DOI: 10.2478/s11686-014-0231-5
© W. Stefański Institute of Parasitology, PAS
Acta Parasitologica, 2014, 59(2), 213–218; ISSN 1230-2821
The effect of initial dose on the recovery and final yields
of Heterorhabditis megidis (Rhabditida: Heterorhabditidae)
in larvae of the great wax moth, Galleria mellonella
Dorota Tumialis1*, Elżbieta Pezowicz1, Anna Mazurkiewicz1, Iwona Skrzecz2,
Elżbieta Popowska-Nowak3 and Agnieszka Petrykowska1
1
Warsaw University of Life Sciences – SGGW, Faculty of Animal Sciences, Department of Animal Environment Biology, Ciszewskiego 8,
02-786 Warsaw, Poland; 2Forest Research Institute, Department of Forest Protection, Sękocin Stary, Braci Leśnej 3, 05-090 Raszyn,
Poland; 3Cardinal Stefan Wyszynski University, Faculty of Biology and Environmental Sciences, Wóycickiego 1/3, 01-938 Warsaw,
Poland
Abstract
The aim of this study was to determine the effect of different initial doses of the infective juveniles (IJs) (50 IJs, 200 IJs, 1000
IJs ) of Heterorhabditis megidis Poinar (Rhabditida: Heterorhabditidae) strain IsM15/09 on recovery, final yields and percent
final yields in larvae Galleria mellonella ( L.). Percent recovery was not directly related to initial dose. Final yields also did
not change with the initial dose. However, percent yields was highly negatively correlated with initial dose of nematodes and
was the highest with the 50 IJs dose. Additional point of the study was to investigate whether the nematodes are able to produce progeny from one hermaphroditic individual. The results showed that the invasive larvae resumed growth and transformed
into hermaphroditic individuals that reproduced without cross-fertilisation.
Keywords
Heterorhabditis megidis, Galleria mellonella, initial dose, recovery, final yields
Introduction
Entomopathogenic nematodes from the families Steinernematidae and Heterorhabditidae and their respective symbiotic
bacteria, Xenorhabdus spp. and Photorhabdus spp., are lethal
parasites of many insects. These nematodes are broadly used
in biological plant protection programmes (Burnell and Stock
2000; Koppenhöfer et al. 2007). One of the advantages of their
use is that these nematodes are easy to culture and may be kept
in vitro and in vivo (Lacey et al. 2001). Caterpillars of Galleria mellonella L. (Lepidoptera: Pyralidae) and larvae of Tenebrio molitor (Coleoptera: Tenebrionidae), which are used in
their production, are easily available, and raising them is easy
and efficient. These hosts are also highly susceptible to infection; hence, it is possible to produce many species of nematodes from these species (Shapiro-Ilan and Gaugler 2002).
In vivo reproductive yields can be increased through the
optimisation of selected parameters, especially at the stage of
host infection. Therefore, it is important to select the appropriate method of infection, optimum initial dose and maxi-
mum host density, which are nematode and insect speciesspecific. Thus, the final effect of in vivo cultures is affected
by many factors that have been the subject of many studies
(Shapiro-Ilan and Gaugler 2002). This study was undertaken
to explain how the initial dose of H. megidis affects its developmental cycle and, consequently, the final yields in G. mellonella larvae.
Materials and Methods
Study material
The strain IsM15/09 of H. megidis used in this study was isolated from Zachodniopomorskie Province in Poland. To
achieve the optimum invasiveness, invasive larvae were kept
for 2 weeks in bottles for tissue cultures filled 1/3 with water
at a temperature of 4°C. Throughout the study period, the nematodes were kept under the same conditions. Prior to the
experiments, the nematodes were kept for 15 min. at room
*Corresponding author: [email protected]
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Dorota Tumialis et al.
temperature. In vivo experiments were performed in G. mellonella larvae with a mean weight of 162 mg cultured at the
Department of Zoology, Faculty of Animal Science, University of Life Sciences-SGGW.
lected and counted. Analyses of reproductive yields were triplicated for each of the initial doses.
