ISRN KTH/IOK/FR--20/57--SE
ISSN 1100-7974
TRITA-IOK
Forskningsrapport 2000:57
KTH
Royal Institute of Technology
Department of Chemistry
Organic Chemistry
CHEMICAL COMMUNICATION IN LEAF
MINING MOTHS OF THE GENUS
PHYLLONORYCTER
Raimondas Mozuraitis
Tekn. Lic.
Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan
i Stockholm framlägges till offentlig granskning för avläggande av filosofie
doktorsexamen i organisk kemi fredagen den 14 januari 2000 kl. 10.00 i
Kollegiesalen, Administrationsbyggnaden, KTH, Valhallavägen 79,
Stockholm. Avhandlingen försvaras på engelska.
Mozuraitis, Raimondas. Chemical Communication in Leaf Mining Moths of the Genus
Phyllonorycter. Royal Institute of Technology, Stockholm, January 2000.
ABSTRACT
Laboratory studies of pheromone release behaviour have revealed that virgin females
of all of the nine species of the genus Phyllonorycter investigated demonstrated the same
pheromone release posture. Their calling activity was registered at the beginning of the
photophase. This is an unusual time for calling in moths. Our hypothesis is, that this
uncommon timing of the pheromone communication may have been caused by the sensitivity
of the males to sex attraction antagonists, released into the environment by females of other
species at other times of the day.
The diurnal calling behaviour of virgin Ph. junoniella females was more pronounced
under a cyclic thermal regime (close to the natural conditions) than under constant
temperature. We assume, that the occurrence of an extra peak in the pheromone release
behaviour as well as the extension of the calling period to cover the larger part of the light
period may be adaptive for Ph. junoniella with a sex ratio strongly shifted towards females
(8:1 females to males), as it may lead to an increased proportion of males mated.
It was found, that the leaf miner moth Ph. emberizaepenella reproduced by
parthenogenesis of the thelytoky type. Despite a complete lack of males, the females
demonstrated a calling posture with a sex pheromone release with the typical diurnal rhythm
of that behaviour. Theoretical speculations that in thelytoky, where there is no need to attract a
sexual partner, the females benefit by reducing their sexual behaviour, was not confirmed for
Ph. emberizaepenella.
Ten compounds used in the sex communication of five phyllonoryctid species were
identified from calling virgin females: Z10-, Z8- and E10-14:OAc for Ph. acerifoliella; E1012:OAc, 12:OAc and E10-12:OH for Ph. blancardella; E8,E10-14:OAc and E8,E10-14:OH
for Ph. emberizaepenella; Z8-14:OAc, 14:OAc and Z8-14:OH for Ph. heegerella; as well as
Z10-14:OAc for Ph. ulmifoliella. The Solid Phase Micro Extraction technique was applied for
the first time to collect a sex pheromone from a single calling microlepidopteran female and
our data clearly demonstrated the advantages of this method.
Our field screening tests in Lithuania disclosed new sex attractants for five
phyllonoryctid species: E10-12:OH for Ph. sorbi; E10-12:OAc for Ph. cydoniella and Ph.
oxyacanthae; Z10-12:OAc for Ph. junoniella; as well as Z10-14:OAc in a 1:10 mixture with
E9-14:OAc for Ph. acerifoliella. The latter mixture was also found to be a potential sex
attractant for Ph. coryli and Ph. heegerella.
In addition, field trapping experiments revealed fourteen sex attraction antagonists for
males of seven Phyllonorycter species: E10-12:OH for Ph. acerifoliella and Ph. cydoniella;
E10-12:OAc and Z10-14:OAc for Ph. heegerella ; Z10-, E10-12:OH and E10-14:OH for Ph.
mespilella; Z10-12:OH for Ph. oxyacanthae; Z7-, Z9- and Z10-12:OAc for Ph. sorbi; as well
as Z8-, Z9-, E9-, E10- and E11-14:OAc for Ph. ulmifoliella.
Schemes of probable interactions by means of allelochemicals acting between Ph.
blancardella and 7 other moth species, Ph. ulmifoliella and 414, Ph. sorbi and 243, Ph.
mespilella and 11 as well as Ph. acerifoliella and 7 other moth species are presented.
Basing both on our own results and on data published by others, we conclude that the
pheromones used by gracillariids have appeared at a period lasting between the formation of
lower and of higher Heteroneura.
Key words: Phyllonorycter, Gracillariidae, Lepidoptera, sex pheromone, sex attractant,
sex attraction antagonist, calling behaviour, calling posture, olfactometer, sex ratio,
parthenogenesis, evolution, ecology, Solid Phase Micro Extraction.
This thesis is based on the following papers, which will be referred to in the text by
their Roman numerals:
I.
Borg-Karlson A-K. and Mozuraitis R. Solid Phase Micro Extraction technique used for
collecting semiochemicals. Identification of volatiles released by individual signalling
Phyllonorycter sylvella moths. Zeitschrift für Naturforschung. 1996, vol. 51C, 599-602.
II.
Mozûraitis R., Bûda V., Borg-Karlson A-K. and Ivinskis P. Chemocommunication in
Phyllonorycter ulmifoliella (Hbn.) (Lepidoptera: Gracillariidae): periodicity, sex
pheromone, and inhibitors. Journal of Chemical Ecology. 1997, vol. 23, 175-189.
III. Mozûraitis R., Bûda V., Borg-Karlson A-K., Ivinskis P., Karalius V., Laanmaa M. and
Plepys D. New sex attractants and inhibitors for 17 moth species from the families
Gracillariidae, Tortricidae, Yponomeutidae, Oecophoridae, Pyralidae and Gelechiidae.
Journal of Applied Entomology. 1998, vol. 122, 441-452.
IV.
Mozuraitis R., Borg-Karlson A-K., Buda V. and Ivinskis P. Sex pheromone of the spotted
tentiform leafminer moth Phyllonorycter blancardella (Fabr.) (Lepidoptera, Gracillariidae).
Journal of Applied Entomology. 1999 (Accepted for publication, manuscript code 98/45).
V.
Mozûraitis R., Bûda V., Jonu aitë V., Borg-Karlson A-K. and Noreika V. Sex
pheromones of Phyllonorycter acerifoliella and Ph. heegeriella and communication
pecularities in three species of leafmining moths. Entomologia Experimentalis et Applicata.
1999 (Accepted for publication, manuscript code ENTO 2237).
VI. Mozûraitis R. and Bûda V. Pheromone release behaviour in females of Phyllonorycter
junoniella (Z.) (Lepidoptera, Gracillariidae) under stable and cycling temperatures.
Manuscript.
VII. Mozûraitis R., Bûda V., Liblikas I., Unelius C.R. and Borg-Karlson A-K. Parthenogenesis,
calling behaviour and sex pheromone identification in the leaf miner Phyllonorycter
emberizaepenella (Lepidoptera, Gracillariidae). Manuscript.
v
CONTENTS
Abstract
iii
List of the papers on which the thesis is based
v
1.
Introduction
1
2.
Principles of the sex pheromone communication system in moths
2
3.
Biological features of Phyllonorycter moths as objects of our studies
6
4.
Methodological aspects of identification of moth sex pheromones
8
5.
Chemical communication in the genus Phyllonorycter
5.1.
5.2.
5.3.
5.4.
5.5.
5.6.
5.7.
Calling posture
Daily rhythm
Sex attractants
Sex pheromones
Antagonists of sex attraction
Ecological aspects
Evolutionary aspects
15
15
16
19
23
27
29
36
6.
Concluding remarks and directions for future work
39
7.
Acknowledgements
41
8.
References
42
9.
Main terms regarding chemical compounds used in insect communication
52
10. List of the moths, caddisflies and plants discussed in the text
56
11. List of the compounds mentioned in the text
58
vii
1. INTRODUCTION
Animals communicate with each other by means of electromagnetic (visual, electric field),
mechanical (acoustic, tactile), and chemical signals. It is believed, that the chemical mode is one of
the oldest means of getting and transferring information (Harper, 1991). Shorey (1976) speculates,
that primitive single-celled organisms first developed a responsiveness to chemicals as a means of
locating other organisms that served as their food. With the advent of sexuality, the organisms were
then preadapted for intraspecific chemical communication, and the strong selective pressure to
come together for the exchange of genetic material led to a rapid specialization of these early sex
pheromone systems. Even at present, chemical communication is probably the major mechanism
allowing single-celled plants and animals as well as the motile gametes of higher animals to orient
themselves toward a sexual partner.
Pheromones are widely used in the animal kingdom in a great variety of species, ranging
from primitive protozoans to higher primates, as one of the principal means of transmitting
information (Shorey, 1976). Among the insect taxa, in which mate-finding is mediated by
pheromones, the Lepidoptera is the best investigated order (Tumlinson, 1988; Löfs tedt and Kozlov,
1997). Up to now, sex pheromones were identified in about 490 species and subspecies of
Lepidoptera, male sex attractants for another 1090 species and subspecies were reported and with
90 species indications of possible attractants were found (based on Arn et al., 1999), whereas the
number of species in the order is probably over 250000 (Nielsen, 1989). Moreover, the largest
number of identifications were made within modern families from the ditrysian lepidoptera, whereas
in the most primitive ones, such as the family Gracillariidae, identifications are known just for 5
species (based on Arn et al., 1999 and Svatos et al., 1999). As for non-ditrysian lepidoptera, only
three literature indications were found on sex pheromone identifications (Tóth et al., 1995, Zhu et
al., 1995 and Kozlov et al., 1996).
