provide them grants-in-aid for purchase of
equipment for this purpose.
Procedure & Minimum Expected Requirements
Each organization desirous of applying for grants-in-aid under the scheme should
apply in the prescribed proforma through any of the recommending officers authorized in
these guidelines. The applicants should have the requisite building for setting up of the biocontrol laboratory. The recommending officer, after inspection of such facilities and
satisfying themselves about the suitability of the organization, shall forward these
applications to the Plant Protection Adviser, Directorate of Plant Protection Quarantine &
Storage, NH IV, Faridabad (Haryana). The Dte. of PPQ&S would process and examine these
application and recommend to Department of Agriculture & Cooperation for granting in
principle approval for release of grants-in-aid to the eligible organizations. Thereafter, the
organizations concerned would purchase the equipment from the list appended to these
guidelines. The actual release of funds would be made after the recommending officer has
inspected the site and furnished a report that the equipment has been purchase and
installed in the laboratory by the organization. The original bills for the purchase of
equipment shall also be enclosed with this report. These organizations shall work in the
field of production of bio-control agents and bio-pesticides for a minimum period of 5 years
from the date of receipt of the grants-in-aid.
General condition for release of funds
1)
Organization should be registered with the State Government.
2)
Organization should have required accommodation in the name
of
organization or person association with organization.
3)
Site of the buildings would be easily accessible to the farmers.
4)
Person heading organization must be Sr. Secondary preferably having experience in
handling bio-agents/bio-pesticides.
5)
Building must have provision of three phase power wiring to run the equipment
efficiently.
6)
Organization must have fair track record.
148
7)
Amount of subsidy is 35% (50% for SC/ST/Women's Organizations) of total cost of
equipment subject to maximum of Rs. 5.00 lakhs per unit. Subsidy will be released
after verification of bills submitted.
8)
Equipment should be of standard quality/ISI marked falling under approved list for
the purpose.
9)
Financial assistance from Government of India is one time only for purchase of
laboratory equipment. The salary of staff/laboratory engaged and other recurring
expenditure for operation of laboratory is to be borne by the organization itself.
10)
Not more than one application from same revenue village will be entertained.
11)
While seeking subsidy under grant-in-aid, application must be accompanied by
building plan where laboratory has been or is to be established.
12)
Organization must have obtained registration of bio-pesticides to be produced from
Central Insecticide Board and Registration Committee (CIB & RC) as per Insecticides
Act, 1968.
13)
Technical persons employed or to be employed must be at least matriculate with
experience in handling bio-control agents/bio-pesticides.
14)
Quality of products is to be maintained as stipulated in registration of biopesticides.
15)
Periodical technical audit of bio-control laboratory will be done by Central/State
Governments to monitor the proper working of laboratory including maintenance of
equipment.
16)
Organization has to submit quarterly progress report in respect of production of biocontrol agents bio-pesticides to Dte. Of PPQ & S for monitoring the progress.
17)
Organization should be willing to provide trainings to farmers/NGOs/Women's
Organizations on mass production of bio-agents/bio-pesticides where desired.
18)
Organization has to maintain proper record in stock register of bio- agents/biopesticides produced and sold for auditing purpose.
19)
Applicants should have recommendation from District Agriculture / Horticulture
Officer / SAUs / Officer – in - charge of CIPMC / KVK / NABARD (List attached).
20)
Application in prescribed format should be routed through central Charge of CIPMC
will verify the case and send
same to Dte. of PPQ & S for acquiring
approval/sanction from Department of Agriculture & Cooperation.
21)
NGOs seeking grant-in-aid from Government will be required to submit an
application
which
organization/institution
includes
such
all
as
relevant
Article
of
information
Association,
regarding
Bye-laws,
the
Audited
Statements of Accounts, Sources, and pattern of Income and Expenditure etc.
149
22)
Organization applying for subsidy under grants-in-aid must fulfil above terms and
conditions.
23)
Government of India reserve the right to accept/reject any proposal submitted for
release of grants-in-aid without assigning any reason.
24)
15% of the total funds will be sanctioned to those NGOs which are headed by
women.
List of Authorized Recommending Officers
SI. No. Department/Institution Name of the Officer
1. Dte. of PPQ&S, NH-IV, Plant Protection Adviser to the Faridabad. Government of India;
Director/Joint Director/Deputy Director/Assistant Director of IPM Division.
2. State Department of Directors, Additional Directors, Joint
Agriculture/Horticulture Directors, District Agriculture/Horticulture Officers.
3. State Agricultural Universities, Director of Extension, Heads (SAUs), Departments of
Entomology/Plant Pathology.
4. Central Integrated Pest Officer-in-charges of CIPMCs of Management Centers respective
States. (CIPMCs)
5. National Bank for Agricultural Branch Manager/Development Officer, and Rural
Development (NABARD)
6. Krishi Vigyan Kendra Officer-in-charge
Proforma for grants-in-aid for setting up of bio-control laboratories by the NGOs etc.
under the Central Sector Plan Scheme "Strengthening and Modernization of Pest
Management Approach in India"
1. Name of the Organization
______________
2. Whether registered under Societies Registration Act, 1860 etc. ___________
3. Registration No. and date
___________
4. Names and addresses of the head of the organization and board of Directors and their
qualifications___________
5. Details of Grants-in-aid received by the organization during the last three years from this
Ministry or any other Ministry and the status of its utilization
6. Details of activities conducted by the organization
_______________
_______________
7. Details of projects relating to agriculture undertaken by organization ___________
8. Project cost of the instant proposal (enclose a detailed Project report also) ___________
9. Address of the site where the said Laboratory is to be established ___________
10. Whether copy of the registration certificate, article of association, bye-laws, audited
statement of accounts for the past three years, Source and pattern of income and
150
expenditure enclosed ___________
Requirement of equipment for establishment of bio-control laboratory
SI. No. Equipment/Store No.
Approx.cost Total
1. Heat convectors
2,000
20,000
30,000
1,20,000
2
20,000
40,000
4. Hot air oven
2
40,000
80,000
5. BOD incubators
2
80,000
1,60,000
6. Centrifuge
1
10,000
10,000
7. Laminar flow station
1
25,000
25,000
8. Autoclave vertical
1
20,000
20,000
9. Steel racks (7X3X18")
10
1,500
15,000
10. Corcyra cages (wooden)
100
500
50,000
11. Chysopa cages
10
500
5,000
12. Laboratory tables
5
7,000
35,000
13. Laboratory stools
20
300
6,000
14. Hygrometers (dial type)
10
500
5,000
15. Thermometer
10
400
4,000
16. Mixer-cum-grinder
2
2,000
4,000
17. Corcyra egg laying cages 10
200
2,000
18. U.V. chamber with UV
2
1,500
3,000 light
19. Exhaust fans
6
1,000
6,000
20. Vaccume cleaners
1
5,000
5,000
21. Water distillation unit
1
3,000
3,000
22. Research microscope
1
10,000
10,000
23. Stereo binocular
1
50,000
50,000
24. Glasswares
-
-
60,000
25. Photocopier
1
20,000
20,000
26. P.C. with accessories
2
50,000
1,00,000
10
Remarks per unit
(in amount (in Rs.) Rs.)
2. Air Conditioners with cooling
& heating arrangements 4
3. Refrigerators 300 Ltr
capacity
microscope
Total
9,48,000
Recommendation of the authorized officer
151
I,
______________
(name),
________________________
(designation)
of
_________________________ (name of authorized organization), hereby certify that I have
personally inspected the site and I am satisfied that the __________(the name of NGO) has
got adequate building for establishing a bio-control lab. I also certify that the NGO is
capable of undertaking the above project.
Signature & Seal of recommending authority
Lecture No. 17 (Unit IV)
152
Genetically Engineered Microbes and other Natural enemies (NEs)
Genetic Engineering
What is biotechnology?
The term ‘biotechnology’ refers to any technological application that uses biological
systems, living organisms, or derivatives thereof, to make or modify products or process for
a specific use.
Biotechnology, in the form of traditional fermentation techniques, has been used for
decades to make bread, cheese or beer. It has also been the basis of traditional animal and
plant breeding techniques, such as hybridization and the selection of plants and animals
with specific characteristics to create, for example, crops which produce higher yields of
grain.
Modern biotechnology, meanwhile, employs advanced techniques such as genetic
engineering or recombinant deoxyribonucleic acid (rDNA) technology whereby researches
can take a single gene from a plant or animal cell and insert it in another plant or animal
cell to give it a desired characteristic, such as a plant that is resistant to a specific pest or
disease.
What is a Genetically Modified
Organism (GMO) ?
In modern science, a Genetically Modified Organism (GMO) is that in which the
basic genetic material (DNA) has been artificially altered or modified to improve the
attributes or make it perform new functions.
Common GMOs include agricultural crops that have been genetically modified for
greater productivity or for resistance to pests or diseases e.g. Bt cotton, incorporating a
gene from a bacterium Bacillus thuriengiensis effective against the American Bollworm, a
major pest on cotton.
What is a Living modified Organism (LMO) ?
The term Living Modified Organism (LMO) is defined as any living organism that
possesses a novel combination of genetic material obtained through the use of modern
biotechnology.
In everyday usage, LMOs are usually considered to be the same as GMOs, but
definitions and interpretations of the term vary widely.
What is Genetic Engineering or recombinant DNA technology (rDNA technology)?
Genetic engineering or recombinant DNA (rDNA) technology involves artificial
transfer of genes or gene fragments from one organism to another to produce novel traits in
the recipient living organism. The important tools used in rDNA technology include :
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
153
a)
Enzymes for DNA manipulation : The first step in the construction of a
recombinant DNA molecule, involves cleaving DNA molecules at specific points and
recombining them together again in a controlled manner. The two main types of
enzymes commonly used for this purpose are restriction endonucleases and DNA
ligases. These enzymes form the backbone of rDNA technology. Restriction
endonucleases cut DNA into defined fragments by targeting junction of specific
sequences of the genetic coding and DNA ligases recombine them by consolidating
loose bonds for creating large fragments. These enzymes are very specific in their
action.
b)
Vectors : The function of the vector is to enable the foreign genes to get introduced
into and become established within the host cell. Naturally occurring DNA
molecules that satisfy the basic requirements for a vector are plasmids and the
genomes of bacteriophages and eukaryotic viruses. They are further classified as
cloning and expression vectors depending on the stage of genetic engineering at
which these vectors are used.
c)
Expression hosts : The functional cell into which the composite DNA molecule
carrying the required gene needs to be introduced is called the expression host.
The choice of the best host-vector system for the expression and large-scale
production of a particular protein is based on considerations of the complexity of
the protein to be expressed and the yield and quantities needed.
d)
Marker genes : Marker genes and reporter genes are utilized for selection and
identification of the clones. These use phenotypic markers, identification from a
gene library and DNA sequencing. DNA sequencing helps in determining the precise
order of nucleotides in a piece of DNA.
How are GMOs developed?
There are four steps in developing a GMO.
1)
Identification of a gene : The first step is to identify a particular characteristic
from any organism (plant, animal or microorganism) and find out which gene or
genes in the organism are responsible for producing that characteristic. This is
followed by the use of molecular biology techniques to isolate and copy the gene of
interest, Identifying and locating genes for the required traits is currently the most
limiting step in the development of GMOs particularly in the most limiting step in
the development of GMOs particularly in plants and animals. For example relatively
little is known about the specific genes required to enhance yield potential, improve
stress tolerance or modify chemical properties in plants Further, identifying a single
gene involved with a trait is not sufficient and it is important to understand how the
154
gene expression is regulated, what other effects it might have on the plant, and how
it interacts with other genes active in the same biochemical pathway.
2)
Designing Genes for Insertion : Once a gene has been isolated and cloned
(amplified in a bacterial vector), it must undergo several modifications before it can
be effectively inserted into a host. A simplified representation of a constructed
transgene, containing necessary components for successful integration
and
expression is given below along with the description of components :
Marker gene
Promoter
Transgene
Termination
sequence
Components of a constructed transgene for integration and expression.