Methods
Recovery (R) – the percentage of invasive larvae which develop to adults (Kaya and Stock 1997), expressed as:
The initial dose of infective juveniles (IJs) (50 IJs, 200 IJs
and 1000 IJs) that was applied to infect the caterpillars of G.
mellonella was the only variable. The same methods of insect
infection were used to determine the number of nematodes in
particular generations and to estimate the final reproductive
yields.
Methods of infection of insects used in experiments
Infection of G. mellonella larvae with initial doses of 50, 200
and 1000 IJs – One millilitre of nematode suspension (50 IJs,
200 IJs or 1000 IJs) was poured on three round pieces of filter papers (45 mm diameter) that were then wrapped around 1
caterpillar of G. mellonella. Wrapped caterpillars placed on
90 mm diameter Petri dishes, 10 larvae per one dish (in total
100 larvae were used). The bundle was wrapped with aluminium foil to create an inoculation cocoon, which was kept
at 25°C for 48 h.
H. megidis nematode development after an initial dose of 1 IJ
To test whether the nematodes are able to produce progeny
from one hermaphroditic individual, 25 larvae of G. mellonella were infected by 1 invasive larva of H. megidis. One
caterpillar of G. mellonella was placed in an Eppendorf test
tube with a scrap of filter paper with one invasive larva on it.
Tubes were incubated at 25°C for 48 h. Insect mortality was
estimated, and the test tubes were transferred again to a growth
chamber. After 5 days, 5 infected caterpillars of G. mellonella
were dissected.
The presence of hermaphroditic individuals at the final phase
of growth cycle of H. megidis
To check whether hermaphroditic individuals appear in the
later phase of the growth cycle of H. megidis occurred in the
body of insect infected by an initial dose of 1000 IJs, 25 caterpillars of G. mellonella were dissected on the 12th day after infection. The test was performed with three replicates of 25
caterpillars three times.
Calculations
R = (H/a) x 100%,
where:
H – number of hermaphroditic individuals,
a – initial dose of nematodes
Final yields (Wk) – number of invasive larvae (IJs)
calculated per 1 larva of G. mellonella
Percent yields (W%) is expressed as:
W% = l.k. IJs /a x 100%
where:
l.k. IJs – final number of invasive larvae (IJs),
a – initial dose of nematodes.
Statistical analysis
Results were calculated with STATISTICA 9.0. Statistical significance of the relationships between the initial dose and studied parameters (final reproductive yields and percent yields of
H. megidis in culture) was estimated with non-parametric
Kruskal-Wallis ANOVA.
Results
Growth of H. megidis after an initial dose of 1 IJ
A high percent of recovery (100%) of nematodes in the host’s
body was observed 5 days after the infection of G. mellonella
with an initial dose of 1 IJ. Invasive larvae resumed growth
and transformed into hermaphroditic individuals that reproduced without cross-fertilisation (in all 25 examined larvae of
G. mellonella the presence of hermaphroditic individuals and
their offspring was found).
The presence of hermaphroditic individuals of H. megidis
in the final phase of the growth cycle
Twelve days after infection, no hermaphroditic individuals
were found in the bodies of G. mellonella infected with an initial dose of 1000 IJs. Only females and young larvae of the
next generation were observed.
Estimating the final yields of nematodes in vivo
The effect of initial dose on recovery (%)
After 48-hour incubation, dead caterpillars of G. mellonella
were transferred into modified White’s traps (White 1929). In
each of the Petri dishes, nematodes were extracted from the
bodies of 8–10 caterpillars of G. mellonella after 14 days at
25°C. The invasive larvae released into the water were col-
No significant effect of the initial dose (50 IJs, 200 IJs, 1000
IJs) on recovery was found (p<0.05) (Fig.1).
