Microsporidian infections in Lymantria dispar larvae: interactions and effects of multiple species infections on pathogen horizontal transmission

The interactions in multiple species infections and effects on the horizontal transmission of three microsporidian species, Vairimorpha disparis, Nosema lymantriae and Endoreticulatus schubergi, infecting Lymantria dispar were evaluated in the laboratory. Simultaneous and sequential inoculations of host larvae were performed and the resulting infections were evaluated. Test larvae were exposed to the inoculated larvae to measure horizontal transmission. Dual species infections demonstrated interspecific competition between Nosema and Vairimorpha in the host larvae, but no observable competition occurred between Endoreticulatus and either of the other microsporidian species. Timing of inoculation was an important factor determining the outcome of competition between Nosema and Vairimorpha. The species inoculated first showed a higher rate of successful establishment; a time lag of 7 days between inoculations allowed the first species to essentially exclude the second. The microsporidia differed in efficiency of horizontal transmission. Nosema and Endoreticulatus were transmitted at very high rates, close to 100%. Horizontal transmission of Vairimorpha was less efficient, ranging from 25% to a maximum of 75%. The patterns of infection observed in inoculated larvae were reflected in the test larvae that acquired infections in the horizontal transmission experiments. Competition with Vairimorpha suppressed horizontal transmission of Nosema after simultaneous and sequential inoculation. In simultaneous inoculation experiments Endoreticulatus had no effect on transmission of Nosema and Vairimorpha.     

Quantifying horizontal transmission of Nosema lymantriae, a microsporidian pathogen of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in field cage studies

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2 ± 3.5% at density of 10 inoculated per 100 larvae to 36.7 ± 5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7 ± 5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20 dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25 dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.     

Influence of the forest caterpillar hunter Calosoma sycophanta on the transmission of microsporidia in larvae of the gypsy moth Lymantria dispar

• 1 The behaviour of predators can be an important factor in the transmission success of an insect pathogen. We studied how Calosoma sycophanta influences the interaction between its prey [Lymantria dispar (L.) (Lepidoptera, Lymantriidae)] and two microsporidian pathogens [Nosema lymantriae (Microsporidia, Nosematidae) and Vairimorpha disparis (Microsporidia, Burellenidae)] infecting the prey.
• 2 Using laboratory experiments, C. sycophanta was allowed to forage on infected and uninfected L. dispar larvae and to disseminate microsporidian spores when preying or afterwards with faeces.
• 3 The beetle disseminated spores of N. lymantriae and V. disparis when preying upon infected larvae, as well as after feeding on such prey. Between 45% and 69% of test larvae became infected when C. sycophanta was allowed to disseminate spores of either microsporidium.
• 4 Laboratory choice experiments showed that C. sycophanta did not discriminate between Nosema‐infected and uninfected gypsy moth larvae. Calosoma sycophanta preferred Vairimorpha‐infected over uninfected gypsy moth larvae and significantly influenced transmission.
• 5 When C. sycophanta was allowed to forage during the latent period on infected and uninfected larvae reared together on caged, potted oak saplings, the percentage of V. disparis infection among test larvae increased by more than 70%. The transmission of N. lymantriae was not affected significantly in these experiments.
• 6 Beetles never became infected with either microsporidian species after feeding on infected prey.
• 7 We conclude that the transmission of N. lymantriae is not affected. Because no V. disparis spores are released from living larvae, feeding on infected larvae might enhance transmission by reducing the time to death and therefore the latent period.

     

Host specificity of microsporidia pathogenic to the gypsy moth, Lymantria dispar (L.): Field studies in Slovakia

Several species of microsporidia are important chronic pathogens of Lymantria dispar in Europe but have never been recovered from North American gypsy moth populations. The major issue for their introduction into North American L. dispar populations is concern about their safety to native non-target insects. In this study, we evaluated the susceptibility of sympatric non-target Lepidoptera to two species of microsporidia, Nosema lymantriae and Vairimorpha disparis, isolated from European populations of L. dispar and applied in field plots in Slovakia. Application of ultra low volume sprays of the microsporidia maximized coverage of infective spores in a complex natural environment and, thus, exposure of non-target species to the pathogens. Of 653 non-target larvae collected from plots treated with V. disparis in 2002, 18 individual larvae representing nine species in four families were infected. These plots were monitored for two subsequent seasons and V. disparis was not recovered from non-target species. Of 2571 non-target larvae collected in N. lymantriae-treated sites, one larva was found to be infected. Both species of microsporidia, particularly N. lymantriae, appear to have a very narrow host range in the field, even when an inundative technique is used for their introduction. V. disparis infections in L. dispar exceeded 40% of recovered larvae in the treated study sites; infection rates were lower in sites sprayed with N. lymantriae. Several naturally-occurring pathogens were recorded from the non-target species. The most common pathogen, isolated from 21 species in eight families, was a microsporidium in the genus Cystosporogenes.     