A promoter sequence must be added for the gene to be correctly expressed (i.e.,
translated into a protein product). The promoter is the on / off switch that controls
when and where in the plant the gene will be expressed.

The termination sequence signals to the cellular machinery that the end of the gene
sequence has been reached.

A selectable marker gene is added to the gene “construct” in order to identify plant
cells or tissues that have successfully integrated the transgene. This is necessary
because achieving incorporation and expression of transgenes in cells is a rare
event, occurring in just a small portion of the targeted tissues or cells. Selectable
marker genes encode proteins that provide resistance to agents that are normally
toxic to plants, such as antibiotics. Only those plant cells that have integrated the
selectable marker gene will survive when grown on a medium containing the
appropriate antibiotic. As for other inserted genes, marker genes also require
promoter and termination sequences for proper function.
3)
Transformation : Transformation is the heritable change in a cell or organism
brought about by the uptake and establishment of introduced DNA. The procedure
for introduction of DNA into a host depends on the type of expression of host. A
brief heat shock, degradative enzymes, electroporation, microinjection of DNA. The
transfer of gene constructs is relatively straight forward in prokaryotic or single
eukaryotic cells, whereas the development of entire multicellular organisms such as
plants or animals is much more complex as these organisms need to carry foreign
genes in all their cells and pass these genes onto their offspring.
4)
Selection : Following the gene insertion process, selection and identification of the
transformed cell is done using marker / reporter genes. For example only the cell
expressing the selectable marker gene will survive in a selective media containing an
155
antibiotic or any other compound, depending on the type of marker used. It is
assumed that these surviving organisms possess the transgene of interest. These
organisms are replicated resulting in a pure culture of recombinant / genetically
modified organisms (GMOs). The methods of replication and regeneration vary
depending on whether it is a microorganism, plant or animal.
What is the status of GMOs?
Healthcare
Global status : The recombinant insulin was the first recombinant biopharmaceutical and
in many biotechnology companies traditional pharmaceutical companies and research
organizations have devoted their resources to discover and develop numerous recombinant
protein therapeutics or biopharmaceuticals.
The initial efforts were towards development of recombinant forms of natural
proteins and biologics derived from natural sources, subsequently several monoclonal
antibody (mAb) based therapeutics have also been developed. It has been reported that
about 60 recombinant products have been approved till 2003 and are being used as
therapeutics in various forms across the world. Notable target indications for approved
therapeutics include diabetes, haemophilia, hepatitis, myocardial infarction and various
cancers.
It may be noted that all recombinant products are considered new if there is a
change in the use of cell lines, major changes in the process techniques and methods of
transformation of hosts, based on which it has been reported that nearly 150 products are
approved for therapeutic use. In addition over 500 candidate biopharmaceuticals are
undergoing clinical evaluation.
Status in India
Regarding the status of approved recombinant therapeutics in India, 20 therapeutically
distinct products have been approved for marketing through imports / indigenous
manufacture.
List of recombinant therapeutics approved for marketing in India
S.No.
Molecules
Therapeutic Indications
1
Human insulin
Diabetes
2
Erythropoietin
Anaemia (Hb weakness)
3
Hepatitis
B
vaccine
(recombinant
Immunization
agaisnt
Hepatitis
B
surface antigen besed)
(Jaundice – Liver infection by Virus)
4
Human growth hormone
Deficiency of growth hormone in children
5
Interleukin 2
Renal cell carcinoma (kidney Cancer)
156
6
Interleukin 11
Thrombocytopenia (Platelets   blood
cells (-) clotting)
7
8
Granulocyte Colony Stimulating Factor
Chemotherapy
induced
neutropenia
(GCSF)
(WBC) due to cancer treatment
Granulocyte Macrophage
Chemotherapy induced neutropenia
Colony Stimulating Factor (GMCSF)
9
Interferon 2
Chronic myeloid leukemia (Blood cancer)
10
Interferon 2
Chronic myeloid leukemia,
Hepatitis B and Hepatitis C
11
Interferon
Chronic
tissue
granulomatous
gall)
and
disease
Severe
(dead
maligant
osteopetrosis (ab@deposits in bone)
12
Streptokinase A
Acute myocardial infarction (Heart attack)
13
Tissue Plasminogen Activator
Acute myocardial infarction
14
Blood factor VIII
Haemophilia type A ((-) blood clotting)
15
Follicle stimulating hormone
Reproductive disorders
16
Teriparatide (Forteo)
Osteoporosis (bone calcium deposits)
17
Drotrecogin (Xigris) alpha
Severe sepsis C
18
Platelet Derived Growth Factor (PDGF)
Bone marrow induction and osteoblasts
proliferation
(bone
forming
calls
multiplication in number)
19
Epidermal Growth Factor (EGF)
1) Mitogenesis and Organ 1) cell division
y mitosis
2) Morphogenesis 2) Organ development
embryonic
20
Eptacogalpha (r-F VIIa)
1)
Haemorrhages,
r-coagulation factor
acquired hemophilia
congenital
or
a
2) Bleeding – blood accumulation
There are more than 30 companies involved in marketing of recombinant therapeutics in
India. In addition, there is significant research and development activity in both private and
public institutions. Some of the products, which are under development, include
recombinant anthrax vaccine, recombinant HIV vaccine, recombinant cholera vaccine,
streptokinase etc.
157
Agriculture
Global Status : Since the introduction of first transgenic crop for commercial use in 1995
in USA i.e. the Flavr Savr tomatoes with delayed ripening, extensive research and
development efforts were initiated all over the world. The areas of crop improvement
currently being targeted using transgenic techniques include resistance to a variety of
pests, pathogens and weed control agents, improvement in nutritional content and
improved survival during environmental stress. Research is also carried out into production
of new and improved raw materials for a wide range of products including medicines.
Approval for commercial planting and use of transgenic crops follows many years of
research involving laboratory and field-testing, peer review and government regulatory
procedures. Twenty one crops have so far been approved in various countries for planting
in various countries across the world incorporating one or more of the basic phenotypic
characteristics such as fatty acid composition, fertility restoration, herbicide tolerance,
insect resistance, male sterility, modified color, mutations, reduced nicotine, delayed
ripening and virus resistance.
List of these products along with the genetically improved trait and countries where
they been approved is given in the table on next page.
Transgenic crops approved for commercial use
S.No.
Crop
Traits
Countries where approved
1
Alfalfa
Herbicide tolerance
USA, Canada, Japan
2
Agrentine
Herbicide
Canola
improved
tolerance
protection
and
against
Canada,
USA,
Japan,
Australia,
European Union, Korea
weeds
3
Carnation
Increased shelf life by delayed
Australia,
rigpening,
Colombia
modified
flower
European
Union,
colour and Herbicide tolerance
4
Chicory
Herbicide tolerance, improved
European Union, USA
protection against weeds and
higher Yields
5
Cotton
Improved
herbicide
6
insect
protection,
tolerance
and
Japan,
Australia,
USA,
China,
Colombia, Mexico, Sourth Africa,
improved protection Against
Argentina,
India,
Weeds
Philippines, Brazil
Flax,
Herbicide tolerance, antibiotic
Canada, USA
Linseed
resistance and improved weed
Indonesia,
protection
158
7
Green
Virus resistance
China
Herbicide tolerance, improved
Canada, Japan, USA, Argentina,
against
European
pepper
8
Maize
weed
resistance
Protection,
to
insects
and
restored fertility of seeds.
Union,
Philippines,
Russia,
South
Africa,
U.K.,
Korea,
China,
Uruguay,
Colombia,
Honduras
9
Melon
Delayed ripening
USA
10
Papaya
Virus Resistance
USA, Canada
11
Polish
Herbicide
Canola
improved weed control
Potato
Improved
12
tolerance
protection
and
from
Canada
USA, Canada, and Russia
insect and leaf roll virus.
13
Rice
Herbicide resistance
USA, Iran
14
Soybean
Improved weed control and
USA, Argentina, Japan, Canada,
herbicide tolerance, increased
Uruguay, Mexico, Brazil and South
cooking quality
Africa,
Czech
Republic,
Korea,
Russia, U.K., Paraguay, Romania
15
Squash
Resistance against watermelon
mosaic
virus
and
USA, Canada
zucchini
yellow mosaic virus
16
Sugar beet
Herbicide tolerance
USA, Canada
17
Sunflower
Herbicide tolerance
Canada
18
Tobacco
Herbicide tolerance
USA, European Union
19
Tomato
Improved
shelf
life,
taste,
USA, Japan, China
colour and texture, improved
insect
resistance,
virus
resistance
20
Petunia
Modified flower colour
China
21
Sweet
Virus resistance
China
Pepper
159
In the eleven year period since the commercial cultivation of transgenic crops
started, the global area under these crops increased by more than 60 fold, from 1.7 million
hectares in 1996 to 102.0 million hectares in 2006 grown by over 10 million farmers
(Figure 2.1). There was a historical landmark (in 2006) in that for the first time more than
100 million hectares of biotech crops were grown in any one year.
Figure 2.1 : Global Area of transgenic crops from 1996 to 2006 (million hectares)
In 2006, more than one third of the global biotech crop area, equivalent to 40.9
million hectares, was grown in developing countries. The increasing adoption of the biotech
crops by five principal developing countries i.e. India, China, Agrentina, Brazil and South
Africa is important in terms of the wordwide acceptance of biotech crops. India, had the
highest percentage year-on-year growth in 2006, with an increase of 192% in Bt cotton area
over 2005, following by South Africa (180%), Philippines (100%), Uruguay (33%), Brazil
(22%) Paraguay (11%), USA (10%), China (6%) and Argentina and Canada at 5%.
In total, there were 22 countries planting biotech crops out of which in 14 countries the
area under biotech crops was 50000 hectares or more. Such countries along with biotech
crops being cultivated and area under these crops have listed in the table on the next page.
Global Area of Biotech Crops in 2006 : by Country (Million Hectares)
S.No.
Country
1
USA
Area (million
hectares)
54.6
GM Crop
Soybean,
maize,
cotton,
canola,
squash,
papaya, alfalfa
2
Argentina
18.0
Soybean, mize, cotton
3
Brail
11.5
Soybean, Cotton
160
4
Canada
6.1
Canola, maize, soybean
5
India
3.8
Cotton
6
China
3.5
Cotton
7
Paraguay
2.0
Soybean
8
South Africa
1.4
Maize, soybean, cotton
9
Uruguay
0.4
Soybean, maize
10
Philippines
0.2
Maize
11
Australia
0.2
Cotton
12
Romania
0.1
Soybean
13
Mexico
0.1
Cotton, Soybean
14
Spain
0.1
Maize
15
Colombia
<0.1
Cotton
16
France
<0.1
Maize
17
Iran
<0.1
Rice
18
Honduras
<0.1
Maize
19
Czech Republic
<0.1
Maize