Mean recovery values obtained for specific initial doses
differed slightly. The highest number of invasive larvae iniUnauthenticated
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The effect of initial dose on the recovery and final yields of Heterorhabditis megidis (Rhabditida: Heterorhabditidae) in vivo
215
Fig. 1. The effect of initial dose on recovery (%)
tiating development (24.2%) was observed with a dose of
1000 IJs.
The effect of initial dose on the final yields of in vivo
culture
There were no significant differences in the effect of initial
dose (50 IJs, 200 IJs, 1000 IJs) on the final yields of in vivo
cultures (p<0.05) (Fig. 2).
There no statistically significant differences between doses
of 50 and 1000, 200 and 1000 (Fig. 2). However, final yields
was found to increase with increasing initial dose. The lowest
mean yields (45 r of invasive larvae obtained after application
of initial doses of 50 IJs and 200 IJs was negligible.
A significant difference was found between the initial dose
of 50 IJs and 1000 IJs. The highest average yield rate (90) was
obtained using 50 invasive larvae, and the lowest rate
(6 228,67%) was obtained with a dose of 1000 IJs. The greater
the number of infectious larvae used per host, the lower the percentage yield obtained in vivo culture. There were significant
differences in the effect of initial dose (50 IJs, 200 IJs, 1000 IJs)
on the percent (%) yields of in vivo culture (p<0.05) (Fig 3).
Fig. 2. The effect of initial dose on the final yields of in vivo culture
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Dorota Tumialis et al.
Fig. 3. The effect of initial dose on the percent (%) final yields of in vivo culture
Discussion
Caterpillars of G. mellonella are hosts that are easily infected
with entomopathogenic nematodes (EPNs). This susceptibility occurs because these caterpillars do not contact nematodes
in their natural environment and, therefore, their immune system does not recognise invasive larvae and release symbiotic
bacteria. On the contrary, the larvae of insects such as Tipula
oleracea, Tipula paludosa and Othiorhynchus sulcatus live in
soil and commonly come into contact with EPNs. Their immune systems have developed many protective mechanisms
such as phagocytosis, encapsulation, synthesis of adhesive
molecules and antibacterial peptides, lysozymes and lectins.
These all factors destroy bacteria and nematodes before they
can cause the host’s death (Li et al. 2007). The nematodes
H. megidis are able to overcome the defences of the potential
host’s immune system. The nematodes may also synthesise
and release numerous enzymes (proteases, α-mannosidase and
α-fukosidase), the activity of which is noted in insects that are
highly infected by virulent nematodes (Simões et al. 2000).
Another feature of the invasive larvae of H. megidis is their
high coefficient of penetration, which is the main source of
the high pathogenicity of this species (Gerritsen et al. 1998).
Studies confirm the ability of H. megidis nematodes to quickly
and effectively penetrate G. mellonella larvae.
H. megidis nematodes develop in the larvae of G. mellonella infected by both low and high doses of invasive larvae.
Hermaphroditic individuals develop from a single invasive
larva and are able to reproduce and to give birth to its offspring
separately. The hermaphroditic individual lays some of its
eggs in the host’s body cavity and retains the remainder of the
eggs inside its uterus. This phenomenon is confirmed by observations of Boff et al. (2001) and Wang and Bedding (1996)
who analyse the growth cycle of H. bacteriophora. Wang
and Bedding (1996) observed the life cycle of H. bacteriophora using an initial dose of 1 IJ. Similar results are found by
Baliadi et al. (2009). They find the occurrence of hermaphroditic individuals on the 3rd day after infection.