1
The chemical communication between insects is a very rich area for exploration. Besides the
identification of pheromones and allelochemicals, a considerable amount of research remains to be
done to elucidate the mechanisms that regulate chemical communication systems in insects as well as
the biochemical and evolutionary causes of the great complexity and diversity of chemical signals
(Löfstedt and Kozlov, 1997; McNeil et al., 1997; Phelan, 1997).
The specificity and nontoxic nature of pheromones and allelochemicals make them useful
tools within integrated pest management programmes for controlling agricultural and forestal pests.
An increased knowledge of the behaviour ecology underlying the pheromone-mediated
communication systems is another goal of the investigations, that could improve the success of pest
management programmes (McNeil et al., 1997).
2. PRINCIPLES OF THE SEX PHEROMONE COMMUNICATION SYSTEM
IN MOTHS
The lepidopteran sex pheromone communication system, which is a typical one, consists of
three components:
production and release of a message . Chemical signals are produced in special sex
pheromone secretory cells. These cells are mostly located in a glandular organ with or without
specialized devices that transfer the chemical molecules into the surrounding medium (Roelofs,
1995). Female sex pheromone glands of moth species have been found in at least 21 of the 38
families of Lepidoptera (Zhu, 1995). They are generally located in one of the following two parts of
the body:
i) in the fifth sternite. Among the less advanced monotrysian families, Lophocoronidae is the
most advanced family possessing these glands in their typical form (Löfstedt and Kozlov, 1997),
2
ii) in the intersegmental membrane between the eighth and ninth abdominal segments. In this
place glands are found in the females of most ditrysian families and in the advanced heteroneuran
lineages (Löfstedt and Kozlov, 1997).
The pattern of the chemical structures of sex pheromone compounds produced by the
glands located in the fifth sternite differs from the structural patterns of those identified from the
glands found on the intersegmental membrane between the eighth and ninth abdominal segments
(Löfstedt and Kozlov, 1997).
It is interesting to note, that in two species within the family Psychidae, Kotochalia junodi
(Bosman and Brand, 1971) and Thyridopteryx ephemeraeformis (Leonhardt et al., 1983), the
sex pheromone appears to be produced in the thorax.
a medium, through which a message is transmitted. For moths, this is mostly air, and
perceiving and processing a message. An olfactory (smell) organ is found on the antennae of
males. The receiving part of the pheromone receptor cell is located within the sensilla trichodea,
whose outer structure can be seen as hairs on the surface of the antenna (Mustaparta, 1984). After
adsorption onto the cuticular surface of the antennae, the hydrophobic pheromones diffuse into the
interior of the sensilla through microscopic pores of the cuticular wall. Later events at the sensilla
level include pheromone binding, transport to a specific receptor site, catabolism, and a process of
signal transduction (Breer, 1997). Without considering all of the steps and morphological structures
in detail we just mention that the sex-pheromonal information proceeds from the receptor cells
through the deutocerebrum and the protocerebrum to descending premotor pathways, and some
type of behaviour reaction can finally be observed (Hildebrand, 1997).
With the vast majority of lepidopterans, sex pheromones are released by females for longrange mate location, whereas the male-emitted chemicals, affecting females, usually act over short
distances and are not typical phenomena in lepidopteran sex communication (Birch et al., 1990;
Phelan, 1992).
3
The long-distance sex pheromone components identified from females of ditrysian moth
species form two major groups. The first group mainly consists of aliphatic C10-C18 straight chain,
saturated and mono-, di-, and tri-unsaturated alcohols, aldehydes and acetates. Conjugated and
non-conjugated double bonds can occur at positions from 2 to 13 in either Z- or E- diastereomers
(Fig. 2.1. Based on Arn et al., 1999).
A
OH
B
O
C
O
O
Fig. 2.1. Representative sex pheromone components identified from females of ditrysian moth
species and attributed to the first group.
(A) E10-12:OH, found in Phyllonorycter blancardella ; (B) E10,E12-16:Al, known in a number
of species from the families Sphingidae, Pyralidae and Noctuidae; (C) Z10-14:OAc, found in the
species Ph. ulmifoliella and Ph. acerifoliella. (Based on Arn et al., 1999 and on Papers II, IV,
and V).
Exceptions to this pattern are present in species from some families: propanoate Lasiocampidae, Noctuidae; butanoate - Noctuidae; isobutanoate - Lymantriidae; pentanoate Lymantriidae; isopentanoate - Attacidae, Lymantriidae; decanoate - Psychidae; dodecanoate Zygaenidae; tetradecanoate - Zygaenidae; carbonyl group - Tortricidae, Lymantriidae,
Geometridae, Carposinidae; triple bond – Thaumetopoeidae; nitrate - Bacculatricidae (based on
4
Arn et al, 1999). A pheromone containing one of these unusual functional groups was identified
only in one species of each family mentioned.
The second major group of moth pheromone components consists of saturated, mono- or
poly-unsaturated (one to four double bonds of Z and E types) straight and branched hydrocarbons
with chain lengths C15-C 29 and their monoepoxides (Fig. 2.2). The pheromones of this type are
most common within the superfamilies Noctuoidea and Geometroidea (based on Arn et al, 1999).
A
B
O
Fig. 2.2. Representative sex pheromone components identified from females of ditrysian moth
species and attributed to the second group.
(A) (Z,Z,Z)-3,6,9-heptadecatriene and (B) (Z,Z)-3,9-(6S,7R)-6,7-epoxynonadecadiene found in
seven and three species of the subfamily Ennominae (of the family Geometridae), respectively.
(Based on Arn et al, 1999).
Pheromones identified from non-ditrysian lepidoptera are C7-C9 straight chain, mono- and diunsaturated alcohols or ketones, with a functional group in position 2 and either S or R configuration
(Fig. 2.3 ). (Based on Arn et al, 1999).
A
OH
B
O
5
C
OH
Fig. 2.3 Representative sex pheromone components identified from monotrysian moth species.
(A) (2R)-(Z)-4-hepten-2-ol found in Eriocrania cicatricella and E. sparrmannella, (B) (Z)-4hepten-2-one found in Eriocrania cicatricella and (C) (S)-(E)-6,8-nonadien-2-ol found in
Stigmella malella. (Based on Arn et al, 1999).
3.
BIOLOGICAL
FEATURES
OF
PHYLLONORYCTER
MOTHS
AS
OBJECTS OF OUR STUDIES
The tentiform leaf miner moths of the genus Phyllonorycter belong to the family
Gracillariidae, one of the oldest families within the taxonomic group Ditrysia of Lepidoptera
(Scoble, 1992). Most of the tentiform leaf miner moths are monophagous or oligophagous species
feeding on one or a few host-plant species. A caterpillar of this genus spends most of its life inside a
leaf, eating the inner part (parenchyma) without damaging the cuticle, and in this way a conspicuous
mark, i.e. a mine, is formed. Characteristic features, such as the form, colour and location of the
mine on a host-plant leaf, make it possible to identify the moths before adult emergence and to
collect single species for pheromone identification.
The leaf size of some host-plant species limits the food resources for caterpillars feeding on
them, because leaf miners of this genus can not move from one leaf to another as many other insect
larvae do. Consequently, only one individual per leaf can develop into an imago, and the capability
of the ovipositing female to avoid leaves with conspecific eggs or caterpillars plays an important role
in decreasing the competition at the larva stage. It is probable that pheromonal signals may by
involved in the recognition of oviposition sites bearing conspecific eggs. The presence of
6
oviposition-deterring (or oviposition-site-marking) pheromones has not yet been demonstrated in
leaf mining moths but was reported in a number of species belonging to the orders Diptera,
Coleoptera and Hymenoptera.
However, the leaf miners are so small (4-8 mm long), that extreme accuracy is needed in
handling these moths during the rearing and collection of pheromones from living individuals and
when recording their electroantennographic (EAG) responses. Besides, when the population density
is high, large numbers of adult males are caught in the sticky traps, which makes the precise
identification of their species a difficult task. These circumstances have probably strongly limited the
pheromone research on phyllonoryctids as well as on other leaf miners.
It is known that individuals of two to a few species of the genus Phyllonorycter may cooccur on the same host-plant forming host-plant-leaf miner complexes (based on Ivinskis et al.,
1985). Competition between species within these complexes is highly probable, which makes the
leaf miners of this genus convenient objects for investigation of interspecific interactions, including
those effected by allelochemicals in adults.
Some species of the genus Phyllonorycter, such as Ph. blancardella, Ph. mespilella, Ph.
pyrifoliella, and Ph. populifoliella, are known to be serious pests of orchards and recreation
parks, and these species are attracting more and more attention in pest monitoring and control
programmes in Europe and North America (Trimble and Hagley, 1988; Dolin and Stobchaty, 1988;
Laanmaa et al., 1991; Gries et al., 1993). As mentioned in the Introduction, pheromones become
widely used within integrated pest management programmes for agricultural and forestal pests and
the need for further detailed investigation of the chemical communication systems of gracillariids
becomes obvious.