20
Portugal
<0.1
Maize
21
Germany
<0.1
Maize
22
Slovakia
<0.1
Maize
Source : International Service for the Acquisition of Agri-biotech Applications (http://www.isaaa.org) +
14 biotech mega-countries growing 50,000 hectares, or more, of biotech crops.
These countries comprised 11 developing countries and 11 developed countries.
Herbicide tolerance has been the dominant trait followed by insect resistance and stacked
genes for the two traits.
These is intensive research going on to develop GM crops with more direct benefits
to consumers. It has been reported in 2004 that 63 countries are in GM crop research and
development programs ranging from laboratory / greenhouse experiments, to field trials, to
regulatory approval and commercial production of 57 plants divided into four groups i.e.
field crops, vegetables, fruits and other plants have been identified for further research.
Status in India
In view of the importance and potential of transgenic crops, extensive efforts have
been initiated in India for development of transgenic crops. As of now, Bt cotton containing
the cry1Ac gene from Bacillus thuringiensis is the only transgenic crop approved for
commercial cultivation in India. The approval was first accorded to M/s Maharashtra
Hybrid Seeds Company Ltd. (MAHYCO) in 2002. Subsequently, several other compaines
have taken sub-licenses from MAHYCO and integrated cry1Ac gene into their hybrids.
161
Taking into consideration the need for introducing diversity in the gene as well as
germplasm as a tool to contain the development of insect resistance, hybrids containing
three new Bt cotton genes/events have been approved in 2006. These are :
1. cry 1Ac gene (event 1) by M/s J.K. Agril Seedcs Ltd.
2. Fusion genes (cry 1Ab+cry 1Ac) GFM by M/s Nath Seds
3. Stacked genes cry1Ac and cry1Ab by M/s Mahyco
As of now 62 hybrids of Bt cotton are approved for commercial cultivation into the
country as listed on the next page and the total area under Bt cotton has increased from
38,000 hectares in 2002 to 38 lakh hectares as depicted in the graph below.
Area under Bt cotton cultivation in India
By Cotton hybrids approved for commercial cultivation in India
Zone
Company
Hibrid
North
Ankur Seed Ltd.
Ankur 2534 Bt.
J.K. Agri Genetics Seeds Ltd.
JKCH 1947 Bt.
MAHYCO
MRC- 630 Bt, MRC 6029 Bt
Nath Seeds Ltd.
NCEH- 6 R
Nuziveedu Seeds Ltd.
NCS 138 Bt.
Rasi Seeds Ltd.
RCH – 134 Bt; RCH 308 Bt., RCH317 Bt
RCH 314 Bt
Central
North
and
Ajeet Seeds Ltd.
ACH-11-2 BT II
Ankur Seeds Ltd.
Ankur – 09
162
Ganga Kaveri Seeds Pvt. Ltd.
GK 205 Bt., GK 204 Bt.
J.K. Agri Genetics Seeds Ltd.
J.K. Varun Bt.
Krishidhan Seeds Pvt. Ltd.
KDCHH 9821 Bt., KDCHH-441
BGII
MAHYCO
MECH 12 Bt*, MRC 7301 BG II
MRC–7326 BG II, MRC–7347BG II
Nath Seeds Ltd.
NCEH-2R
Pravardhan Seeds Ltd.
PRCH – 102 Bt.
Rasi Seeds
RCH 377 Bt., RCH – 144 Bt, RCH
– 118 Bt. RCH – 138 Bt
Central
and
South
Vikki Agroctech Pvt. Ltd.
VCH – 111 Bt.
Ankur Seeds Ltd.
Ankur-651 Bt
MAHYCO
MRC-6301 Bt
Ajeet Seeds Ltd.
ACH-33-1 Bt., ACH-155-1
Emegrent Genetics
Brahma Bt.
Krishjidhan Seeds Pvt. Ltd
KDCHH 9810 Bt., KDCHH 9632
Bt.
North/Central
MAHYCO
MECH 162 Bt., MECH 184 Bt*
Nuziveedu Seeds Ltd.
NCS–207 Mallika,NCS-145 Bunny
Prabhat Seed Ltd
NPH 2171 Bt.
Rasi Seeds Ltd
RCH 2 Bt.
Tulasi Seeds Pvt. Ltd.
Tulasi 4 Bt., Tulasi 117 Bt.
Vikram Seeds Pvt. Ltd.
VICH 5 Bt., VICH 9 Bt.
Nuziveedu Seeds Ltd.
NCS-913 Bt.
Ganga Kaveri
GK – 209 Bt., GK -207 Bt
J.K. Agri Genetics Ltd.
J.K. Durga Bt. JKCH – 99 Bt.
MAHYCO
MRC – 6322 Bt., MRC – 6918 Bt,
South
South
MRC-7351 BG II, MRC 7201 BG II
Nath Seeds Ltd.
NCEH – 3R
Prabhat Seeds Ltd.
PCH – 2270 Bt.
Rasi Seeds Ltd.
RCH – 20 Bt, RCH – 368 Bt, RCH
111 BG I, RCH – 371 BGI, RCHB –
708 BGI
163
*Approval not renewed for Andhra Pradesh, North Zone: Haryana, Punjab and Haryana, Central Zone:
Gujarat Madhya Pradesh and Maharashtra, South Zone : Andhra Pradesh, Karnataka and Tamil Nadu.
Besides, Bt cotton, ten food crops were under contained limited field trials in India
in 2006. The trials are being conducted by both public and private sector institutions and
are mainly for insect resistance using Cry genes.
Transgenic crops approved for conducting contained limited fielf trials (including multilocation field trials)
S.No.
Crop
Organization
Transgene
1.
Brinjal
IARI, New Delhi
cry1Aa and cry1Aabc
MAHYCO, Mumbai
cry1Ac
Sungro Seeds Research Ltd., Delhi
cry1Ac
2.
Cabbage
Nunhems India Pvt. Ltd., Gurgaon
cry1Ba and cry1Ca
3.
Castor
Directorate of Oilseeds Research
cry1Ac
(DOR), Hyderabad
cry and cry1Ec
Sungro Seeds Research Ltd., Delhi
cry1Ac
Nunhems India Pvt. Ltd., Gurgaon
cry1Ba and cry/Ca
4.
Cauliflower
5.
Cron
Monsanto India Ltd., Mumbai
cry1Ab gene (Mon 810 event)
6.
Groundnut
ICRISAT, Hyderabad
chitinease
gene
from
rice
(Rchit)
7.
Okra
MAHYCO, Mumbai
cry1Ac, cry2Ab
8.
Potato
Central Potato Research Institute
RB
(CPRI), Shimla
Solanum bulbocastanum
MAHYCO, Mumbai
cry1Ac and cry2Ab
Tamil Nadu Agricultural University
rice chitinase (chi 11) or
(TNAU) IARI, Pusa New Delhi
tobacco osmotin gene
9.
Rice
gene
derived
from
cry1B-cry1Aa fusion gene
10.
Tomato
IARI, Pusa New Delhi
antisense replicase gene of
MAHYCO, Mumbai
tomato leaf curl virus cry2Ab
Source : Department of Biotechnology, Government of India
What are the safety concerns related to GMOs?
The safety concerns associated with use of GMOs and products thereof broadly fall
under three categories: risk to human health, environmental concerns and social and
ethical grounds.
What are the risks of GMOs to human health?
Risks to human health are related mainly to toxicity, allergenicity and antibiotic
resistance of the new organisms / products.
164
The risk of toxicity may be directly related to the nature of the product whose
synthesis is controlled by the transgene or the changes in the metabolism and the
composition of the organisms resulting from gene transfer. Most of the toxicity risks can be
assessed using scientific methods both qualitatively and quantitatively.
The introduction of newer proteins in transgenic crops from the organisms which
have not been consumed as foods, sometimes has the risk of these proteins becoming
allergens. However, it may be noted that there is no evidence that transgenic crops pose
more risks than conventional products in triggering allergies. Further, the new transgenic
crops can be tested for allergens prior to their commercial release. For example, when it
was found that the consumption of transgenic soybean with a methionine producing gene
from brazilnut could trigger an allergic response in those allergic to brazilnut, the product
was not released for sale.
The use of genes for antibiotic resistance as selectable markers have also raised
concerns regarding the transfer of such genes to microorganisms and thereby aggravate the
health problems due to antibiotic resistance in the disease causing organisms. Although,
the probability of such transfer is extremely rare, steps are being taken to reduce this risk
by phasing out their use.
What are the risks of GMOs to environment?
Risks to environment due to release of GMOs include impact of imparted traits on
other related species, the potential build up of resistance in insect populations, effect on
biodiversity and unintended effects on non-targeted organisms.
As occurs between conventional plant varieties, cross breeding between transgenic
and non-transgenic varieties of the same or closely related species can happen. This may
lead to the establishment of the novel traint in conventional or land race varieties or
transfer of the trait into weedy relatives. The environmental consequences associated with
such gene transfer depends on the trait and whether it will confer a selective advantage in
the recipient population. However, these risks can be anticipated easily and then evaluated
by experiments prior to any commercial release. For example in case of Bt crops, it was
suspected that insecticidal proteins can persist in the environments but experiments have
proved that they were degraded in the soil.
Bacillus thruringiensis
Bacillus thruringiensis var. kurstaki, which kills the caterpillar stage of wide array
of butterflies and moths. Advances in biotechnology improved the prospects for placing Bt
toxins within crop plants in a variety of ways. Genes responsible for production of Bt toxins
is incorporated into soil dwelling bacteria. When these altered bacteria grow and multiply
with in the plant, the Bt toxin is expressed with in the plant. So current plans are in
165
progress to develop transgenics in other words genetically modified crops that show
resistance to different crop pests.
Bt srains are highly specific in their activity like for examples
Bt Var. kurstaki – caterpillars
Bt Var. aizwai – wax moth larvae
Bt Var. israelensis – mosquito, black fly and fungus gnat larvae
Bt Var. tenebrionis-beetles (Colorado beetle larvae and elm leaf beetle larvae)
Table of Crystal proteins
Crystal proteins
Orders Effective
1. Cry-I
Lepidoptera
2. Cry-II
Lepidoptera and Diptera
3. Cry-III
Coleoptera
4. Cry-IV
Diptera
5. Cry-V
Lepidoptera and Coleoptera

First commercial product of Bt is Sporeine form France in 1938

Kurstaki isolated highly potent strain in France (1962)

First field release of Bt cotton was in 1996

In India, the Bt cotton was released for commercial cultivation in 2002

The estimated area of global area of genetically modified crops is 67.7 M. ha (2003

World leaders in overall biotech crops are Brazil, S. Africa, USA, Argentina, Canada
and China

Among Asian countries, Phillipines ranks first to grow 20,000 ha of maize

The GM crop which is widely grown is soybean for herbicide tolerance against
Glyphosate (Roundup)

The maize transgenic crop is cultivated for the character of insect tolerance against
European corn borer, Ostrinia nubilalis which is a major pest in USA and other
European countries but this pest is not found in India

The cotton transgenic crop is cultivated for the character of insect tolerance against
American bollworm, Helicoverpa armigera which is a major pest in cotton growing
areas

Bt gene, Cry1Ac is inserted into cotton crop which protects cotton crop from
American bollworm attack

Agrobacterium was considered as the vector for plant DNA transfer which was
successful for cotton gene transformation
166
Table 1 : Widely grown transgenics crop in world
Crop
Area (Million hectares)
Percent of global GM area
Rank of crop
Soybean
41.4
61
1
Maize
15.5
23
2
Cotton
7.2
11
3
Canola
3.6
5
4
Potato
NA
~1
5
Source : Economic times, 14th January, 2004
Table 2 : Transgenics produced for different trait
Character
Percent of transgenics so for produced for
this character
Herbicide tolerance
71
Insect tolerance
28
Quality traits
1
Status of genetically modified plants in India

The first experiment on transgenic plants in the field was started in 1995 when
Brassica juncea plants containing Barnase, Barstar and Bar genes were planted at
Gurgaon in Haryana

Mahyco introduced Monsanto’s Bollgard® Bt gene into the Indian cotton hybrids by
backcrossing with a transgenic line. This Bt cotton confers protection against Indian
cotton bolloworm, Helicoverpa armigera.