The number of invasive larvae of H. megidis penetrating
the host’s body increase with a greater initial dose, but recovery remained almost stable. Regardless of the initial concentration of nematodes in the insects’ hemocoel, 22.0–24.2% of
invasive larvae initiated further development, and the highest
recovery was observed at an initial dose of 1000 IJs. Fan
and Hominick (1991), who expose insects to various nematode
densities (10 IJs–300 IJs), also find that the percentage of penetrating invasive larvae of Heterorhabditis was independent of
the initial dose. According to Mannion and Jansson (1993),
Heterorhabditis nematodes maintain their invasive abilities for
several days, and the higher the initial nematode concentration,
the greater the percentage of larvae entering host’s body and
initiating further development. Quite a different tendency is
observed by Boff et al. (2000), who find that an increase of initial dose results in a decrease of the percentage of nematodes
initiating further development in G. mellonella larvae. They
obtain the highest recovery (38.3%) at the smallest initial dose
(10 IJs) and the lowest recovery (16%) with 1000 IJs. A similar relationship is also found by Selvan et al. (1993) when
studying the effect of various initial doses (10 IJs–6400 IJs) on
recovery in H. bacteriophora and S. carpocapsae. They conUnauthenticated
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The effect of initial dose on the recovery and final yields of Heterorhabditis megidis (Rhabditida: Heterorhabditidae) in vivo
clude, as do Fan and Hominick (1991), that in a given time period, only part of the initial dose of invasive larvae is able to infect and penetrate the host’s body. Lewis et al. (2006) suggest
that a high density of nematodes in insects’ hemocoel is a
source of signals that are sent to the external environment.
These signals hamper the penetration of invasive larvae from
outside; thus, the nematodes avoid “overcrowded” hosts that
would provide poor food resources. Bonifassi et al. (1999)
demonstrate that invasive larvae of EPNs may retain numerous
bacterial cells on the surface of their external cuticular layer. A
high number of nematodes penetrating insect’s hemocoel may
be a source of many pollutants, making the resumption of larval development impossible. Those pollutants could be another
reason for the low recovery observed at high initial doses of
200 IJs and 1000 IJs.
The number of offspring produced (final yields) in H.
megidis does not depend on the initial concentration of nematodes. Nevertheless, the number of invasive larvae released
from the bodies of G. mellonella caterpillars infected with
1000 IJs is the highest. The number of offspring obtained at 50
and 200 IJs doses are similar and slightly smaller than that obtained at the highest nematode concentration. No effect of the
initial dose on the production of invasive larvae is also demonstrated by Shapiro-Ilan and Gaugler (2002) for H. bacteriophora and S. carpocapsae, by Flanders et al. (1996) for H.
bacteriophora and by Elawad et al. (1999) for S. abbasi. A
strong effect of a high concentration of nematodes (3000 IJs)
on final yields is, however, shown by Boff et al. (2000). A
slight increase of the number of invasive larvae that are observed with increasing dosages remains until the application of
a dose of 300 IJs. Increasing nematode density to 1000 IJs and
3000 IJs results in a decline in final yields. Comparable results are obtained by Selvan et al. (1993), who study final
yields of H. bacteriophora and find an increase of the number
of produced nematodes with increasing concentration up to
the dose of 100 IJs. According to Zervos et al. (1991), final
production of H. bacteriophora achieves its maximum level
when infecting hosts with 25 invasive larvae and then decreases with increasing initial dosages. In liquid cultures, the
initial dose affects final yields in various ways. In H. bacteriophora, the initial concentration of invasive larvae affects
total production (Han 1996), while in H. indica, the number of
offspring produced does not depend on the initial dose (Ehlers
et al. 2000).
These studies suggest that for some species of EPNs, there
is an “optimum” initial dose that results in a density of nematodes in the insects’ hemocoel at which point no competition
for food is observed. An increasing number of nematodes and
decreasing food resources cause the proliferation of invasive
larvae that do not continue further growth and leave their
host’s body. If the concentration of nematodes in the insect’s
body is too high, strong competition exists not only for food,
but also for space and oxygen, even in the first generation. Intense competition decreases the health of parasitic nematodes,
their reproductive abilities and, consequently, the number of
217
offspring. Based on studies and observations of Boff et al.
(2000), it could be concluded that H. megidis are more resistant to overcrowding than other species of EPNs, such as H.
bacteriophora. The final efficiencies found for H. megidis
were higher than those noted by Boff et al. (2000), despite the
fact that both experiments were performed under the same
thermal conditions (25°C). Mason and Hominick (1995) found
that nematode production in the body of G. mellonella larvae
is higher at 15°C and 20°C than at 25°C.
The initial dose did not affect final yields but, as shown in
this study, it determined the percent yields of in vivo culture.