Hostplant,larval age,and feeding behavior influence midgut pH in the gypsy moth (Lymantria dispar)

Summary The midgut pH of late instar gypsy moth (Lymantria dispar L.) larvae is strongly alkaline, and varies with diet, larval stadium, and time since feeding. Midgut pH rises with time since feeding, and does so more quickly, reaching greater maximum values, on some diets than others. Leaf tissues of 23 tree species resist increases in alkalinity differentially; this trait and differing initial leaf pH may explain the impact of diet on gut pH. Third instar larvae may have gut conditions favorable for tannin-protein binding shortly after ingesting certain foods, but with time midgut alkalinity becomes great enough to dissociate tannin-protein complexes. Older instars rarely exhibit gut pHs low enough to permit tannin activity. Alkaline gut conditions may explain the gypsy moth's ability to feed on many tanniniferous plant species, especially in later instars. Consequences for pathogen effectiveness are discussed.     

Effects of the ant Formica fusca on the transmission of microsporidia infecting gypsy moth larvae

Transmission plays an integral part in the intimate relationship between a host insect and its pathogen that can be altered by abiotic or biotic factors. The latter include other pathogens, parasitoids, or predators. Ants are important species in food webs that act on various levels in a community structure. Their social behavior allows them to prey on and transport larger prey, or they can dismember the prey where it was found. Thereby they can also influence the horizontal transmission of a pathogen in its host's population. We tested the hypothesis that an ant species like Formica fusca L. (Hymenoptera: Formicidae) can affect the horizontal transmission of two microsporidian pathogens, Nosema lymantriae Weiser (Microsporidia: Nosematidae) and Vairimorpha disparis (Timofejeva) (Microsporidia: Burenellidae), infecting the gypsy moth, Lymantria dispar L. (Lepidoptera: Erebidae: Lymantriinae). Observational studies showed that uninfected and infected L. dispar larvae are potential prey items for F. fusca. Laboratory choice experiments led to the conclusion that F. fusca did not prefer L. dispar larvae infected with N. lymantriae and avoided L. dispar larvae infected with V. disparis over uninfected larvae when given the choice. Experiments carried out on small potted oak, Quercus petraea (Mattuschka) Liebl. (Fagaceae), saplings showed that predation of F. fusca on infected larvae did not significantly change the transmission of either microsporidian species to L. dispar test larvae. Microscopic examination indicated that F. fusca workers never became infected with N. lymantriae or V. disparis after feeding on infected prey.     

Pheromonotropic activity in the gypsy moth Lymantria dispar: evidence for a neuropeptide

The brain-suboesophageal ganglion complex of the gypsy moth, Lymantria dispar, contains pheromonotropic activity detectable using a Helicoverpa zea in vivo bioassay for pheromone-biosynthesis-activating neuropeptide. Pheromonotropic activity was detected as early as the third larval instar and was present throughout development and through day 6 post-eclosion. Activity in the adult is presumably associated with pheromone production, while it is speculated that larval activity may be related to melanization. Adult pheromonotropic activity is associated with a peptide of approximately 3.500 kDa. It is heat labile and only partially stable when incubated at 35°C or exposed to freeze-thawing. Isolation of L. dispar pheromonotropic factor should facilitate the elucidation of the mechanism of pheromone production in this insect pest.Abbreviations ED ₅₀ dose at which one-half maximal response is observal - eq equivalent - MRCH melanization and reddish colorization hormone - MW molecular weight - PBAN pheromone biosynthesis activating neuropeptide - SOG suboesophageal ganglion - TFA trifluoroacetic acid - Z11-16: Ald (Z)-11-hexadecenal     

Effects of transferring gypsy moth, Lymantria dispar, larvae between artificial diet and Quercus rubra foliage

Fourth instar gypsy moth larvae, Lymantria dispar, from a laboratory colony were transferred between artificial diet and three ages of leaves from a preferred tree, Quercus rubra. Mortality after transfers was generally minimal (<13.3%) and did not differ from mortality of larvae remaining on the same food. Larvae that were transferred to a new food accepted artificial diet and the early season foliage that is normally eaten by early instars as readily as larvae that were not transferred. Pupal weights from larvae continually fed artificial diet or transferred from artificial diet to early season foliage were not significantly different. For trials beginning during June and later, 12-day weight gain and pupal weight were greater for individuals transferred to or reared continually on artificial diet than for individuals transferred to or reared continually on mature foliage.First instars of wild larvae fed artificial diet gained less weight after 3 weeks than foliage-fed first instars. Pupal weights of wild larvae transferred from June foliage to artificial diet as fourth instars were not significantly different from pupal weights of wild larvae remaining on foliage. Thus, the response of laboratory colony larvae to artificial diet was superior to that of wild larvae.