The area under Bt cotton in India is 1,00000 ha in 2003 (Economic times, 15 th
January, 2004)

The “Refuge” strategy is followed to avoid resistance development in Helicoverpa
armigera where 20% of the area cultivated should be under non Bt cotton

Mahyco in collaboration with Monsanto (USA) released Bt cotton varieties as MECH
(Mahyco Early Composite Hybrid) series for different regions of India like MECH-12,
MECH-162, and MECH-184 for south and central India whereas MECH-912 for
north and central India.

Generic Engineering Approval Committee (GEAC) is responsible for monitoring of
genetically modified crops cultivated in India which comes under Ministry of
Environment and Forests
167
Development of Transgenic plants against various insect pests
Crop
Gene
Target pest
Rice
cry 1Ab
Chilo supressalis
Rice
cry 1Ac
Yellow stem borer :
Scirpophaga incertulas
Potato
cry 13a
Colorado potato beetle : Leptinotarsa decemilineata
Tomato
cry 1Ac
Fruit borer : Helicoverpa armigera
Brinjal
cry 1Ab
Shoot and fruit borer :
Leucinodes orbonalis
IMPORTANCE OF BIO-TECHNOLOGY IN BIO-CONTROL
Insect – pests have been the major scourge of the agriculture. Approximately 14 per
cent of crop productivity is lost due to insect-pests on a global scale. Application of
pesticides had resulted into the management of Insect-pests but ultimately ended with the
adverse effects on human health and environment. To ward off these ill effects, effective
alternatives are available in the form of ecofriendly strategies such as bioagents.
The field of bio-technology has experienced a sea change in the last one decade. A
number of sensitive techniques have emerged to detect the slightest variation in genomic
DNA. PCR and automated sequencing facilities have opened up newer opportunities for
taxonomist to clearly define intra-specific, interspecific and genus specific boundries.
Entomologists have realized the potential of these techniques recently. Molecular markers
are being used to define the speciation in the insect population. Molecular marker which is
based on actual primary nucleotide sequence, the resolution demarketing the individual
species becomes finer. Greater level of polymorphism will be detected using DNA based
techniques. Protein based technique depends upon manifestation of changes at nucleotide
level in the expression of protein, the DNA based techniques help in detecting the changes
at the nucleotide itself There are many molecular markers available for use in a wide range
of entomological studies. Judicious application of them would definitely help in correctly
identifying the bio-control agents. Success of any biocontrol programme with parasitoids
relies on correct identification of them in the habitat where it has been released. Moleculer
markers anlaysis not only help in correctly identifying them but also may help in studying
their evolution.
A fundamental requirement of agricultural research programme is the ' accurate
identification and systematic understanding of the organisms that are causing immediate
damage to crop plant/products either in the field, in storage and their natural enemies.
168
Due to minute size and relatively uniform morphology of Trichogramma, precise
identification of the spp./strain is difficult.
Arbitrarory primers have been extensively use,d in the taxonomy of aphids, moths,
and Hymenoptera parasitoids. The method is relatively quick and reveals great genetic
variability.
Management of insect-pests and weeds may be attained effectively by integrating
traditional and modern bio-technology. Few technologies like monoclonal antibiodies, bioprocessing and recombinant DNA, genetic improvement of natural enemies, behavioural
modification by synthetic pheromones, sterile male release and autocidal genetic control
have paramount importance in biological control programmes.
Monoclonal antibodies
Monoclonal antibodies can be used for the identification of biota difficult to be
identified by other simpler traditional means (Campbell, 1984) various disease causing
microbes are being detected by this technique (Mayer and Walker, 1987). Monoclonal
antibodies are also being used to detect the presence/absence of microbial or viral
insecticides while other methods require microscopic analysis and some knowledge of
taxonomic characters. Morphological similarity is more in the pre-adult stages of related
insects e.g. Heliothis armigera and Heliothis punctigera (Daly and Gregg. 1985). Accurate
and timely identification is pre-requisite for adoption of management practices as H.
armigera is potentially resistant to a number of pesticides while H. punctigera is relatively
succeptible one. Under such situations monoclonal 'antibodies’ diagnostic kits are useful.
Biotechnology in entomopathogenic nematode/bacteria complex
The nematodes contain specific symbiotic bacteria which enter into the insect-blood
and cause septicaemia which is responsible for death of insects with in a day or two (
Pionar and Thomas, 1966). The nematodes are highly specific to insects, have no harmful
effects on vertebrates or plants (Obendorf et al., 1983). The efficacy of such association is
less, so genetic engineering can play an important role in enhancing the efficacy by genetic
engineering
of
bacteria
mutually
associated
with
entomopathogenic
nematodes.
Engineering to stablize these bacteria in the preferred form would greatly facilitate the
commercial exploration of nematode/bacteria complex.
Bio-technology in entomopathogenic bacteria
Bacillus thuringiensis is one of the most important bacteria exploited commercially
against lepidopterans, and is produced in liquid fermenters. The drawback of the B. t. is
that these are inactivated by ultra-violet light in the field in one to five days. Due to this
problem, repetitive release is required to have the desired level of control which ultimately
enhances the management cost. Production by liquid fermentation technique and repetitive
application has restricted the use of B. t. on large scale. Bacteria like Pseudomonas spp.
169
have desirable properties in "respect of mass production and environmental stability
(Obukowiez et al., 1986). Under such situations bio-technology can play a role. Genes
responsible for toxicity may be transferred to Pseudomonas spp. to achieve the desired
control of pests as well as wide spectrum application.
Biotechnology in fungal pesticides
The major drawbacks in the use of myco-insecticides in,field are the requirement of
very moist conditions for persistence and infectivity and slow rate of kill. To overcome the
problem of slow rate of kill, some improvements may be made by judicious selection among
naturally occurring variants and their incoporation into commercial strains by protoplast
fusion.
Genetic engineering of insect viruses
Hundreds of viral isolates have been obtained from different insect orders.
Baculoviruses are generally pathogenic to small and medium sized larvae and relatively
harmless to adults. It may persist, as long as several years if protected frpm UV or extreme
heat or a few hours if not (Evans and Harrap, 1982). Baculoviruses have narrow host
range, time of infection may be two to three weeks. Due to this reason a few numbers have
been exploited for large scale pest control. Viruses in entomopoxviruses group has not been
tried as insecticides. Although these viruses are host specific and environmentally stable
but are insufficiently pathogenic to give economic control (Arif, 1984; Mitchell and Smith,
1981). This is the problem that genetic engineering could solve. By the application of this
technology, more pathogenic EPVs can be developed.
Genetic engineering of insect-pests
Genetic procedures may utilize the deleterious and marker genes on chromosomes
to have a genetic killing system (Whitten, 1985). Recombinant DNA and genetic engineering
technology may offer autocidal system of genetic control.
Genetic engineering of natural enemies and other beneficials
Here the aim is to augment the fitness of natural enemies and other beneficial
insects so that these may be effectively used to manage the insect-pests. Cloned genes are
available. Genes encoding carboxylesterases that can detoxify chemical insecticides are now
being cloned from selected pests (Field et al., 1988) and can be transferred into succeptible
beneficials. It. may also be possible to engineer resistance to some microbial, viral diseases
into beneficial insects.
Genetic improvement of natural enemies
Bio-control agents are normally more sensitive to pesticides than the target pest.
Laboratory selection can eliminate factors that delay or prevent development of resistance
of natural enemies. For example, green lacewing, Chrysoperla carnea were selected in the
170
laboratory for resistance to carbaryl. Thus, the mortality of larvae decreased from 98 per
cent to between 10 and 20 perent (Carwell et al., 1986 ).
Predatory mite, Metaseivlus accidentallis, was
methomyl,
selected
against abamectin
dimethoate and carbaryl which resulted in the increased rate of survival of
predatory mite (Roush and Hov, 19Si; Hoy and Ouyang, 1989).
No doubt bio-iechnology field has revolutionised (he field of science in every respect
but the generation of genetically modified oreanism has increased concerns that they may
pose risks'of any sort that might require particular steps to ameliorate those risks.
171
Lecture No. 18 (Unit IV)
Genetics of Ideal Treaits in BC agent
Genetic improvement of Baculovirus
Baculoviruses have been studied extensively for their potential as microbial
insecticides. Through genetic improvement programmes, their stability, field persistence.,
and efficacy can be improved greatly. Viruses with desirable attributes may be selected by
searching in nature for new virus strains or by genetic manipulation of existing isolates.
Variants of baculoviruses with heritable variations and virulence arise spontaneously in
nature. Variants may also arise when virus infects an alternate host. Mutants and in vivo
recombinarits with enhanced virulence, desirable host range, UV resistance, and increased
environmenrai persistence may be selected and further improved upon.
Introduction : Enlomopalhngenic viruses, particularly the baculoviruses, have great
potential in the microbial control of agricultural pests (Jayaraj and Rabindra, 1989). However, their relatively slow action, specificity to a single insect pest, high level of UV
inactivation as well as inactivation by glandular secretion of leaves, and poor storage
capability limit their efficacy and large-scale commercial production and use. It is possible
now to overcome these drawbacks by genetic improvement of these baculoviruses. Because
of their small genomes and comparatively simple molecular biology, insect viruses are
prime targets for genetic improvement.
A virus with desirable attributes may be developed by two approaches: by searching
in nature for new virus strains or by genetic manipulation of existing isolates. A virus may
be genetically improved by selecting for desired traits or by using the techniques of genetic
engineering.
Selection of Desirable Strains
Viruses from members of the same host species from different geographical
locations show variability in virulence abid biological characteristics and their DNA differs
in restriction endonuc'lease and SDS-PAGE protein patterns (Brown, 1982). Variant of
bacuioviruses with heritable variations in virulence and host range arise spontaneously in
nature. Field isolates collected in diverse geographic locations are often genetically
heterogenous. Hukuhara "(1968) observed that a tetragonal strain, of nuclear polyhedrosis
virus was more virulent than a hexahedron strain to Hyprantna cunea and in addition
multiplied more rapidly. Cameron (1962) suggested that an automatic selection of virus
strains in, an insect in the-field might occur. accounting for great variations in mortality.
Recent studies have shown the 'Variation of NPV Heliothis armigera obtained from different
agroclimaltic regions had variation in their Virulence. An isolate obtained from tiie Nilgiris
recorded the lower LCs:
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
172
Smirof (1961) selected a strain of NPV of increased virulence to Neodiprion spp. from
the first larvae to die after inoculation. The virulence of the NPV of gyps;y moth, Lymantria
dispar, increased five times above that of the original strain aerial passages through a new
host (Fedorintchik, 1964). Variants of baculoviruses may also arise when a virus infects an
alternate host. An isolate of Choristoneura fimiferana NPV was fed to neonate larvae of the
cabbage looper and the wax. moth Choristoneura fumiferaa. After passage, the virus was
able to infect new hosts (Stairs et al., 1981). Studies arc in progress in the Tamil Nadu