4. METHODOLOGICAL ASPECTS OF IDENTIFICATION OF MOTH SEX
PHEROMONES
7
The principal pathway of identification of the sex pheromone of a moth consists of the
following main steps:
collection of the pheromone,
determination of the composition of the pheromone and of the chemical structures of
its components,
synthesis and purification of the chemicals identified (unless they are commercially
available),
evaluation of the biological activity of the synthetic pheromone components.
The collection step will be discussed in some detail here, but the discussion of the later steps will be
limited to the most important features.
Collection of the pheromone. Some considerations should be taken into account when
moth pheromones are to be collected. Most pheromones are produced by the females in
nanogramme quantities. Most pheromones are multicomponent blends, in which the ratios of the
components are precisely controlled. The contents of minor components may be even lower than
one percent of the blend, but they may still be critical in eliciting key behaviour interactions among
the individuals (Tumlinson, 1988). Moreover, the pheromone should be collected at the time when
moths are calling. This is important for several reasons: just before and during the calling time, the
pheromone glands contain the largest amount of pheromone. Moreover, during the non-calling time
the glands may contain many other substances, including precursors, some of which may be
inhibitors (Teal et al., 1984).
There are basically two methods of collecting a pheromone: (1) solvent extraction of the
moth pheromone glands (residue analysis) and (2) adsorption of the moth-released airborne
volatiles (entrainment).
8
Extraction is the most common method for collecting moth pheromones. Preferred solvents
are dichloromethane, hexane, and diethyl ether, since they differ in polarity and are sufficiently
volatile for extracts to be concentrated at room temperature. There are many modifications of this
method, depending on the size of the moths, on whether or not the site of pheromone production is
known, and on others factors.
There are many variants of the entrainment technique (Golub and Weatherston, 1984;
Heath et al., 1989; Borg-Karlson, 1990), but despite many differences among the entrainment
techniques, the basic principle of volatile collection is the same. The compounds are adsorbed, via
an air stream (dynamic headspace) or passively (static headspace), on a porous polymer, such as
Porapak Q, Tenax GC, or active charcoal. After collection, the volatiles are desorbed from the
polymers by heat or by rinsing the polymer with an organic solvent (Borg-Karlson, 1990).
However, one important consideration should be taken into account. Part of the compounds
found in the sex pheromone gland may not be released into the air. Examples are the species
Adoxophyes orana, Cacoecimorpha pronubrana, and Choristoneura parallela of the family
Tortricidae as well as the species Chrysodeixis chalcites, Pseudoplusia includens, Tichoplusia
ni, Spodoptera exigua, and Agrotis ipsilon of the family Noctuidae (based on Arn et al., 1999).
The ratios of the components released into the air may differ from the ratios found in the gland, as
for the pyralid Eoreuma loftini. In some cases the actual pheromone may not be stored in the
gland but be produced only just before release. This process has been found to occur when the
pheromone compounds are aldehydes, as in some Noctuidae species: Heliothis virescens and H.
zea (Teal et al., 1986; Tumlinson, 1988), Sesamia nonagrioides and Mamestra suasa as well as
in the Tortricidae species Choristoneura fumiferana, Ch. occidentalis and Croesia curvalana
(based on Arn et al., 1999). The methods of collecting the emitted volatiles from the air may then
become very important, but factors like the need for long periods of entrainment, the possible
discrimination of compounds of low volatility, and the large amount of solvent needed limit the range
9
of detectable volatiles, specially from weakly scented organisms, as well as the
possibility to study short time changes in the proportions of the volatiles (Borg-Karlson,
1990).
The Solid Phase Micro Extraction (SPME) technique (Fig. 3.1) had already been
developed and made commercially available (Zhang and Pawliszyn, 1993) at the time
when we started our experiments. Volatiles, collected by this method on the SPME fibre,
are desorbed by heat in a GC injector. This makes the collection procedure simple, short
and efficient, because the desorption step of the volatiles trapped does not require the use
of a solvent (Fig. 3.2). A number of applications of this method had been published,
dealing with environmental research, volatiles in food, fungi, bioremediation and
beverages as well as with water contaminants (Pelusio et al., 1994; Zhang and Pawliszyn,
1995; Pawliszyn, 1997; Eriksson et al., 1998 and others).
We proved that the SPME technique was highly efficient for the collection of sex
pheromones of even quite small moths, such as phyllonoryctids. Three components of the
sex pheromone from one calling Ph. acerifoliella (=Ph. sylvella) female were collected
during three hours and identified in this species for the first time (Paper V). The amount
of volatiles released by one calling female during three hours and collected on a
polydimethylsiloxane fibre, was as large as the amount extracted from the sex pheromone
glands of about 20 females (Paper IV). It was also shown, that the SPME technique
makes it possible to investigate individual variations of pheromone compositions and we
believe that differences in the proportions of the sex pheromone components may be
determined among different geographical or host-plant races, among populations and on
other conspecific levels. The collection of volatiles from the same female without
disturbing the calling makes it possible to collect the pheromone during the entire
signalling period of a female, which may last for several days.
B
A
Plunger
Barrel
Hub-viewing window
Colour-coded screw hub
Retaining Screw
Tensioning spring
Retaining nut
Ferrule
Colour -coded screw hub-viewing window
Adjustable depth gauge
B
Needle guide
Septum piercing needle
Fibre attachment needle
Coated SPME fused silica fibre
Fig. 3.1. Solid Phase Micro Extraction fibre holder installed with fibre assembly. ( A) general view,
(B) profile.
We have demonstrated another advantage of the SPME technique in determining the sex
pheromone composition of Bonagota cranaodes. Only one compound, 3E,5Z-12:OAc,
A
Adsorption via
air stream
Volatiles
Impurities
Desorption of collected
volatiles with solvent
Impurities from
large amount of
solvent
Partial loss of
compounds
Concentration
of the sample
Sampling
for analysis
Partial loss of
compounds
Partial loss of
compounds
B
Adsorption
Volatiles
Impurities
Desorption of collected volatiles by
heat in GC injector
Partial loss of
compounds
Fig. 3.2. Comparison of principal steps in the entrainment collection. (A) common method, (B)
Solid Phase Micro Extraction method.
was obtained from a calling virgin Bonagota cranaodes female by the SPME technique, while six
additional acetates were obtained by extraction of the sex pheromone glands of the females (Eiras
et al., 1999). Experiments with synthetic acetates in ratios and amounts close to those found in the
extract demonstrated that the amounts of all of these compounds were above the detection limits of
the SPME technique (unpublished data). This proved that a combination of the SPME technique
11
with the extraction method makes it possible to distinguish between the compounds actually
released by the female into the air and those occurring only in the sex pheromone gland.
The SPME technique enables one to analyse volatiles collected exclusively from calling
females without preceeding GC-EAG analyses. The chromatographic comparison of the volatiles
collected from females during their sex pheromone releasing and non-releasing periods allows us to
determine the chemical structures of those compounds, which are released only by the calling
female. Moreover, allelochemicals released by a female to ensure the specificity of her sex
pheromone blend can usually not be detected by EAG analysis using the antennae of conspecific
males.
We have used the SPME technique to collect the sex pheromones from the females of
species which differ with respect to the type of the sex pheromone release posture as well as the
timing of their calling. The SPME technique was easiest to apply to the females of those species
which stay stationary during the calling, bend their abdomen dorsally and signal during the light
period (all species of the genus Phyllonorycter that we have investigated, the family Gracillariidae
(Papers I, II, IV, V and VII) as well as females of the species Sesia apiformis, Synanthedon
scoliaeformis, and S. tipuliformis belonging to the family Sesiidae; unpublished data). It was much
more difficult to place the SPME fibre close to the abdominal tip of the calling Bonagota
cranaodes females (family Tortricidae), because they bent their abdomen ventrally and released
their pheromone at night (Eiras et al., 1999). Under the red light, which did not disturb the calling
females, the SPME fibre was practically invisible.
The SPME technique is so rapid and easy to use, that despite its disadvantages, it will no
doubt enhance the collection of sex pheromones from calling insects.
Determination of the composition of the pheromone and of the chemical structures of
its components. In determining the composition of a pheromone it is important to separate its
constituents. Gas chromatography (GC) with various capillary columns, depending on the nature of
12
the compounds to be analysed, is normally used for this purpose. The chemical structures of the
pheromone components are determined by comparing their retention times with those of synthetic
standards on at least two types of columns. In our analyses we used polar and unpolar fused silica
capillary columns (Papers I, II, IV, V and VII). It was found to be rather difficult to separate
some of the double bond position isomers of dodecenyl and tetradecenyl acetates, and for these
purposes we had to optimize the temperature programmes of the GC runs.
Mass spectrometry combined with gas chromatography is another widely used method to
determine the chemical structures of compounds. The molecular fragmentation pattern of the
compound analysed gives possibilities to determine the functional groups, the degree of unsaturation
and, for some compounds, double bond positions, molecular weights as well as other structural
features of the substance. A large library of EI spectra of compounds allows quick preliminary
comparisons of the spectrum obtained with those stored in the computer memory.
Besides pheromone components, many non-pheromone compounds are usually present in
the sample to be analysed. It is necessary to know, which compounds will elicit behaviour
responses, i.e. which of them are true pheromone components and should be studied further. A
helpful tool for solving this problem is a gas chromatograph with a capillary column connected with
an electroantennographic detector. This instrument gives simultaneous records of sample analyses
by flame ionization and electroantennographic responses, i.e. it shows which of the compounds in
the pheromone mixture elicited a signal in the insect antenna.