These results suggest that the most effective initial dose of
H. megidis grown in the body of G. mellonella larvae at 25°C
is 50 IJs. Boff et al. (2000) calculate the coefficient of reproduction, i.e. the ratio of final yields to the number of nematodes that managed to penetrate the insect’s hemocoel, which
was also negatively correlated with the size of initial dose.
Studies show that initial dose is a parameter that significantly affects the percent yields of culture, selection of appropriate host species in which nematodes multiply is also
important (Shapiro-Ilan and Gaugler 2002).
Conclusions
1. Hermaphroditic individuals of H. megidis do not need other
hermaphrodites for reproduction, thus exhibiting an ability
to self-fertilise.
2. Final yields of H. megidis culture does not depend on the
size of initial dose. Regardless of the initial concentration of
nematodes, the number of invasive larvae produced was
high (>45 000 IJs/G. mellonella larva).
3. The initial dose significantly affects the percent yields of in
vivo culture. The best results were obtained after application of an initial dose of 50 IJs.
4. Percent of invasive larvae initiating further growth (recovery) regardless of the initial dose ranged from 22.0 to 24.2%.
References
Baliadi Y., Kondo E., Yoshiga T. 2009. The continual forming and
contribution of infective juveniles produced via endotokia matricida of entomopathogenic nematodes in the family of Steinernematidae and Heterorhabditidae. Indonesian Journal of
Agricultural Science, 10, 26–33.
Boff M.I.C., Wiegers G.L., Gerritsen L.J.M., Smits P.H. 2000. Development of the entomopathogenic nematode Heterorhabditis megidis strain NLH-E 87.3 in Galleria mellonella.
Nematology, 2, 303–308. DOI: 10.1163/156854100509178.
Boff M.I.C., Wiegers G.L., Smits P.H. 2001. Host influences on the
pathogenicity of Heterorhabditis megidis. BioControl, 46, 91–
103. DOI: 10.1590/S1519-69842013000200003.
Bonifassi E., Fischer-Le Saux M., Boemare N., Lanois A., Laumond
C., Smart G. 1999. Gnotobiological study of infective juveniles
and symbionts of Steinernema scapterisci: A model to clarify
the concept of the natural occurrence of monoxenic associations in entomopathogenic nematodes. Journal of Invertebrate
Pathology, 74, 164–172. DOI: 10.1098/rspb.2001.1795.
Unauthenticated
Download Date | 7/12/17 11:59 PM
218
Burnell A.M., Stock S.P. 2000. Heterorhabditis, Steinernema and
their bacterial symbionts lethal pathogens of insects. Nematology, 2, 31–42. DOI: 10.1603/029.102.0348.
Ehlers R.U., Niemann I., Hollmer S., Strauch O., Jende D., Shanmugasundaram M., Mehta U.K., Easwaramoorthy S.K., Burnell A.M. 2000. Mass production potential of the bactohelminthic biocontrol complex Heterorhabditis indica – Photorhabdus luminescens. Biocontrol Science and Technology,
10, 607–616. DOI: 10.1007/s10123-003-0144-x.
Elawad S.A., Gowen S.R., Hague N.G.M. 1999. The life cycle of
Steinernema abbasi and S. riobrave in Galleria mellonella.
Nematology, 1, 762–764.
Fan X., Hominick W.M. 1991. Efficiency of the Galleria (wax moth)
baiting technique for recovering infective stages of entomopathogenic rhabditidis (Steinernematidae and Heterorhabditidae) from sand and soil. Revue de Nématologie, 14,
381–387.
Flanders K.L., Miller J.M., Shields E.J. 1996. In vivo production of
Heterorhabditis bacteriophora ‘Oswego’ (Rhabditida: Heterorhabditidae), a potential biological control agent for soilinhabiting insects in temperate regions. Journal of Economic
Entomology, 89, 373–380.
Gerritsen L.J.M., Wiegers G.L., Smits P.H. 1998. Pathogenicity of
New Combinations of Heterorhabditis spp. and Photorhabdus luminescens against Galleria mellonella and Tipula olerace. Biological Control, 13, 9–15.