Agricultural University, Coinbatore, to identify baculoviruses from different species of
insects that would infect H. armigcra and Spodoptera litura and also insect pests that would
be susceptible to the NPVs of H. armigera'and S. litura. After passage through the alienate
hosts, the viruses can be studied for enhanced virulence to the original host.
Seiection for UV Resistance
A strain of Cydia pomonella GV which was 5.6 times more resistant to UV light than
the original isolate and remained infective for twice as long in the field was selected by
Brassel and Benz (1979). Such investigations in our agroecosystem are bound to yield
fruitful results as there is a greater biodiversity under tropical conditions.
Mutants of Baculoviruses
A direct application of genetics to viral insecticides is to generate stable variants of
baculoviruses which arc more effective in the field. Wood et al, (1981) could isolate a
mutant strain of AcMNPV designated HOB which produced a'large number of occlusion
bodies in infected cells and which had higher virulence in insects than the parent strain.
Mutants can be generated in tissue culture by growing the wild type virus in the presence
of a chemical mutagen. The spruce budworm virus was grown in the presence of the
mutagen nitrosomethyl guanidine and the surviving virus was cloned (Ireland, 1982). Of
the 34 plaque isolated and examined, one isolate. cfNTG 29, was more virulent than the
standard.
Recombinations
In vivo recombination has been carried out by mixedly infecting Galleria mellonella
larvae with two closely related baculoviruses (AcMNPV and GmMNPV). REV analyses of
pooled virus DNA extracted from occlusion bodies harvested after five in vivo passages in
the wax moth showed that rccombinants were present {Croizicrand and Quiot, 1981).
Gcnetic recombination between viruses wilh similar but not identical REN patterns occurs
in vitro and gene reassortment has been demonstrated (Summers et a!., 1980).
Recombinants with desirable attributes like enhanced virulence, desirable host range, UV
resistance, and increased environmental persistence may be selected and further improved
upon.
173
THE ROLE OF GENETIC ENGINEERING AND TISSUE CULTURE FOR
MICROBIAL CONTROL OF INSECT PEST MANAGEMENT IN INDIA
Introduction
Most increases in crop production have been achieved by scientists whose goals
have been directly aimed at increasing plant yield. Within the past several decades,
however, new knowledge and techniques have been developed in the science of plant
protection, especially in microbial control of insect pests, that hold promise for alternative
methods of increasing crop production through crop protection. Although the pathogens or
microbial products currently used to control insects offer certain advantages over chemical
insecticides, they also have certain disadvantages that have limited their applications. The
advent of invertebrate cell c ulture and recombinant DNA technology offers the possibility of
developing entirely new biological insecticides based on the construction of genetically
modified microorganisms that retain the advantages of the classical control agents but
suffer fewer of their drawbacks.
Tissue Culture
At present, several baculoviruses are produced commercially by the insectary
method. The advantages of producing baeuloviruses in vitro rather than in vivo are the
following-. Cell cultures can be monitored to assure that they are free from undesirable
viral or microbial contamination. It is comparatively easy to maintain insect cell lines
continuously and virus replication in such a lines can be monitored. Baculoviruses
obtained from in virtro cultures are free from insect debris and contain low amounts of
insect proteins that can be removed easily. Insect cells can be cloned and stored and
provide stable and uniform production of polyhedral. The existing large-scale equipment for
vetertnary and human virus vaccine production can be used with slight modification to
produce baculoviruses in vitro. Therefore, the application of invertebrate techniques are of
interest even though insects can be grown easily in rather primitive conditions on natural
or artificial diets with little or no sterility precautions ( Maramorosch 1987 kompier et al.
1988 )
In the case of the insect virus, development and use of insect tissue culture for
studying certain promising baculoviruses of natural pests like Heliothis armigera and
Spodoptera liiura is a must for their cheap and large-scale production free from other
pathogen contamination as well as to satisfy the recent registration protocol (Narayanan,
1991 d). The need for vitro rather than in vivo multiplication of insect viruses reported for
dormant pests like hairy caterpillar of groundnut, Amsacta albistriga, legume pod borer,
Adisura atkinsoni, and the quarantine pest, the coding moth of apple, Cydia pomonella, is a
must for the reason that A. albistriga and A. atkinsoni are seasonal pests and rearing them
174
throughout the year is a problem due to their pupal diapause, as also of the codling moth
of apple, Cydia pomonella (Narayanan, 1980 ).
Since viruses can be cultivated in living cells, cell culture has proved beneficial for
in-depth studies. The general users of cell cultures include.: (1) isolation of virus from
suspected specimens, (2) Identification of virus, (3) study of cytopathic effect of virus, and
(4) study of the stage of multiplications of virus.
The life cycle of certain parasitic hymenopteran insects requires as an integral
component the participation of polydna viruses which replicate in the ovary nf the female
wasps. Some physiological and immunological effects on parasitised larva have been
attributed to the presence of polydna virus. For example, inliibition of phenoloxidase
activity has been shown in parasitised larvae of Trichoplusia ni. Similarly, it has been
reported that the virus component of the calyx fluid of Compoletis sonorensis is capable of
inhibiting encapsulation of parasite eggs by the host Heliothis virescens (Stoltz, 1986).
Insect tissue culture for mass multiplication and use of polydna viruses of certain females
of ichnumonid and braconid parasitic insects to overcome the abrogation of their defense
mechanism and thereby increase the efficiency of biological control crop pests goes a long
way towards increasing the efficiency of biological control agents.
For proper identification of viruses isolated froml the lab -lab pod borer, Adisura
atkinsoni, and Heliothis armigera, a serological technique like macrophage
migration
inhibition (Narayanan, unpublished) has become useful In view of their cross-infectivity
(Narayanan, 1985) as well as similarity in their polyhedral sizes, shapes, and virion
occlusion patterns, the above two species of nuclear polyhedrosis viruses are virtually
indistinguishable.
For detection and diagnosis of new variants and strains of baculovirus as well as
testing for safety to non-target organisms in the environment, the development and use of
the hybridoma – technique in insect cell culture by way of producing monoclonal antibodies
has been suggested because it has been proven to be easy. Sensitive, and rapid in use in
the medical and veterinary fields (Marmorosch 1987 ).
Baculovirus as a Vector for Genetic Engineering
The need for proper molecular characterisation of virus through genome analysis (
Narayanan. 1991 a, 1991 b. Narayanan and Gopalakrishnan, 1990) using various bacterial
restriction enzymes (REN) especially in certain cross-infective studies of NPV of A atkinsoni
(Narayanan, 1985) and NPV of certain plusine larvae ( Rabindraand Subramaniam, 1976 )
has become most important. Isolation of highly virulent and broad-spectrum baculoviruses
from insects with extreme habitats and use of recombinant DNA technology for the
development of desired and needed novel viral pesticidal agents in the only way to enhance
175
the efficacy of various microbial agents including insect viruses as suggested by
Kirschbaum (1985 ).
The objective of genetic engineering of baculoviruses is to improve their speed of
action. This is desirable since, during the normal infection process of a permissive host, a
baculovirus undergoes several cycles of replication. These cycles take time — several days
or weeks, depending on the virus, the host, and the environmental conditions ( such as
temperature ). By contrast, most chemical insecticides act quickly, killing the target insect,
and often other, beneficial insects, in a matter of hours. By means of genetic engineering
procedures it will be possible to minimize the time taken by a viral insecticide to exert an
effect on a target species by incorporating into the viral genome genes for other, quickly
acting products such as insect specific neurotoxins ( Carbonel et al. 1988.)
Excellent prokaryotic host-vector systems are currently available for cloning and
propagating portions of eukaryotic DNA. In addition, some eukaryotic proteins require
modification (for example, glycosylation) for natural biological activity and appropriate
modification of eukaryotic proteins is unlikely in a prokaryotic cell environment. Thus
genetic engineering may find it advantageous to use baculoviruses as a eukaryotic hostvector system (Miller, 1988). The success of baculovirus expression systems owes much to
the fact that baculoviruses are helper – independent viruses, easy to work with, safe to
handle, and non-pathogenic to vertebrates or plants. These viruses have a large (100-125
kd), circular, double-stranded DNA genome with an extendable rod-shaped capsid.
Furthermore, the polyhedrin, the major protein gene, is non-essential for viral replication
and it is a late gene with a strong promoter (Miller,1989 Maeda. 1989)
Applications of baculoviruses as efficierit expression vectors so far have been aimed
at producing foreign proteins as abundantly as possible in the medical and veterinary
fields. Foreign gene expression in baculovirus is also of interest in the development of a
more effective baculovirus. Merryweather et al. (1990) have expressed Bacillus thuringiensis
(B.t.) toxin in a baculovirus vector. A primising technique in agricultural plant protection is
the use early polyhedrin promoter of baculovirus as vector, or the use of multiple cloning
site vectors in order to derive the expression of a foreign gene, the Insect-specific
neurotoxin. B.t. toxin so that the target pest can be killed quickly, while retaining the
polyhedra production which is essential for horizontal transmission of virus within the field
population.
A future approach will be to synthesise biologically active and authenticated insect
pheromones and other behaviour-modifying proteins (O' Reilly and Miller, 1989) which are
used in insect pest management practices, by way of expressing the genes responsible for
pheromones in insect cells through baculovirus vector instead of depending on in vitro
production technique, which is cost-prohibitive and time consuming.
176
Genetically Engineered Suicidal Viruses
Baculoviruses are fragile creatures, but nature has given them the ability to make a
tough protein coat called polyhedrin that protects them in the environment. Without this
coat, these viruses cannot survive for long. To arrest the multiplication o'f these viruses in
the environment, by genetic engineering, one can shut off the geneticsystem that makes the
protective coat, thereby crippling the viruses. Unable to make polyhedrin, these viruses will
die within hours after infecting and killing the insect host. For environmental monitoring of
the persistence and spread of pathogens, release of such ‘suicidal viruses’ i.e., genetically
engineered recombinant baculovirus containing an oligonucleotide marker, paves the way
for future microbial control of virus in an environmentally safe manner (Anonymous, 1990).
Bacteria
Bacillus thuringiensis and a number of its subspecies are important in agriculture
because they kill leaf-eating caterpillars. Similarly, B. popilliae is important for the reason
that it effectively checks some of the soilborne coleopteran insect pests. A number of
positive attributes of some species of entomopathogenic bacteria are used as a model
system in recombinant DNA manipulations and merit emphasis. First, many of these
bacteria, especially B. popilliae and varieties of B. thuringeiensis, are soil bacilli that are
non-pathogenic to non-target organisms, but are primarily species or genus-specific insect
pathogens, and several have been tested for safety to vertebrate and invertebrate non-target
organisms. Secondly, they are agriculturally important as biological control agents with
potential to replace a number of hazardous chemical insecticides (Narayanan. 1991 ).
Though the use of B. thuringiensis for the control or various agricultural pests