Synthesis and purification of the chemicals identified (unless they are commercially
available). The compounds that we used in the field tests were all commercially available. The
receptors, by which a moth perceives a pheromone, are specific and sensitive. A few percent or
even parts of a percent of impurities in a synthetic pheromone blend may decrease or totally
obliterate the attraction of males. For this reason, it is important to use very pure compounds for the
biological tests. The medium presser liquid chromatography (MPLC) was used to purify some
13
synthetic compounds. For more details, see Paper III .
Evaluation of the biological activity of the synthetic pheromone components. The
most common bioassay for evaluating the biological activity of synthetic pheromone components is
the field-trapping test. Sticky traps, containing dispensers impregnated with mixtures of pheromone
components in different ratios, are placed in the habitat area of the species, for which the lures may
be attractive. It is important to develop formulations that will release the synthetic compounds at the
same rate and in the same ratio as the natural pheromone is emitted by the females and to prevent
any isomerization or degradation of the pheromone components. To study the behaviour stages of
the male sex attraction, elicited by various pheromone components, a wind-tunnel bioassay is
usually arranged for flying insects (Baker and Linn, 1984).
5. CHEMICAL COMMUNICATION IN THE GENUS PHYLLONORYCTER
5.1. Calling posture
The pheromone release behaviour in most moth species consists of a few stages, one of
which is usually characterised by the distinctive body position of the female, which is known as the
calling or pheromone release posture. The calling postures vary among lepidopterans in that they
hold their abdomen, wings, antennae, legs and other parts of the body differently.
Females of all those species of the genus Phyllonorycter, which we investigated, clearly
preferred to stay on the underside of the host-plant leaves, where they remained stationary while
releasing their pheromone. The most typical elements of their calling posture were the following:
wings slightly spread and lowered; abdomen curved dorsally and distal abdominal segments
telescopically protruded; antennae not raised, but placed along the wings (Paper VI , Fig. 1, a).
The intermediate postures (Paper VI, Fig. 1, b, c) between the calling and the resting one
14
(Paper VI, Fig. 1, d) were also noted. These intermediate postures differed from the calling one in
that the abdomen was less curved and the distal segments were not protruded. Thus, the
pheromone gland was probably not exposed and pheromone was not actively released.
The calling postures of phyllonoryctid females are quite similar to those of the pyralids
Ephestia kuhniella (=Anagasta kuhniella) (Traynier, 1968) and Plodia interpunctella (Brady
and Smithwich, 1968). The most pronounced difference is found in the position of the antennae.
The antennae of calling pyralids are raised (Traynier, 1968, Barrer and Hill, 1977), whereas those
of phyllonoryctids are placed along the wings.
5.2. Daily rhythm
Female moths release their pheromones under certain favourable conditions and the males
that perceive the pheromones have their greatest tendency to respond when exposed to the same
conditions. The great majority of moths release their sex pheromones and copulate during the dark
period (see reviews: Dreisig, 1986, and McNeil, 1991, and papers: Kou, 1992; Kamimura and
Tatsuki, 1993; West and Bowers, 1994; Kinjo and Arakaki, 1997 and others). Only a few of
those moth species which use distant chemical communication call during the light period. Most of
these are active in the second half of the day and their activity ceases in the dusk (Sanders, 1971;
Sanders and Lucuik, 1972; Baker and Cardé, 1979; Solomon, 1979; Greenfield and Karandinos,
1979; Buda and Karalius, 1985; Dreisig, 1986; Schal and Cardé, 1986, and others) and one
species, Lymantria dispar, is active during the entire light period (Charlton and Cardé, 1982). A
calling activity in the morning is found in only a few species (Gentry et al., 1977; Hendriksen, 1979;
Solomon, 1979; Greenfield and Karandinos, 1979; Webster and Conner, 1986; Dreisig, 1986;
Tóth et al., 1995). Our data on the daily calling periodicity indicate that the species of the genus
Phyllonorycter belong to the latter group. In all species that we have investigated the peak of the
15
pheromone release behaviour was registered 0.5-1 hour after the beginning of the photophase and a
high level of the activity was maintained for about 2-3 hours (Papers II, IV-VII and Fig. 5.1).
An interesting pattern of the female diurnal pheromone release behaviour was found in Ph.
junoniella. When the females were held under the constant temperature of 20 oC , only one peak of
calling activity was registered at the beginning of the photophase. Under the cycling thermal regime
(14 oC in the scotophase and 20 oC in the photophase), a second peak of calling activity was registered
at the end of the photophase in addition to the pheromone release peak occurring at the beginning of the
photophase. We have not found any publication, reporting that a lepidopteran female pheromone
release behaviour consists of two calling peaks, occurring on the same day under conditions close
to the natural ones. Moreover, the sex ratio in this phyllonoryctid was quite different from 1:1, the
common ratio in many other species. It was strongly shifted towards females, the ratio being 8:1
(females to males). In our opinion the occurrence of the second peak of calling activity could be
adaptive for species with a sex ratio strongly shifted towards females, as in Ph. junoniella. If the
refractory period between two copulations in males is shorter than 24 h (or 8 h in Ph. junoniella),
calling virgin females can be fecundated during both the morning and the evening periods of their
activity. This could compensate significantly for a small proportion of males in a population (Paper
VI).
An even more unexpected finding was the discovery of a calling behaviour in the
parthenogenetically reproducing Ph. emberizaepenella species. According to Gilyarov (1982)
16
and Went (1984) the parthenogenetic reproduction in animals has a number of drawbacks in
comparison with the usual amphimixis, but it also has some advantages. One of these is the
reduction of the complex processes of finding a sexual partner, since with the move towards
parthenogenesis (particularly in thelytoky) these processes are not necessary. We established, that
despite a complete lack of males, parthenogenetically reproducing females demonstrated calling
postures in which sex pheromone was released with the diurnal rhythm of such behaviour (Paper
VII). Both the behavioural and the chemical characteristics of this process were similar to those
known for Lepidoptera with the usual amphimictic mode of reproduction. Theoretical speculations
that in thelytoky, where there is no need to attract a sexual partner, the individuals obtain certain
advantages due to reduction in their sexual behaviour, was not confirmed for Ph. emberizaepenella.
5.3. Sex attractants
About 150 species of the genus Phyllonorycter are known in the European part of the
Palaearctic region (Medvedev, 1981) and over 1000 species have been found altogether
(Kuznetzov, 1979). In spite of the abundance of species in the genus Phyllonorycter, only 14 or
15 compounds, acting as sex attractants, alone or in mixtures, have so far been known for only 13
phyllonoryctid species. All of those substances, except Z8-14:OH, E11-16:Al and Z11-16:Al, are
aliphatic C12-C14 straight chain mono- or di-unsaturated acetates with Z- or E- double bonds
occurring in positions 4, 7, 8, 10 or 11. E10-12:OAc and Z10-14:OAc are the most frequently
occurring sex attractant components among phyllonoryctids, each of them being found in at least 4
species (see Table 5.1).
In the spring of 1994, about 40 monounsaturated dodecenyl and tetradecenyl acetates and
a few corresponding alcohols were tested in tube olfactometers as possible components of sex
attractants to males of Ph. blancardella , Ph. sorbi, Ph. dubitella and Ph. strigulatella , which are
the most abundant phyllonoryctids in Lithuania. On the basis of the results obtained (Paper III), we
selected only E10-12:OAc for tests on Ph. blancardella (the corresponding alcohol had not
been tested in the olfactometer); E10-12:OH, the
Table 5.1. List of the sex attractants determined for males of the genus Phyllonorycter.
18
Species
Compound
Ph. acerifoliella
E9-14:OAc
Z10-14:OAc
Ph. blancardella
E10-12:OAc
Roelofs et al., 1977
E10-12:OAc
E4,E10-12:OAc
Voerman & Herrebout, 1978
Gries et al., 1993
Ph. coryli
Ph. corylifoliella
Ph. cydoniella
E9-14:OAc**
Z10-14:OAc**
E4,Z7-13:OAc
E4-12:OAc
E10-12:OAc
Ratio
Reference
10
1
Paper III
10
1
Paper III
Voerman & Herrebout, 1978
Voerman, 1991
Paper III
Ph. elmaella
Ph. heegerella
E10-12:OAc
E9-14:OAc**
Z10-14:OAc**
Ph. junoniella
Z10-12:OAc
Ph. kleemannella
Z10-14:OAc
E11-14:OAc
Ph. mespilella
Ph. orientalis
E10-12:OAc
Z10-14:OAc
Hrdy et al., 1989
Ando et al., 1977
Ph. oxyacanthae
Ph. pulchrum
Ph. pygmae
Ph. pyrifoliella
Ph. ringoniella
E10-12:OAc
E8,E10-14:OAc
Z8-14:OH
E10-12:OAc
Z10-14:OAc
E4,Z10-14:OAc
10
1
Paper III
Ando et al., 1987
Ando et al., 1977
Laanmaa et al., 1991
Sugie et al., 1986
E11-16:Al
Z11-16:Al
7
3
Su & Liu, 1992
Z10-14:OAc
E4,Z10-14:OAc
6
4
Boo & Jung, 1998
Ph. saportella
E8-14:OAc
E10-14:OAc
1
1
Voerman, 1988
Ph. sorbi
E10-12:OH
Paper III
Ph. ulmifoliella
Z10-14:OAc
Voerman, 1988
Ph. watanabei
Z10-13:OAc
Ando et al., 1977
10
1
Shearer & Riedl, 1994
Paper III
Paper III
*
Booij & Voerman, 1984
* the ratio was not presented; ** potential sex attractant; bold text indicates the present
investigations.