Han R.C. 1996. The effects of inoculum size on yield of Steinernema
carpocapsae and Heterorhabditis bacteriophora in liquid culture. Nematologica, 42, 546–553. DOI: 10.1163/004625996X
00045.
Kaya H.K., Stock S.P. 1997. Techniques in insect nematology. In:
(Ed. L.A. Lacey) Manual of techniques in insect pathology.
Academic Press, London, 281–324.
Koppenhöfer A.M., Grewal P.S., Fuzy E.M. 2007. Differences in penetration routes and establishment rates of four entomopathogenic nematode species into four white grub species. Journal
of Invertebrate Pathology, 94, 184–195. DOI: 10.1016/j.jip.
2006.10.005.
Lacey L.A., Frutos R., Kaya H.K., Vail P. 2001. Insect Pathogens as
Biological Control Agents: Do they Have a Future? Biological Control, 21, 230–248. DOI: 10.1006/bcon.2001.0938.
Li X.Y., Cowles R.S., Cowles E.A., Gaugler R., Cox-Foster D.L.
2007. Relationship between the successful infection by entomopathogenic nematodes and the host immune response.
Dorota Tumialis et al.
International Journal of Parasitology, 37, 365–374. DOI:
10.1016/j.ijpara.2006.08.009.
Lewis E.E., Campbell J., Griffin C., Kaya H., Peters A. 2006. Behavioral ecology of entomopathogenic nematodes. Biological
Control, 38, 66–79. DOI: 10.1016/j.biocontrol.2005.11.007.
Mannion C.M., Jansson R.K. 1993. Infectivity of five entomopathogenic nematodes to the sweetpotato weevil, Cylas formicarius (F.), (Coleoptera: Apionidae) in three experimental arenas.
Journal of Invertebrate Pathology, 62, 29–36. DOI: 10.1006/
jipa.1993.1070.
Mason J.M., Hominick W.M. 1995. The effect of temperature on infection, development and reproduction of Heterorhabditis.
Journal of Helminthology, 69, 37–347. DOI: 10.1017/S00
22149X00014929.
Saunders J.E., Webster J.M. 1999. Temperature Effects on Heterorhabditis megidis and Steinernema carpocapsae. Infectivity to Galleria mellonella. Journal of Nematology, 31,
299–304.
Selvan S., Campbell J.F., Gaugler R. 1993. Density – dependent effects on entomopathogenic nematodes (Heterorhabditidae and
Steinernematidae) within an insect host. Journal of Invertebrate Pathology, 62, 278–274. DOI: 10.1006/jipa.1993.1113.
Shapiro-Ilan D.I., Gaugler R. 2002. Production technology for entomopathogenic nematodes and their bacterial symbionts. Journal of Industrial Microbiology and Biotechnology, 28,
137–146. DOI: 10.1038/sj/jim/7000230.
Simões N., Caldas C., Rosa J.S., Bonifassi E., Laumond C. 2000.
Pathogenicity Caused by High Virulent and Low Virulent
Strains of Steinernema carpocapsae to Galleria mellonella.
Journal of Invertebrate Pathology, 75, 47–54. DOI: 10.
1006/jipa.1999.4899.
Wang J., Bedding R.A. 1996. Population development of Heterorhabditis bacteriophora and Steinernema carpocapsae in
the larvae of Galleria mellonella. Fundamental and Applied
Nematology, 19, 363–367.
White G.F. 1929. A method for obtaining infective nematode larvae
from cultures. Science, 66, 302–303. DOI: 10.1126/science.66.1709.302-a.
Zervos S., Johnson S.C., Webster J.M. 1991. Effect of temperature
and inoculum size on reproduction and development of Heterorhabditis heliothidis and Steinernema glaseri (Nematoda:
Rhabditoidea) in Galleria mellonella. Canadian Journal of
Zoology, 69, 1261–1264. DOI: 10.1139/z91-177.
Received: May 29, 2013
Revised: January 9, 2014
Accepted for publication: January 29, 2014
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Download Date | 7/12/17 11:59 PM