hasten encouraging, continued work on it has become static and its field testing is highly
restricted. Where sericulture is being practised it is highly toxic to silkworms, causing
general paralysis. Because of the fear that B. thuringiensis applied in the field migh spread
and trigger epizootics in silkworms, the use of B.t. is restricted, even though the silkworm
is highly confined and domesticated and mulberry berry plantations are devoid of most of
the biting and chewing lepidopteran pests.
A spore-negative and crystal-positive mutant of the pathogen developed through
various chemical mutagens (Narayanan, 1987) and use of recombinant DNA techniques to
insert B. thuringiensis endotoxin gene into other prokaryotes and eukaryotes including
plants to have transgenic plants with microbial toxin B.t. so that they can protect
themselves from the ravages of pest attack offer the exciting Possibility of developing a
pcsticidal plant ( Vaeck et al., 1987 ).
Nematodes
Steinernematid and heterorhabditid ncmatodes mutually associated with the
bacteria, Xenorhabdus spp., are highly effective insect pathogens. This bacterium is the kcy
177
to the culture of steinernematid and heterorhabditid nematodes. Phase variation is a
common nature of Xenorhabdus spp. The primary phase, which is the phase naturally
occurring in the infective stage nematodc, provides better growth conditions for the
nematodes than the secondary. Although either phase may be carried within the infective
stage nematode, infectives formed in the presence of both phases contain only the primary
(Akhurst, 1986). The primary phase symbiont is of major importance in the nematodebacteria association. Either phase can convert a variety of media into suitable nutrient for
the nematodes so that they can be mass produced in vitro. However, the primary phase is
required for the maximisation of mass production that is necessary for commercial scale.
only the primary form is usually isolated from infective stage juveniles, but this form tends
to be unstable and production on the secondary form is greatly reduced (Woodring and
Kaya, 1988). In the case of entomophilic nematodes, the need to identify the gene
responsible for retention ot primary form instead of changing to secondary form in the case
to mutalistic bacterial pathogens like Xenorhabdus nematophilus and X. luminescense
associated, with certain steinernematids and Rhabditis in connection with their cheap and
large-scale multiplication and use, has been stressed.
In conclusion it may be indicated that most of the genetic engineering work has
been carried out with one baculovirus, Autographa californica, on only one cultured insect
cell line derived from the ovarian tissue of Spodoptera frugiperda. Since import of
entomopathogenic organisms is restricted in India, there is a tremendous scope in future
for studying the level of expression and the protein, processing, targeting and transport of
recombinant proteins / toxins as a whole in cell lines derived from other tissues and other
susceptible host insect available in India.
GENETIC ENGINEERING OF BACULOVIRUS FOR PEST CONTROL
The idea of biological control of phytophagous insect pests gained momentum due to
aggravating problems associated with chemical pesticides. Biological control can be
achieved by the use of naturally occurring parasites, predators, microbial pathogens
and/or pest resistant varieties of host plants, Viral pathogens of lepidopterans, namely
nuclear polyhedrosis viruses (NPV) are considered one of the promising biological control
agents (Cunningham, 1982). Nuclear polyhedrosis viruses of the group Baculoviridae
exclusively infect arthropods. The baculoviruses are mostly specific to their host and bring
out an effective control of the pests and are termed 'viral insecticides' and 'living
insecticides'. Reviewing the literature.Evans (1986) reported that viral insecticides are
realistic alternative' to chemical pesticides in crop protection. A number of successful viral
products have been registered as viral pesticides. The following may be cited as examples:
Elcar (Heliothis NPV. Ignoffo and Couch, 1981); Gypchek (Lymantria dispar NPV; Shapiro,
178
1982), TM Biocontrol (Orgyia pseudosugata NPV; Martignoni, 1978), Virox (Neodiprion
sertifer NPV; Cunnmgham and Entwistle, 1981), and Lecontvirus (Neodiprin iecontei NPV;
Cunningham and Entwistle, 1981). Payne (1988), while listing the baculbvirus candidates
for insect pest control, suggested about one dozen NPVs infecting the target pest (mostly
lepidopterans). Present formulations of viral pesticides are preparations of wild type NPVs
isolated and amplified from infected larvae.
Accepting NPV as a biological control agent, laboratories in the world are involved in
manipulating the genome to enhance the efficacy of the virus (Bishop et al. , 1988). This
brief review intends to bring out the existing important information on the use of gentic
engineering to increase the efficiency of NPV for biocontrol.
Genetic Engineerhig of NPV Genome
The genome of baculovirus is composed of double stranded DNA which exists as a
covalently closed genome whose size ranges from from 128 – 135 Kbp. It has been
calculated that baculvirus genome contains approximately 80 genes of which a maximum
of about 30 are probably involved in the synthesis of structure of virus inclusion bodies
(Kelly. 1982). The remaining genes are involved in the production of polyhedral bodies.
However, these genes are not essential for infection and replication of the virus in the host
system (Smith el al., 1983). Furthermore, they are normally expressed during the late
phase of development. Hence, they are termed (non essential late gene). By genetic
engineering techniques, it has been shown that non-essential genes can be removed and
foreign gene can be cloned under the control of non-essential gene promoter (Lucknow and
Summers, 1988; Miller, 1988; Maeda. 1939a). The foreign gene can be introduced into the
genome of wild type NPV using a transfer vector, which serves as a vehicle for the transfer
of the foreign gene.
Transfer vector has already been constructed for the lepidopterans Autographa
californica (Ac NPV; Smith et al., 1983; Summers and Smith, 1987) and Bombyx mori NPV;
Maeda et al., 1985). These transfer vectors provide a site for the cloning of foreign gene
under the control of polyhedrin promoter, p 10 promoter or IE (immediate early gene
promotor). The scope of cloning and gene under the control of these virus promoters has
resulted in the production of recombinant viruses.
Cloning of Neuropeptides for Pest Control
Neuropeptides, growth regulators, and toxic of prokaryotic and eukaryotic origin are
some of the potential genes for the production of recombinant virus. For the first time
Maeda (1989 b ) has shown that the gene for insect diuretic hormone can be cloned in Bm
NPV and the expression of the gene results in strong alteration in the larval body fluid
metabolism and increases the efficiency of larval killing by about 20%. Body fluid
regulatory mechanisms of insects are regulated by diuretic and antidiuretic hormone.
179
Based upon the sequence of the diuretic gene of Manduca sexta, Maeda (1989 b )
synthesised diuretic hormone gene of 41 amino acid composition and cloned it into the Bm
NPV transfer vector under the control of polyhedron promoter. He produced the
recombinant Bm NPV carrying diuretic hormone gene and studied the haemolymph volume
and mortality of B. mori larvae infected with wild type and recombinant NPV. His
observations are quite interesting that the haemolymph volume of the larvae infected with
recombinant NPV has been decreased by 30% in comparison to wild type NPY infected
larvae. Furthermore, he showed that within four days after infection all the larvae infected
with recombinant NPV were killed while the wild type NPV infected larvae required five to
six days to achieve the same level of mortality. These observations demonstrate that the
genetically engineered NPV has the ability to change the water metabolism and increase the
mortality of the larva.
Hammock and his co-workers (1990) showed the scope for the introduction of
neuropeptide into the NPV to regulate the development of the target host. It is known that
juvenile hormone (JH) is associated with larval phase of the insect and reduction in the
titre of this hormone resulted in moulting or metamorphosis. Continuous production or
addition of JH results in the prolonged larval growth phase. Concentration of JH is reduced
as the juvenile hormone esterase (JHE) titre increases in the haemolymph. Production or
introduction of additional quality of JHE at an early stage nf development could result in
the reduction of JH titre Thus, the production of JHE during the early phase of
development resulted in the cessation of feeding and decreased growth of the larva.
Considering this concept, Hammock and his co-workers (1990) cloned the JHE gene
in the transfer vecior of AcNPV and produced the recombinant NPV carrying the JHE gene.
On measuring the feeding efficiency and final body weight of the larvae of Trichoplusia ni
infected with wild type and recombinant NPV, Hammock el al. (1990) reported a reduction
of final body weight by about five times in the larvae infected with recombinant NPV
compared to control Further-more, the feeding efficiency decreased in recombinant NPVinfected first instar larvae. They are also concentrating on the control of Spodoptera exigua
by a similar recombinant virus.
Virus Genes for Pest Control
The NPV genome contains a number of genes with varied functions. A gene that
interferes with ecdysteroid UDP glucosyl transferase of the host was identified by O'Reilly
and Miller (1989). This particular gene of viral origin has been identified in AcNPV genome
and showed to interfere with the normal development of the host insects. The published
data showed that the baculovirus AcNPV specifically interrupts the normal development
and moulting of host insects by producing ecdysteroid UDP-glucosyl transferase. This
observation suggests that a similar type of gene interfering with the growth and
180
development of host insects may exist in other baculoviruses. Efforts are needed to identify
the viral genes that interfere in the normal development and growth of the insects and to
make use of them in active pest management.
Combination of Viral and B. thuringiensis Genes for Pest Control
Insecticidal properties of delta endotoxin gene of Bacillus thuringiensis and its
commercial value are well known (Dean, 1984; Andrews et al., 1987). The idea of cloning
the B.t. (toxin gene in the NPV genome was contemplated in different laboratories. Bacillus
thuringiensis aizawai endotoxin gene was cloned into the polyhedron locus of ACNPV and
the expression of the gene was recorded in the Spodoptera frugiperda cells (Martens et al.,
1990). Spodoptera frugiperda cells infected with recombinant virus produced the endotoxin
crystal protein. The cells were fed to Pieris brassica second instar larvae. LC 50 volume of
3.4 x 102 cells was recorded while the same concentration of S frugiperda cells infected with
wild type virus did not produce harmful effect in the larvae. Thus, the above observation
showed the production of delta endotoxin in the cells infected with recombinant virus.
Mathavan and Maeda (1991) cloned the B. thuringiensis aizawai gene in the BmNPV
transfer vector and expressed the toxic protein in vivo. They obtained the Haemolymph
protein from the control larva (uninfected) and larvae infected with wild type NPV as well as
recombinant NPV carrying B. thuringiensis aizawai gene. Immunoblot of haemolymph
protein of these larvae revealed a single positive hybridisation in recombinant virusinfected
larvae showing that recom-binant virus produced endotoxin. This protein size is in
accordance with that of endotoxin protein of B. thurinziensls aizawai. This information
shows the production of endotoxin protein in the infected larva systems.
During field application of baculovirus for pest control, the presence of polyhedral
bodies is ideal for the survival of the virus in the field. Hence, it was assumed that a
recombinant virus with polyhedrin (+) may be better than polyhedrin (-) virus. Keeping this
concept in mind Merryweather et al (1990) cloned B. thuringiensis krustaki delta endotoxin
gene under the control of a copy of AcNPV p10 promotor positioned upstream of the
polyhedral gene and produced a polyhedrin (+) recombinant virus. By fluorographic and
immunoblot analysis, these showed the expression of the endotoxin protein under p10
promoter. They also showed the reduction in the feeding of the larvae fed on a diet
containing recombinant virus infected cells. The reduction in the feeding has been