19
corresponding acetate and three binary mixtures of these compounds for tests on Ph. sorbi; E914:OAc, Z10-14:OAc and three binary mixtures of these substances for tests on Ph. dubitella as
well as some additional compounds for field tests. For Ph. strigulatella males some other
compounds should, probably, be added to Z10-14:OAc to elicit wing fanning, the highest type of
behaviour, but we did not prepare an appropriate mixture for this species during the field test.
In the field screening tests in 1993 and 1994, twentyone saturated and monounsaturated
straight chain C 12 and C 14 alcohols and acetates and some binary mixtures of them were
investigated in dosages of 1 and 0.2 mg/dispenser (Paper III). New sex attractants were
discovered for six moth species of the genus Phyllonorycter (Gracillariidae) as well as for five
species of the family Tortricidae, two species of Gelechiidae and one species of each of the families
Yponomeutidae and Oecophoridae.
E10-12:OH, alone and in a binary mixture with the corresponding acetate in the ratio 10:1,
was significantly more attractive than all of the other lures tested on males of the species Ph. sorbi
in all localities (Paper III , Tables 2 and 3). Twentynine and 37 males of Ph. cydoniella were
attracted to the traps containing E10-12:OAc in the localities I and II, respectively, (Paper III ,
Table 2) and 20 males were trapped in locality IV in 1994 (Paper III, Table 3). For the males of
Ph. oxyacanthae, E10-12:OAc alone or in a 10:1 mixture with E10-12:OH showed the highest
attraction (Paper III , Tables 2 and 3). Z10-14:OAc and E9-14:OAc in the ratio 1:10 was the
most attractive blend for the males of Ph. acerifoliella as well as for the males of Ph. heegerella
and in the locality II for the males of Ph. coryli (Paper III , Table 2). Males of the species Ph.
dubitella were captured in the traps baited with various compounds, their binary mixtures and the
control as well. No catches differed significantly from that of the control in locality II and only the
number of the males trapped by a 1:10 mixture of Z10-14:OAc and E9-14:OAc differed
significantly from that of the control in locality III (Paper III, Table 2). Due to the small number of
males captured, we consider the data on the attraction of this phyllonoryctid species as preliminary.
20
For the males of Ph. junoniella , Z10-12:OAc was the best attractant in locality I in 1993 (Paper
III , Table 2) and in locality IV in 1994 (Paper III , Table 3). Males of Ph. strigulatella were
attracted by various lures as well as by the control, but the catches did not differ significantly from
the 0 level.
According both to our results and to literature data, two groups of Phyllonorycter species
could be distinguished according to their sex attractants. The first group includes the species Ph.
blancardella, Ph. cydoniella , Ph. elmaella, Ph. mespilella, Ph. oxyacanthae, Ph. pyrifoliella
and Ph. sorbi, the males of which are attracted to E10-12:OAc or the corresponding alcohol
(Table 5.1 ). We point out that E10-12:OH is the most attractive compound for Ph. sorbi males
but acts as a sex attraction antagonist for Ph. mespilella and Ph. cydoniella , providing one of the
potential isolation barriers in cross-attraction between Ph. sorbi and the two species last
mentioned.
The second group includes Ph. kleemannella, Ph. orientalis, Ph. ringoniella, Ph.
acerifoliella, Ph. ulmifoliella and Ph. heegerella , the males of which are attracted to Z1014:OAc alone or in blends with other componds (Table 5.1). The question of how the specificity of
the pheromone signal is achieved between leaf miner moths of the Ph. acerifoliella , Ph.
ulmifoliella and Ph. heegerella species will be discussed later, in the subchapter 5.6: Ecological
aspects.
It remains to be analysed whether or not the two groups belong to different taxonomic ranges
within the genus.
5.4. Sex pheromones
21
According to literature data, sex pheromones have been known for only three species of the
genus Phyllonorycter: Ph. crataegella, Ph. mespilella and Ph. ringoniella . In all these species,
the sex pheromones consist of two components (Table 5.2 ).
Table 5.2. List of the sex pheromone components and other compounds released by calling virgin
females of the genus Phyllonorycter.
Ratio
Species
Compound
Ph. acerifoliella
Z10-14:OAc
Z8-14:OAc
E10-14:OAc
Ph. blancardella
E10-12:OAc
12:OAc
E10-12:OH
Z10,Z12-14:OAc
E10,E12-14:OAc
Ph. crataegella
Reference
Extract
SPME
90
8
2
92
6
2
Paper V
79
8
13
10
0.1-1
80
8
12
Paper IV
Ferrao et al., 1998
Ph. emberizaepenella
E8,E10-14:OAc
E8,E10-14:OH
>99
<1
Paper VII
Ph. heegerella
Z8-14:OAc
14:OAc
Z8-14:OH
E10-12:OAc
E4,E10-12:OAc
88
2
10
Paper V
Trace
Ph. ringoniella
E4,Z10-14:OAc
Z10-14:OAc
6
4
Ph. ulmifoliella
Z10-14:OAc
Ph. mespilella
Gries et al., 1993
Boo & Jung, 1996
Paper II
Bold text indicates present investigations
The compounds extracted from the sex pheromone glands of Ph. crataegella females were
not detectable by GC with a flame ionization detector but were identified by coupled GC-EAD
analyses, involving retention index calculations of EAD-active compounds and comparative GCEAD analyses of female-produced and synthetic compounds. In field experiments, Z10,Z1214:OAc alone attracted conspecific males. Addition of E10,E12-14:OAc to Z10,Z12-14:OAc in
ratios from 0,1:10 to 1:10 significantly enhanced the attractivity of the lure (Ferrao et al., 1998).
22
The proportion of E4,E10-12:OAc in the sex pheromone gland extract of Ph. mespilella
females was below the detection threshold of GC with an FID detector but was determined by the
GC-EAG method. However, this pheromone component was two to four times more attractive
than E10-12:OAc under field conditions and no additive or synergistic effect between the two
components was detected (Gries et al., 1993).
Boo and Jung (1998) demonstrated that the optimum ratio of E4,Z10-14:OAc and Z1014:OAc for attraction of Ph. ringoniella males in Korea was 6:4, which differed from the ratios
previously reported in Japan (2:8) and China (3:7 to 4:6).
We determined the sex pheromone composition for 5 phyllonoryctid species, Ph.
acerifoliella, Ph. blancardella, Ph. emberizaepenella, Ph. heegerella and Ph. ulmifoliella.
Three compounds, Z10-, Z8- and E10-14:OAc (Fig. 5.2) in the ratio (92±1):(6±1):(2±0.3)
were collected by means of the SPME technique from single calling virgin Ph. acerifoliella females
as well as extracted from the sex pheromone gland of the females in the similar ratio 90:8:2 (Paper
I). The first two compounds were found to be essential for the attraction of conspecific males and
are sex pheromone components of this species. When tested separately, Z10- and Z8-14:OAc did
not attract Ph. acerifoliella males. Thus, to be active these acetates must be present together in
ratios close to 10:1. The role of the third compound, E10-14:OAc, released by the female remains
undetermined (Paper V).
Our finding that Ph. acerifoliella males were attracted to a binary mixture of Z10-14:OAc
and E9-14:OAc in the ratio 1:10 ( Paper III, Table 2) might be explained by E9-14:OAc acting as
a mimic of Z8-14:OAc. Another explanatio n could be, that E9-14:OAc, when applied at a dosage
of mg/lure, contained a small amount of Z8-14:OAc, an essential
23
A
B
O
O
O
O
12:OAc
14:OAc
C
D
OH
O
O
E10-12:OH
E10-12:OAc
E
F
OH
O
O
Z8-14:OH
Z8-14:OAc
G
H
O
O
O
O
Z10-14:OAc
E10-14:OAc
I
J
OH
O
O
8E,10E-14:OH
8E,10E-14:OAc
Fig. 5.2. Chemical structures of the compounds released by virgin calling females of five
Phyllonorycter species and identified by us.
Ph. acerifoliella females released the compounds F, G and H; Ph. blancardella A, C and D; Ph.
emberizaepenella I and J; Ph. heegerella B, E and F; Ph. ulmifoliella compound G.
component for the sex attraction of Ph. acerifoliella males. 100 ng of E9-14:O Ac had been used
to determine the chemical purity of this acetate, but Z8-14:OAc could have been present in a
proportion below the GC detection limits.
Another three compounds, E10-12:OAc, 12:OAc and E10-12:OH (Fig. 5.2), in the ratio
(80±3):(8±3):(12±1) were identified in the volatiles collected by means of the SPME technique
from single virgin females of the spotted tentiform leaf miner moth, Ph. blancardella. The same
24
compounds in the ratio 79:8:13 were extracted from the sex pheromone glands of calling females.
Trapping results obtained in the field tests demonstrated that only one compound, E10-12:OAc,
was essential for the attraction of conspecific males and should be considered as a sex pheromone
(Paper IV). According to the preliminary data, the other two compounds, 12:OAc and E1012:OH might be responsible for the specificity of the pheromonal signal of Ph. blancardella .
Z8-14:OAc, 14:OAc and Z8-14:OH (Fig. 5.2) in the ratio (88±3):(2±0.6):(10±5) were
collected, using the SPME technique, from single calling virgin Ph. heegerella females. Field
trapping experiments revealed, that the first two compounds are important for the attraction of
conspecific males and are sex pheromone components of this phyllonoryctid species. Z8-14:OAc
was found to be attractive when tested separately, while 14:OAc acted as a synergist. The role of
the third compound, Z8-14:OH, released by the females remains undetermined ( Paper V ).
Two compounds were collected from parthenogenetically reproducing calling Ph.
emberizaepenella females by means of the SPME technique and were identified as E8,E1014:OAc and E8,E10-14:OH (Fig. 5.2). The second compound occurred at the trace level (Paper
VII). The chemical structures of the compounds identified corresponded to the general pattern of
those known as sex pheromones and sex attractants from other Phyllonorycter species. The
attractivity tests of the compounds were not carried out because this species reproduces by
parthenogenesis of the thelytoky type, where all ancestry and progeny are female.
Z10-14:OAc (Fig. 5.2) was extracted from the sex pheromone glands of virgin Ph.
ulmifoliella females and the biological activity of this acetate as a sex pheromone was confirmed in
field tests ( Paper II).
All compounds released by calling females of the five phyllonoryctid species, except E1012:OAc and Z10-14:OAc, are new in the family Gracillariidae. Moreover, E10-12:OH, E8,E1014:OAc and E8,E10-14:OH have not been identified earlier from any species of the order
Lepidoptera. Thus, our identification extends the number of taxons, in which these compounds are
released by calling moth females.
25
5.5. Antagonists of sex attraction
Our field trapping experiments not only showed the sex attraction of Phyllonorycter males
exerted by some of the compounds tested, but also revealed thirteen sex attraction antagonists for
males of seven Phyllonorycter species ( Table 5.3).
Table 5.3 List of the sex attraction antagonists determined for males of the genus
Phyllonorycter.
Species
Compound
Reference
Ph. acerifoliella
Ph. crataegella
Ph. cydoniella
Ph. heegerella
Ph. mespilella
Ph. oxyacanthae
Ph. sorbi
E10-12:OH
E10,Z12-14:OAc*
E10-12:OH
E10-12:OAc; Z10-14:OAc
Z10-, E10-12:OH, E10-14:OH
Z10,Z12-14:OAc
Z10-12:OH
Z7-, Z9-, Z10-12:OAc
Paper V
Ferrao et al., 1998
Paper III
Paper V
Paper III,
Ferrao et al., 1998
Paper III
Paper III
Ph. ulmifoliella
Z9-, E9-, E10-*, E11-14:OAc
Paper III
Z8-14:OAc
Paper V
* weak or potential sex attraction antagonist; bold text indicates present investigations.
All of the sex attraction antagonists that we have found are hereby reported for the first time
on the level of the family.
The molecular structures of sex attraction antagonists (Fig. 5.3 ) are usually similar to those of
sex pheromones (Fig. 5.2) and sex attractants. Sex attraction antagonists significantly
26
decrease or even obliterate the attraction of males when applied in the proportion of 0.1-10% of
the sex pheromones or sex attractants. The role of a sex attraction antagonist is to contribute to the
specificity of a pheromone signal.
The data that we have obtained about the chemical communication systems of three
phyllonoryctid species clearly demonstrate the role of sex attraction antagonists in the achievement of
species-specific sex pheromone signals. Z8-14:OAc was identified as a sex pheromone component
both for Ph. acerifoliella and Ph. heegerella. However, the attraction of Ph. heegerella males to
Ph. acerifoliella females was reduced by Z10-14:OAc, which is present in the sex pheromone of
Ph. acerifoliella (Paper V). Z10-14:OAc was found to be a sex pheromone component both in
Ph. acerifoliella and Ph. ulmifoliella. The attraction of Ph. ulmifoliella males to Ph. acerifoliella
females was reduced by the Z8-14:OAc released by the females of Ph. acerifoliella. For attraction
of Ph. acerifoliella males, Z8-14:OAc and Z10-14:OAc must be present together in the
appropriate ratio (10:1) and because Ph. heegerella and Ph. ulmifoliella females do not release a
mixture of Z8-14:OAc and Z10-14:OAc (essential components in Ph. acerifoliella females), neither
Ph. ulmifoliella nor Ph. heegerella females were attractive for Ph. acerifoliella males.
5.6. Ecological aspects
Interaction by allelochemicals can occur (i) when individuals of one species perceive and react to
compounds emitted by organisms of another species, (ii) when the species are dispersed within
distances allowing communication, and (iii) when the periods of communication activity of the
species overlap.
E10-12:OAc, 12:OAc and E10-12:OH were identified as sex pheromone components of
Ph. blancardella (Paper IV), Z10-14:OAc was identified as a sex pheromone component
of Ph . ulmifoliella (Paper II), E4,E10-12:OAc and E10-12:OAc were found to be sex
28
pheromone components of Ph. mespilella (Gries et al., 1993) and Z8-, Z10- and E10-14:OAc
were identified as sex pheromone components of Ph. acerifoliella (Papers I, II ). In addition,
E10-12:OH was determined as a sex attractant for Ph. sorbi (Paper III). Almost all of the
compounds that we have found to be sex attraction antagonists affecting the species
Ph. ulmifoliella, Ph. sorbi, Ph. mespilella and Ph. acerifoliella (Table 5.3), have been known
earlier as components of sex pheromones or attractants in lepidopteran species belonging to other
families. On the basis of available data on moth sex pheromones, sex attractants and their inhibitors,
we present schemes of probable interactions by means of allelochemicals acting between
Ph. blancardella and 7 other moth species (Fig. 5.4, A), Ph. ulmifoliella and 414 (Fig. 5.4, B),
Ph. sorbi and 243 (Fig. 5.4, C), Ph. mespilella and 11 ( Fig. 5.4, D ) and Ph. acerifoliella and 7
other moth species (Fig. 5.4, E).
The host-plants of the tentiform leaf miner species Ph. blancardella and Ph. mespilella
(Malus sp.), Ph. ulmifoliella (species of the genus Betula), Ph. sorbi (Sorbus aucuparia and
Padus avium ) and Ph. acerifoliella (Acer platanoides) are common and widespread in the
temperate climatic zone in extremely varying habitats. Thus, the phyllonoryctids are found at
distances short enough to allow interactions by allelochemicals with other moth species in case their
activity periods overlap.
However, the crucial question, to what extent communication periods overlap so as to allow
these interactions, remains to be answered, because, unfortunately, data on pheromone
communication periodicity are as yet available for a limited number of species only. It is known, that
females of most moth species, for which pheromone release periods have been determined, call at
night. Our investigation of the daily calling activity of six tentiform leaf miner species showed, that
females of all of the species examined, including Phyllonorycter ulmifoliella (Paper II), Ph. sorbi
(Fig. 5.1, B), Ph. acerifoliella (Paper V) and, through preliminary results, Ph. blancardella,
released sex pheromones during the light period of the day, early in the morning.
29
A
0 (0)
E 10
:OH
-12
1
1
1
3
E 10
Gracillariidae
Tortricidae
Pyralidae
Total
12:OAc
31
-12
:OA
c
Phyllonorycter blancardella
Gracillariidae 3
Tortricidae
1
Total
4
Fig. 5.4. Possible interactions by allelochemicals between (A) Phyllonorycter blancardella, (B) Ph. ulmifoliella, (C) Ph.
sorbi, (D) Ph. mespilella and (E) Ph. acerifoliella.
Each arrow indicates, that a chemical functions as a sex pheromo ne in one species and as a sex attraction antagonist in
another species; the arrow points towards the species perceiving the chemical. The number following the name of the family
indicates the number of species within that family, for which the compound is known to be a sex pheromone component; the
number of species, for which the compound is known as a sex attractant (i.e. potentially can be a pheromone constituent), is
indicated in parenthesis.
B
-14
:OA
c
Z9
Ac
:O
14
1E1
Ac
4:O
0 -1
E1
Gracillariidae 2 (0) Tineidae
0 (1)
Tortricidae
8 (4) Gracillariidae
0 (1)
10 (4) Oecophoridae
0 (2)
Total
Gelechiidae
0 (2)
Yponomeutidae 0 (1)
Plutellidae
0 (1)
Argyresthiidae 0 (1)
Tortricidae
20 (13)
Zygenidae
0 (5)
Pyralidae
13 (5)
Noctuidae
39 (110)
Total
72 (142)
c
Z10 -14:OA
32
Z
Ac
O
:
14
8
E9-14:OA
c
Phyllonorycter ulmifoliella
Fig. 5.4. (Continued).