attributed to the production of endotoxin by recombinant virus.
181
Figure 1 : Analysis of B.I. aizawi toxin in the haemolymh of Bombyx mori larvae.
Haemolymph was collected from the control wild type and recombinant virus-infected
larvae. Proteins were separated in SDS-PAGE( 10%), transferred to nitrocellulose filter, and
immunoblotted against B.t. aizawai antibody.
Scope of IE Promotor and Chimeric Clones for Pest Control
Both the polyhcdrin and p 10 or late gene promators and the genes cloned under
the control of these promoters will start expressing after 18-24 hours of post infection. It
was thought that it is ideal to clone a gene under the control of early gene promotor.
Recently, Jarvis el al. (1990) showed the use of early gene promotor for the continuous
expression of foreign product. It will be exciting if the endotoxin gene/neuropeptide gene
can be cloned under the immediate early gene prornotor and the cloned gene product can
be produced even during the early phase of viral infection. This area of research needs more
intensive work.
One of the disadvantages of the baculovirus pathogen is the host specificity of the
virus. This is mainly because of the presenc of specific midgut binding domain in the virus.
Similarly, B. thuringiensis genes have host specificity; some strains of B. thuringiensis are
toxic to dipterans, some to lepidopterans, and some lo coleopterans. This is also due to the
specific midgut binding domain of B. t. toxin genes. Sivasubramanian et al ( 1990) have
recently cloned an insect virus surface protein gene of wide host spectrum with strong
midgut binding domain and B t. delta. exotoxin gene. Using the chimeric clone they have
shown that the host range and the work in this line can produce a viral vector with wide
host range increased toxicity.
182
Conclusion
Potential use of wild type NPV for pest control has already been demonstrated. The
double-stranded, circular nature of the NPV genome has offered scope for genetic
engineering and production of recombinant NPV
Results obtained so far on the use of
recombinant NPV for pest control encouraging. The possibility of cloning the toxic gene or
neuropeptides growth regulators under IE promotor opens the door to the production of
polyhedrin (+) recombinants with early expression of cloned genes. Attempts on the use of
chimenc clones with wide host spectrum further suggest the advantages of adoption of
genetic engineering as a major new technology for pest control.
183
Glossary of Recent Trends in Biological Control
1) Accretive Release : A method of periodic introduction of biotic agents in which annual
early season liberations against fairly abundant pest populations allow the beneficial
organisms population to increase naturally in response to rising pest densities as the
season progress.
2) Antagonists : The saprophytic fungi. Trichoderma viride is parasitic on other disease
causing fungi and produce antibiotics ( Gliotoxin and viridian ) which kill the root-rot
diseases of pulses and oilseeds.
3) Appressorium: A suw.r.veiling, produced at the end of germ tube from conidiophores of
some entomogenous fungi which attaches the host cuticle and penetrated the integument
with "infective pegs".
4) Agroecosystem : The modified and simplified system of plants, animals and habitat
used for human agricultural purposes.
5) Arrhenotoky: In parasitic hymenoptera the fertilized eggs are diploid and give rise to
females, whereas the azygotes from the unfertilized eggs are haploid and arc males e.g.
many hymenoptera like Trichogramima exiguum, T. minutum, Telenoumus remus.
6) Artificial diet : An unfamiliar food which hasTeen formulated, synthesized, processed
and /or concocted on which an insect in captivity can develop through all or part of its life
cycle.
7) Augmentation: It is the process which involves to improve the effectiveness of natural
enemies by manipulating either mass production, periodic colonization or by genetic
improvement.
7) Autoparasitism or Adelphoparasitism: A special type of parasitism in which the
female develops as a primary parasitoid but the male of Coccoplagoides utilis Doutt develop
as a hyperparasite from unfertilized egg laid by parent female of C. utilis a primary
parasitoid of purple citrus scale, Paralotoria oleae.
8) Axenic : Rearing of one or more individuals of a single known species in or on non living
medium of synxenic.
9) Balance of nature: The natural tendency of plant and animal population, resulting from
natural regulative processes in an undisturbed environment, to neither decline in numbers
to extinction, nor increase to infinite density.
10) Biological check method: A method of evaluating the effects of introduced natural
enemies of pest population in which biological agents (natural enemies) are excluded from
one plot and encouraged in another for comparison of their effectiveness (De Bach el al.
1951).
Compiled by : Dr. U.P. Barkhade & Dr. A.Y. Thakare
184
11) Biological control : It is the study and utilization of parasitoids. predators and
pathogens for the suppression of pest population of plants or animals.
12) Biological insect pest suppression : The use encouragement by man. of living
organisms their products for the population reduction of insect pest.
13) Biotype : A biological strain of an organism, morphologically indistinguishable from
other members of the species, but exhibiting different physiological characteristics.
14) Baculoviruses : Polyphagous pests like gram pod borer (Helicoverpa) tobacco
caterpillar (Spodoptera) and red hairy caterpillar (Amsacta) destroy majority of crops and
may he reasonably kept under check by the viruses.
15) Classical biological control: It involves the deliberate introduction and establishment
of natural enemies into areas where they did not previously occur and employed largely
against pests of exotic origin, e.g. Rodolia cardinalis (Muls) an Australian ladybird was used
against Iceryai purchasi Mask in India which gave spectacular control.
16) Cleptoparasitism : A type of parasitism is which the adult parasitoid preferentially
appropriates for its own progeny the previously paralysed and parasitized host of another
parasitoid.
17) Cocoon : The silk-like protective covering spun by same species prior to quiescence^
"diapause or pupation.
18) Colonization : It is the attempted establishment of a community of organisms in a
new locality. It includes the field release of the imported species and such manipulation as
is necessary for favoring their general increase and spread.
19) Comb: A layer of brood cells or cocoons crowded together in a regular arrangement
such as those produced by many species of wasps and bees.
20) Confusant: The term is used for a pheromone or pheromone-like analog when it is
used in a program of disruption of communication for insect pest suppression.
21) Contamination: The presence of unwanted ogranims or microorganisms in food, air or
insects.
22) Culture : A laboratory population maintained usually under controlled conditions.
Although usually
reserved
for populations of micro-organisms, it
may
be
used
synonymously with colony or sub-colony when referring to insect populations.
23) Capsule: Proteinaceous elongate^round covering on virus rods.
24) C. I. B. C. : Common wealth Institute of Biological Control. It is an international
organisation head quartered at Trinidad, West Indies. It supplies beneficial organisms in
various parts of the world on contract basis, provider of useful bioconlrol review and
documents.
185
25) Conservation: It is the process involved in manipulation of environment to favour
natural enemies either by removing or modifying the adverse effects or by providing the
lacking prerequisites.
26) Colonizations : The controlled release of a quality of biological control agents in a
favorable environment for the purpose of permanent or temporary establishment.
27) Density dependent factors : It refers to mortality factors or processes
in
the
environment which destroy an increasing percentage of the subject population as the
numerical population density increases and vice-versa (Smith. 1935).
28) Density independent factors : Refers to mortality factors or processes in the
environment which destroy a relative constant percentage of the subject population
regardless of changes in its density (Smith, 1935).
29) Definite or definitive host : An insect in which a parasite attains sexual maturity.
30) Dcutorotoky (ampherotokous) : There are parasitoids exhibiting thelyotoky. but also
produce
few
males,
such
individuals
are
again
uniparental. e.g.
Trichograinma
brasiliensis when reared at temperatures exceeding 25°C also produces males while beyond
30°C mainly males.
31) Ectoparasite : An insect parasite which develops externally on its arthropod host.
32) Egg batch: A group of eggs that have been laid singly or in a group by the same female
at about the same time.
33) Egg sheet : A piece of a paper, cardboard, polythene, or other artificial substrate on
which eggs have been deposited.
34) Enhancement : The term denotes the use of measures that increase the longevity and
reproduction of natural enemies in field.
35) Environment : The sum total of all of the physical, biological, and chemical
surroundings in which an insect lives.
36) Environmental resistance : The integration of physical and biological factors which
limit the growth of an insect population.
37) Exotic : An organism brought in/introduced from abroad.
38) Entomogenous: Refers to organisms (usually micro-organisms) growing in or on the
bodies of insects.
39) Entomopathogenic: Capable of causing disease in insects.
40) Entomophagous: Refers to the consumption of insects or their parts.^insectivorous.
41) Entomophilic: Insect loving.
42) Epizootic: An outbreak of a disease (or sometimes a pest) in which unusually high
number of individuals of the pest are affected.
43) Endotoxin: A toxic substance formed by certain bacteria (e.g. B. t) and retained within
their vegetative cells.
186
44) Exotoxin: A soluble toxic substance produced by certain bacteria (e.g. B. t) and found
in their surrounding growth medium.
45) Factitious host: An unnatural but acceptable host used in laboratory for propogation
of beneficial organisms.
46) Factitious (Surrogate) host : An insect that is not the preferred host but is used as a
host for the production of parasites. Such hosts are usually used because they are easier to
rear in large numbers than the preferred host e.g. Corcyrc cephalonica st. is a factitious
host of Trichogramma spp.
47) Flareback (Pest resurgence): The rapid increase of a target insect pest population
subjected to an insecticidal treatment which also destroys its associated and possibly
regulative natural enemies.
48) Fortuitous biological control : The accidental movement of exotic natural enemy to
new
area
lepidosaphes
where
pest
Comp.
indigenous to the
on
population
purple
suppression
scale
area of China has
of citrus
spread
to
eventually
results,
Lepidopsaphcs
California
and
e.g.
Aphytis
heckii
(Newm).
other/ countries
alongwith the import of citrus plantings.
49) Frass : The solid excrement produced by larvae, nymphs or adults.
50) Generation: The period from a given stage in the life cycle (usually the egg) to the same
stage in the offspring.
51) Gregarious parasitism: It is the phenomenon of parasitization of an{ individual host
by more than one larva of single parasitic species but all survive is gregarious parasitism,
e.g. Apanteles flavipes Cameron is a gregarious parasitoid of jowar stem borer chilo
partelluls (Swinhoe).
52) Group rearing: The rearing of more than one individual (usually of the same species)
in a single rearing container of individual rearing.
53) Head capsule measurement: The width of the head capsule (usually in mm)
measured laterally acrossed the widest part of the gena or subgena. Such measurements
are used to distinguish between larval instars.
54) Healthy insect : An individual that is able to develop normally, and is fertile and
disease free.
55) Hold back : To retard or prevent the development of an immature stage using low
temperature.
56) Host : This is an organism which harbor another organism, e.g. the rice meal moth,
Corcyra cephalonicas st. is a host of many natural enemies.
57) Host : The organism in/on which a parasite lives, and the plant on which an insect
feeds.
187
58) Hyperparasitism: It is the phenomenon of parasitization of a host individual when a
parasite attacks and develops on primary parasite e.g. Aphanogmus fijiensis ferr, is a
hyperparasitoid on cocoons of Apanteles flavipes Cameran, a primary parasitoid of Chilo
partellus (Swinhoe)
59) Hyperparasitoid : An insect parasite of another parasitoid.