Gracillariidae 0 (1) Gracillariidae 1 Gracillariidae 1 (1) Gracillariidae
0 (2)
Gelechiidae
0 (2) Tortricidae
Tortricidae
3
(5)
Ethmiidae
0 (2)
1
Tortricidae
1 (2) Total
1 (2)
4 (6) Oecophoridae
2 Total
Pyralidae
2 (3)
Cosmopterigidae 0 (1)
Noctuidae
1 (2)
Gelechiidae
0 (3)
Yponomeutidae 6 (2)
Total
4 (10)
Plutellidae
1 (0)
Tortricidae
56 (59)
Zygenidae
0 (2)
Pterophoridae
0 (4)
Pyralidae
4 (9)
Drepanidae
0 (1)
Noctuidae
3 (2)
Total
71 (89)
C
Fig. 5.4. (Continued).
-12:O
H
E 10
Gracillariidae 3
Tortricidae
1
Total
4
Cosmopterigidae 0 (1)
Yponomeutidae 1 (0)
Argyresthiidae
0 (1)
Tortricidae
20 (38)
Pyralidae
0 (3)
Noctuidae
11 (7)
Total
32(50)
Ac
2:O
0 -1
Z1
Oecophoridae
0 (2)
Xyloryctidae
0 (1)
Coleophoridae
0 (7)
Cosmopterigidae 0 (1)
Gelechiidae
0 (3)
Plutellidae
0 (3)
Argyresthiidae
0 (3)
Tortricidae
2 (9)
Zygenidae
0 (5)
Pterophoridae
0 (4)
Pyralidae
0 (3)
Geomertidae
0 (2)
Noctuidae
26 (78)
Total
28 (118)
c
Z9-12:OA
33
Z7
-12
:O
Ac
Phyllonorycter sorbi
Gracillariidae
0 (1)
Cosmopterigidae 0 (1)
Tortricidae
0 (9)
Total
0 (11)
D
Fig. 5.4. (Continued).
-12:
OH
12
:O
H
Z 10
E1
0-
0 (0)
Ac
4:O
2 -1
, Z1
Z10
0 (0)
OH
0 (0)
E10-14:
34
Gracillariidae 0 (2)
Tortricidae
0 (1)
Total
0 (3)
E4,E10
-12:OAc
Phyllonorycter mespilella
E1
012
:O
Ac
Gracillariidae 1 (0)
Tortricidae
3 (1)
Total
4 (1)
Gracillariidae
Tortricidae
Pyralidae
Total
1
1
1
3
E
Fig. 5.4. (Continued).
Phyllonorycter acerifoliella
Z8 -1
4:OA
c
E1
0-
c
OA
14:
Gracillariidae 1
Total
1
0Z1
OAc
-14:
Gracillariidae 1
Total
1
E 10
12
:O
H
35
Gracillariidae 0 (2)
Tortricidae
0 (1)
Total
0 (3)
Gracillariidae 1
Tortricidae
1
Total
2
Our hypothesis is, that this uncommon timing of the pheromone communication in
phyllonoryctids may have been caused by the sensitivity of the males to sex attraction antagonists,
which females of many other species emit into the environment at other times of the day. The
communication during the period free of sex attraction antagonists should be adaptive for the
species. Phyllonoryctids are small moths (length of the body 4-8 mm), and flying males are not as
easily detectable by birds as the males of the other lepidopteran species. We suppose, that this
feature allows tentiform leaf miners to use the light period of the day to find calling females, while the
males of bigger moth species have to search for females at night when birds are not active.
5.7. Evolutionary aspects
There is probably no animal group that has a more thoroughly studied pheromone
communication system than the Lepidoptera, and yet the evolution of these systems remains little
understood (Phelan, 1992; Löfstedt, 1993; Löfstedt and Kozlov, 1997). Likewise, there are just a
few reports on the occurrence of any pheromone compounds during evolution, even though there
are reports on other characters.
The families of Lepidoptera, representatives of which use the compounds known as sex
pheromones or sex attractants in gracillariids, belong to the taxonomic group Ditrysia. As was
mentioned in Chapter 3, the family Gracillariidae si one of the first lineages within the ditrysian
lepidopterans (Scoble, 1992).
During the evolution, a type of structure most probably evolved once in a common ancestor
of two closely related groups rather than turning up independently in both of them. As regards the
gracillariid sex pheromones and sex attractants, some of which may later on be identified as
pheromones, the ancestor first using such compounds should be common to the entire Ditrysia
group. Thus, one can conclude that the first appearance of these
35
compounds as sex pheromones most probably did not take place later than with the appearance of
Ditrysia. For terms and taxonomic relationships between the groups, see Fig. 5.5 .
MONOTRYSIA
DITRYSIA
HETERONEURA
LEPIDOPTERA
Fig. 5.5. Cladogram of Amphiesmenoptera insects, indicating the position of taxa discussed in the
text in relation to the evolution of gracillariid sex pheromones. Adapted from Kristensen, 1989;
Kristensen and Skalski, 1998.
Can these pheromones have evolved much earlier?
The chemical structures of the compounds used in the sex communication of monotrysian
moths of the families Eriocraniidae (Zhu et al., 1995 and Kozlov et al., 1996) and Nepticulidae
(Tóth et al., 1995) (Fig. 2.3) correspond more closely to those of sex pheromones and sex
attractants of Trichoptera species (Fig. 5.6), a sister order of Lepidoptera, than to the general
pattern of those known from ditrysian lepidoptera (Fig. 2.1, 2.2). It is evident, then, that the
pheromones used by gracillariids could not evolve earlier than Heteroneura.
Only one chemical identification of a potential sex pheromone is known in the family
Incurvariidae. It indicates that Lampronia capitella females produce a compound, Z11-14:OAc
36
(Löfstedt, 1990), which is similar to those occurring in the taxonomic group Ditrysia. Another
compound, Z7-14:Al was found to be attractive to the males of Antispila treitschkiella, a species
belonging to Heliozelidae, a sister family of Incurvariidae (Tóth et al., 1992). This aldehyde was
previously known as a sex pheromone component and sex attractant in the ditrysian families
Plutellidae and Noctuidae (Arn et al., 1999). If sex pheromones following the general pattern of
those known from ditrysian lepidoptera species should be found in other species of the families
belonging to the same taxonomic group as Incurvariidae and Heliozelidae, it would be evident that
pheromones used by gracillariids have appeared at a period lasting between the formation of lower
Heteroneura (Nepticulidae, Opostegidae) and the formation of higher or more advanced
Heteroneura (Heliozidae, Incurvariidae and four other families).
A
B
O
HO
C
D
O
HO
E
O
Fig. 5.6. Representative sex pheromone components identified from females of Trichoptera
species.
(A) heptan-2-ol, (B ) heptan-2-one and (C) nonan-2-ol found in Rhyacophila fasciata; (D) nonan2-one found in Rhyacophila fasciata and Hydropsyche angustipennis as well as (E) 6methylnonan-3-one found in Hesperophylax occidentalis (based on Löfstedt and Kozlov, 1997
and Bjostad et al., 1996).
6. CONCLUDING REMARKS AND DIRECTIONS FOR FUTURE WORK
37
Our investigations of the pheromone release rhythms of phyllonoryctids revealed that females
of these leaf miners call at the beginning of the photophase. Ph. junoniella females showed a
different periodicity of the female pheromone release behaviour than the other phyllonoryctids that
we have investigated. A second (evening) calling peak occurred in addition to the usual morning one
and we concluded that this second peak could be adaptive for this gracillariid, since the sex ratio in
this species is shifted towards females. In future work we would like to test our conclusion by
investigating the ability of males to fecundate virgin calling females during both of the peaks of
pheromone release activity.
Despite a complete lack of ma les, parthenogenetically reproducing females of the Ph.
emberizaepenella species demonstrated both the calling posture typical of other species of the
genus Phyllonorycter and the diurnal rhythm of that behaviour. Two components of a possible sex
pheromone collected from a calling female were identified. The nearest goal of the future work
would be to determine the sex ratio in the second (summer) generation more precisely. If males of
this species should not be found, the theoretical speculations that in thelytoky, where there is no need
to find a sexual partner, the females benefit by reducing their sexual behaviour, will not be confirmed
for Ph. emberizaepenella.
If the thelytoky type parthenogenesis could be found in Ph. emberizaepenella and the shift in
the sex ratio towards females in Ph. junoniella could be shown to be caused by cytoplasmic sex
ratio distorters, another goal of our future studies would be reached.
Basing both on our results and on published data, two groups of Phyllonorycter species
could be distinguished according to their sex pheromones and sex attractants. The direction for the
future work would be to reveal the mechanisms, by which the specificity of the pheromone signal is
achieved in species within these groups. We suppose that a determination of the compounds used in
the sex communication of other phyllonoryctid species would provide an additional character for
more precise grouping of the species within the genus. Our data demonstrated the advantages of the
38
SPME technique for the collection of compounds released by single females of microlepidopteran
species and SPME will help us to collect compounds used in the sex communication in
phyllonoryctids without extracting hundreds of leaf miners of different species. It will increase our
ability to investigate individual variations in the pheromone composition as well as the variation of
pheromone components among different geographic or host-plant-determined races, among
populations or on other conspecific levels.
Another direction for the future work could be to test the hypothesis, that the uncommon
timing of the pheromone communication in phyllonoryctids may have been caused by the sensitivity
of the males to sex attraction antagonists, which females of many other species emit into the
environment at other times of the day, because the communication during the period free of sex
attraction antagonists should be adaptive for the species. We hope to test the efficiency of sex
attraction antagonists when applied in separate dispensers placed at some distance from those
containing sex attractants.
39
ACKNOWLEDGEMENTS
I am glad to express my warm gratitude to my supervisor, Associate Professor Anna-Karin
Borg-Karlson, for excellent help in moth pheromone analysis and for her suggestions and comments
during writing. I also thank Dr G. Aulin-Erdtman for valuable discussions and improvements of my
manuscripts, through hours of attentive work. Special thanks go to Dr V. Buda, the head of the
Laboratory of Chemical Ecology at the Institute of Ecology, Vilnius, Lithuania. He started his
supervision when I took my first steps in chemical ecology and he still provides excellent scientific
advice. My thanks are also directed to everybody else at the Department of Chemistry, Organic
Chemistry (Stockholm), and at the Institute of Ecology (Vilnius), who helped me to complete my
work.
I sincerely thank Professor I. Neretnieks for providing financial support from the Fund for
Bilateral Cooperation between Sweden and the Baltic Countries (Royal Institute of Technology,
Stockholm). The financing support from the Swedish Institute, the Carl Trygger Foundation and
Mistra are gratefully acknowledged.
40
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50
9. MAIN TERMS REGARDING CHEMICAL COMPOUNDS USED IN INSECT
COMMUNICATION
A few systems of dividing semiochemicals have been suggested by various authors.
Below we present a scheme of terms categorized on the basis of information available to us and
short definitions of the main terms characterizing chemical compounds used in insect
communication.
Semiochemicals
Independent of communication level
Attractants
According to communication level
Allelochemicals
(interspecific)
Pheromones
(intraspecific)
Repellents
Kairomones
Arrestants
Primers
Releasers
Allomones
Sex
Aggregation
Invitation
Dispersal
Alarm
Trail
Territorial
Surface
Synomones
Deterrents
Apneumones
Stimulants
Funeral
Fig. 1. Categorization of main terms regarding chemical compounds used in insect
communication. (Based on Mohl, 1985 as well as on Ali and Morgan, 1990)
Any chemical that conveys information between organisms is termed a semiochemical
(Law and Regnier, 1971), after the Greek semion, which means signal.
Chemicals that modify the behaviour of animals can be categorized according to Mohl,
(1985) in terms of the type of behaviour they induce independently of the communication level.
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There are five such categories:
attractant, a chemical that causes insects to make movements orientated towards its
source,
repellent, a chemical that causes insects to make movements orientated away from its
source,
arrestant, a chemical that causes insects to aggregate into contact with it, the mechanism
of aggregation being kinetic,
deterrent, a chemical that inhibits feeding or oviposition when presented in a place
where, in its absence, insects would feed or oviposit,
stimulant, a chemical that elicits either feeding, mating or oviposition.
Another way is to divide semiochemicals into two subcategories, allelochemicals and
pheromones, according to the communication level and the type of behaviour that they induce.
Allelochemicals (the Greek allelon means each other) are a chemicals that convey
information between organisms of different species (i.e. interspecifically). Allelochemicals are
categorized according to the advantage of the behavioural response caused by them:
kairomone, a chemical that benefits the receiving individual, as with a predator locating
its prey by the prey odour,
allomone, a chemical that serves the odour-releasing individual, as with species using
secretions for defensive purposes,
synomone, a chemical that benefits both the releaser and the recipient, for example scents
that attract pollinating insects,
apneumone, a term applied to signals from non-living emitters.
Pheromones. The term is derived from the Greek pherein, transfer (carry), and horman,
excite. Pheromones are defined as substances which are secreted to the outside by an individual
and received by a second individual of the same species, in which they release a specific
53
reaction (Karlson and Butenandt, 1959; Karlson and Lücher, 1959). Wilson and Bossert (1963)
proposed the dividing of pheromones into two categories according to their modes of influence,
primer and releaser pheromones:
primer pheromone, which induces relatively long-lasting physiological changes in
receiving individuals, for example a chemical secreted by a queen-bee and inhibiting the
formation of other queens (caste inhibitors),
releaser pheromone, which stimulates the receiving individual to perform immediate
behaviour responses. The pheromones of this group mediate a wide variety of behaviours and
are classified according to the function or the behaviour that they elicit in the receiving insects
(based on Ali and Morgan, 1990) into:
sex pheromone, usually released by a female insect to advertise her presence, i.e. to lure
the male into successful mating. The chemical which facilitates courtship or prepares the
opposite sex for copulation is called an aphrodisiac and is usually released by males,
aggregation pheromone, a chemical compound which releases a behaviour response
leading to congregation, i.e. to an increase in the insect density in the vicinity of the source of
the pheromone,
invitation pheromone, attracts members of the species to a site for feeding or oviposition,
but, in contrast to aggregation pheromones, does not lead to the accumulation of individuals at
that site,
dispersal or spacing pheromone, one that stimulates behaviour leading to increasing
space between individuals (dispersion), with consequent reduction in intraspecific competition.
The compounds may be considered to act as deterrents or repellents. Oviposition deterring
pheromone might also be included in this category,
alarm pheromone, a compound producing accelerated movement (or rapid flight in
flying insects) and attack. Alarm and defensive behaviour often go side by side,
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trail pheromone, a chemical substance which is at first applied to a surface by one
individual and then detected and followed by other individuals. This type of a pheromone is best
known in ants,
territorial or home range pheromone, a chemical by which animals mark and recognize
their own territories (home range) for gathering or defence,
surface pheromone, is a substance that seems to be mainly adsorbed on the body surface
and perceived by direct contact or at most over a very short distance. Examples are recognition
pheromones (caste-recognition, brood recognition), releasers of grooming and courtship
behaviour as well as secretions that stimulate food exchange,
funeral pheromone, a compound produced by dead ants that stimulate a live ant to
remove a dead congener to a refuse pile outside the nest.
The system of grouping chemicals that modify behaviour into allelochemicals and
pheromones and of subdividing these categories is one that has stood the test of expanding
knowledge. Few primer pheromones are yet known, largely due to the lack of simple bioassays
by which they can be recognized for isolation, and the large time interval between receipt of the
pheromone message and production of the physiological effect. On the other hand, the short
response time and the availability of many good bioassays has led to the isolation and
identification of a vast number of releaser pheromones. The list of categories is clearly not
complete and as the behaviour knowledge grows, additions and subdivisions of the present list
are expected (Ali and Morgan, 1990).
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10. LIST OF THE MOTHS, CADDISFLIES AND PLANTS DISCUSSED IN THE TEXT
Moth species:
Gracillariidae:
Incurvariidae:
Phyllonorycter acerifoliella (Z.) = (Ph. sylvella (Hw.)) Lampronia capitella (Cl.)
Ph. blancardella (F.)
Ph. coryli (Nick.)
Heliozelidae:
Ph. corylifoliella (Hbn.)
Antispilla treitschkiella (F.v.R.)
Ph. crataegella (Cl.)
Ph. cydoniella (Den et Schiff.)
Psychidae:
Ph. dubitella (H.-S.)
Kotochalia junodi (Heyl.)
Ph. elmaella (Doganlar et Mutuura)
Thyridopteryx ephemeraeformis (Hw.)
Ph. heegerella (Z.)
Ph. junoniella (Z.)
Pyralidae:
Ph. kleemannella (F.)
Eoreuma loftini (Dyar.)
Ph. mespilella (Hbn.)
Ephestia kuhniella (Z.) = (Anagasta
Ph. orientalis (Kumata)
kuhniella Z.)
Ph. oxyacanthae (Frey)
Plodia interpunctella (Hb.)
Ph. populifoliella (Tr.)
Ph. pulchrum (Kumata)
Lymantriidae:
Ph. pygmaea (Kumata)
Lymantria dispar L.
Ph. pyrifoliella (Gram)
Ph. ringoniella (Matsumura)
Tortricidae:
Ph. saportella (Dup.)
Adoxophyes orana (F.v.R.)
Ph. sorbi (Frey)
Bonagota cranaodes (Meyr.)
Ph. strigulatella (Lien. et Z.)
Cacoeicimorpha pronubrana (Hb.)
(Ph. sylvella (Hw.)) = Ph. acerifoliella (Z.)
Choristoneura fumiferana (Clm.)
Ph. ulmifoliella (Hbn.)
Ch. occidentalis (Frm.)
Ph. watanabei (Kumata)
Ch. parallela (Rb.)
Croesia curvalana (Ker.)
Eriocraniidae:
Eriocrania cicatricella (Zt.)
Sesiidae:
E. sparrmannella (Bosc.)
Sesia apiformis (Cl.)
Synanthedon scoliaeformis (Brkh.)
Nepticulidae:
S. tipuliformis (Cl.)
Stigmella malella (Stt.)
56
Noctuidae:
Agrotis ipsilon (Hfn.)
Chrysodeixis chalcitis (Esp.)
Heliothis virescens (F.)
H. zea (Boddie.)
Mamestra suasa (Den. et Schiff.)
Pseudoplusia includens (Wlk.)
Sesamia nonagrioides (Lef.)
Spodoptera exigua (Hb.)
Trichoplusia ni (Hb.)
Caddisfly species:
Hesperophylax occidentalis (Banks)
Hydropsyche angustipennis (Curtis)
Rhyacophila fasciata (Hagen)
Plants:
Acer platanoides L.
Betula species
Malus sp.
Padus avium L.
Sorbus aucuparia L.
57