60) Inoculation : The act of introducing an insect often at the egg or larval stage on to a
food source in a rearing container.
61) Inundative release : A method of periodic introduction of biotic agents which is
analogous to insecticide treatment in that a greater amount of the liberated material is
used than is actually effective^ repetition may be necessary and the effect is more or less
immediate.
63) Innoculative release :
The repeated colonization of relatively small
numbers of
natural enemy for purpose of building up a population over several generations.
64) Indigenous : Born in or natural to a country.
65) Inundalive Release : The production and release of large numbers of beneficial insects
against a particular pest population.
66) Inoculative release: The establishment of a population of exotic entomophagus insects
for severaTgenerations per season using laboratory reared insects.
67) Insect rearing laboratory : Any room or place used to rear insects, an insectary.
Normally there is some degree of environmental control.
68) Insectary: Means a place wherein insects are housed or propagated.
69) Insect rearing management (IRM): It is the efficient utilization of resources for the
production of insects of standard quality to meet the research goals.
70) Insect rearing management system (IRMS) : The integration of insect rearing
management techniques and production processes, and their application to rearing a
particular insect species.
71) I.O. B. C. : International Organisation of Biological Control head quartered at Zurich
Switzer land. Disseminate information, co-ordinate and promote research on iocontrol.
72) IU (International Unit) : An arbitraty set basis for comparing the efficacy on insect
pathogenic Bacillus thuringiensis preparations. An IU=1/1000 insecticidal activitymg of
primary standard E-61 strain of B. t. with potency of 1000 lU/mg. Secondary USA
standard, HD-l-S-1971 has the potency of 18000IU/mg.
73) Life table : A device for expressing in an orderly fashion observations on the changing
density of an insect population in time and space and the processes which direct those
changes, especially in relation to the age specific distribution of mortality and its causes.
74) Microbial insecticide: A pathogenic microorganism or its products, (e.g.toxins) used
by man to suppress insect pest.
188
75) Mass culture: A term usually reserved for large scale production of bacteria, viruses or
other biological control agents using insect hosts. It is also synonymous with factory
rearing (mass rearing in millions per unit time or a regular basis).
76) Multiparasitism : It is the phenomenon of simultaneous parasitizalion of a host
individual by two or more different species of primary parasites at the same time e.g.
Parasitization of a lepidopteron egg by two primary parasitoids namely (Trichogramma spp.
and Telenomus spp.).
77) Multiple parasitism : A condition resulting from the simultaneous use of a single host
individual by two or more species of primary parasitoids.
78) Natural enemies: A term commonly used for all parasitoids, predators and pathogens
associated naturally with a specific wild population of plants or animals causing premature
death.
79) Natural host: The plant or animal species on which an insect normally feeds and
develop.
80) Nucleus culture: A small population from which any colony begins. Usually refers to a
population obtained from a culture in another insectary but synonymous with parental
colony if the individuals are collected from the wild.
81) Natural control : It is the process of dynamic equilibrium which maintains the
characteristic mean density of a wild population within particular upper and lower limits,
over a period of time, by a complex combination of all the additive. conditioning and
subtractive process striking upon that wild population.
82) Parasite : It is an organism that is usually much smaller than its host, and a single
individual usually does kill the host. Numerous individuals may irritate, weaken or
otherwise debilitate the host, and occasionally cause its death e.g. tapeworm, lice. fleas,
mosquitoes, etc. on animals including human beings.
83) Parasite : An organism which lives & feed in/or other organism (the host), contributing
nothing to the relationship, & frequently destroying the host in the process if it's
development.
84) Parasitoid : It is a special kind of predator that is often about the same size as its
host. kill its host, and require only one host (prey) for development into a free-living adult
e.g. Trichagmmma spp., Bracon spp., Telenomus., etc.
85) Parasitoid : It is only an insect of an arthropod parasitic only in its immature stages,
often destroying its host in the process of its development, and free living as an adult.
86) Predator : An animal which feeds upon other animals (prey) that are usually smaller
^and weaker than itself, frequently devouring them completely and rapidly. A predator most
often is required to seek out and attack more than one prey to reach maturity.
189
87) Predators : Predators (Chrysopids and Coccinellides) may voraciously feed on aphids.
Mealyhuge and other insects attaeking cotton tobacco, sunflower, groundnut, mustard
guava. citrus, grapes, etc.
88) Predator: It is a free living organism through out its life, normally larger than prey
(host) requires more than one host to develop and are not very specific unlike parasitoids
e.g. Ladybird beetles (Coccinella septcmpunctata Menochilus sexmaculatus) and green lace
wing etc. (Chrysoperla spp. Mallada spp.)
89) Parasitism: It is the relationship (temporary or permanent) between two species in
which one (parasite) derives the benefit while the other (host) may or may not feel the loss
or injury caused by the parasite.
90) Parthenogenesis: Reproduction without exchange of gametes.
91) Periodic
release : It involves repeated liberations to artificially maintain high
population levels of indigenous biotic agents in situations where such levels are
unattainable naturally.
92) Pest upset: A condition in which innocuous species become man made pests as a
result of insecticide use which unintentionally destroys their population-regulative natural
enemies.
93) Polyemhryonic parasitism: It is the phenomenon of parasitization of an individual
host by single larva of a single parasitic species, but develops into several parasitoids
within the host body e.g. Copidosoma koehleri on potato tuber moth operculella.
94) POBs: Polyhedral (many faced) occulusion body. It is the proteinaceous outer covering
on virus rods.
95) Prey : An animal that is fed upon or destroyed by a predator.
96) Primary parasitoid : An insect parasite of any arthropod host which is not itself
parasitic.
97) Protelean parasitoid : An insect species in which only the immature stages are
parasitic. (Askew 1971)
98) Quarantine :
The isolation of an individual insect or group of insects in order to
determine or eliminate any associated or potentially hazardous organisms.
99) Recovery: It is referred as to obtain a simple qualitative assessment of the presence or
absence of the species soon after its initial release in a new locality, with little or no
reference to its probable efficiency.
100) Shipping: The sending of insects from one place to another by land, air or sea.
101) Solitary parasitism: It is the phenomenon of parasitisumip of an individual host by
more than one larva of a single parasitic species but only one survives e.g. Campolelis
chlorideae, Uchida parasitic on the larva of Helrcoverpa armigera ( Hubner ).
102) Secondary parasitoid : An insect parasite of a primary parasitoid.
190
103) Solitary parasitoid : An insect parasite which normally develops at a rate of one
individual per arthropod host.
104) Superparasitism : A condition resulting from the use of a single host individual by
more individual parasitoids of the same species than it can successfully sustain to maturity
because of nutritional limitations.
105) Superparasitism: It is the phenomenon of parasitisation of an individual host by
more larvae of a single parasitic species that can mature in the host e.g. Trichogramma
chilonis.
106) Synexenic : The rearing of one or more individuals of single species in association
with
one
or more
known
species of organisms.
Conditions
are
referred
to
as
monoexenic. dixenic, trixenic. or polyxenic depending on whether the number or associated
species is one, two, three, or more, respectively of axenic.
107) Thelyoloky :
Some species are obligatorily parthenogenetic and each generation
consists entirely of females. Such individuals are termed as impaternate or uniparental e.g.
Chelonus blackburni an egg-larval parasitoid of cotton bollworms.
108) Trichocard : It is a paper card of 15 cm x 7.5 cm size perforated into 36 equal blocks
(2.5cm x 1.25cm) and has capacity to withhold 1 cc eggs of rice meal moth, normally
sprinkled evenly over it for rearing the Trichogramma spp. Telenomus remits etc.
Terminology related to pest suppression: : Government may also consider the following
suggestion for future work which may help farmers in adopting the biocontrol technology.
1. Judicious, as well as, restricted import of biotic agents from other countries.
2. Emphasis on exploration of indigenous biotic agents.
3. Preparations of "Field Guide For Biotic Agents" alongwith their visible stages and
natural hosts;
4. Establishment of commercial factories to ensure supply of potential biotic agents.
5. Establishment of National Institution on conservation of biotic agents, alongwith its
network at district level.
6. Studies on biotie
agents in
relation
to intercropping, trap cropping, cultural
practices and otlier forms ot organic farming.
191
Referred Books for RTBC-606 course
1) Chillar B.S., Kaushik H.D., Malik V.S. and Ombir (2003) : Classical biological control,
Emerging Trends in Biological Control of Insects, Pests and Weeds. Book pp. 154161.
2) Chillar (2003) : Kaushik H.D., Malik V.S. and Ombir : Dynamics of biocontrol agenc
vis-a-vis target pest population. Emerging Trends in Biological Control of Insect,
Pests and Weeds Book pp. 75-80.
3) Paul De Bach (1964) : Nutrition of Entomophagous insects and their hosts.
Bilogical Control of Insects, Pests and Weeds Book pp. 356 – 363 and 371-372.
4) Ghorpade S.A., Chandele A.G. and D.S. Pokhakar (2004) Mass culturing techniques.
Production and Use of Bioagents in Crop Pest Managemue Book pp. 105-107.
5) Paul De Bach (1964) : Insectary facilities and equipment. Biological Control of Insect,
Pests and Weeds Book. pp. 381-401.
6) R.D. Gautam (1994) : Basic standard of insectary and good insectary practies. Bilogical
Pest Suppression Book pp. 27-31.
7) Paul De Bach (1964) : Good insectary practices” Biological Control of Insect, Pests and
Weeds Book pp. 350-353.
8) R. D. Gautam (1994) : Colonization techniques of release of natural enemies, recovery
and evaluation of natural enemies, conservation and augmentation of natural
enemies. “Biological Pest Supperession Book p.p. 57-72.
9) Paul De Bach (1964) : Augmentation and Conservation of natural enemies. Biological
Control of Insect, Pests and Weeds Book pp. 17-20.
10) Paul De Bach (1964) : “Method of colonization, Recovery and evaluation” Biological
Control of Insect, Pests and Weeds Book pp. 402-426.
11) Coppel H.C., James W. Mertin (1977) : Release and Colonization, follow up, recoveries
and evaluation : Biological Insect Pest Suppression Book pp. 58-63.
12) Paul De Bach (1964) : Colonization procedure. Biological Control of Insect, Pests and
Weeds Book pp. 432-436
13) Paul De Bach (1964) : Inoculative releases of Natural enemies. Biological Control of
Insect Pests and Weeds Book pp. 436-441.
14) Ananthakrishnan T.N. : Augmentation / Augmentation of natural enemy, bio-control
insecticides. Emerging Trends in Biological Control of Phytophagous Insect Book pp.
213-222.
192
15) Ghorpade S.A., Chandele A.G., and D.S. Poldrarar (2004) : For large scale production
of bio-agent, mass production and field utilization of bio-agents in biological
suppression of crop pests. Production and Use of Biologent in Crop Pest
Management Book pp. 94-121
16) Chillar B.S., Kaushik H.D., Malik V.S. and Ombir (2003) : For genetic engineering of
microbes (viruses). Emersing Trends in Biological Control of Insect, Pests and Weeds
Book pp. 118-121.
17) Coppel H.C., James W. Mertin (1977) : For genetics of ideal trait in biological agent
for introgression and for progeny selection. Biological Insect Suppression Book pp.
211-216.
18) Ananthkrishnan T.N. : Genetic improvement of baculoviruses and genetic
engineering
of
baueloviruses.
Emerging
Trends
in
Biological
Control
of
Phytophagous Insects Book pp. 183-198.
19) Paul De Bach (1964) Breeding technique of biocontrol agent. Biological Control of
Insect, Pests and Weeds Book pp. 452-458.
---------
193