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Résumé Des chenilles du quatrième stade de L. dispar, élevées au laboratoire sur régime artificiel et sur feuilles de différents stades de leur chêne préféré, Q. rubra, ont été transférées de l'un à l'autre de ces régimes alimentaires. La mortalité après transfert est généralement faible (<13,3%) et de même importance que pour les chenilles maintenues sur le régime artificiel. Les chenilles transférées sur un nouvel aliment ont accepté le régime artificiel ou le feuillage jeune,- normalement consommé par les premiers stades larvaires-, aussi facilement que les chenilles qui n'avaient pas été transférées. Les poids des chrysalides des chenilles élevées continuellement sur régime artificiel ou transférées sur feuillage jeune n'ont pas présenté de différences significatives. Pour des expériences commencées en juin ou ultérieurement, les gains de poids à 12 jours et pour les chrysalides sont plus élevés pour les insectes maintenus ou transférés sur régime artificiel que pour ceux transférés ou maintenus sur feuillage âgé.Le poids des chrysalides, provenant de chenilles de la nature tranférées au quatrième stade, d'un feuillage de juin à un régime artificiel, n'a pas différé significativement du poids des chrysalides provenant de chenilles maintenues sur feuillage. Les premiers stades de chenilles de la nature élevées sur régime artificiel ont pris moins de poids en trois semaines que les premiers stades ayant consommé du feuillage.

     

New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach

Autophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by an F.A.R. grant from the University of Modena and Reggio Emilia (D.M. and E.O.) and by an “Experimental approaches to the study of evolution” grant from the Department of Animal Biology of the University of Modena and Reggio Emilia (D.M.).     

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 μM, for (+)-D was 5.32 ± 0.11 μM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 μM and 5.54 ± 0.04 μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.     

Deposition and germination of conidia of the entomopathogen Entomophaga maimaiga infecting larvae of gypsy moth,Lymantria dispar

Germination of conidia of Entomophaga maimaiga, an important fungal pathogen of gypsy moth, Lymantria dispar, was investigated on water agar and larval cuticle at varying densities. Percent germination was positively associated with conidial density on water agar but not on larval cuticle. When conidia were showered onto water agar, the rate of germination was much slower than on the cuticle of L. dispar larvae. From the same conidial showers, the resulting conidial densities on water agar were much higher than those on larval cuticle in part because many conidia adhered to setae and did not reach the cuticle. A second factor influencing conidial densities on larval cuticle was the location conidia occurred on larvae. Few conidia were found on the flexible intersegmental membranes in comparison with the areas of more rigid cuticle, presumably because conidia were physically dislodged from intersegmental membranes when larvae moved. Conidia were also exposed to heightened CO(2) to evaluate whether this might influence germination. When conidia on water agar were exposed to heightened CO(2) levels, germinating conidia primarily formed germ tubes while most conidia exposed to ambient CO(2) rapidly formed secondary conidia.     

Effects of maternal nutrition and egg provisioning on parameters of larval hatch,survival and dispersal in the gypsy moth,Lymantria dispar L.

North American gypsy moths disperse as newly hatched larvae on wind currents in a behavior called ballooning. Because ballooning occurs before neonates begin to feed, resources used in dispersal are limited to those carried over from the egg. We show that nutritional experience of the maternal parent can influence the tendency of offspring to disperse, and that resource provisioning of eggs by the maternal parent affects the duration of the window for disperal. Offspring of females from defoliated sites had a lower tendency to balloon in a wind tunnel than larvae from females which had not experienced nutritional stress associated with host defoliation. The number of eggs in an egg mass, a reflection of the maternal parent's nutritional experience, also contributed to the predictive model for dispersal that included defoliation level. Egg weight and the levels of two yolk proteins, vitellin (Vt) and glycine-rich protein (GRP), however, had no influence of the proportion of ballooning larvae. The length of survival without food, and thus the maximum period of time for dispersal, was correlated with levels of Vt and GRP, but not with egg weight. The level of defoliation at the site from which the maternal parent was collected was not related to the longevity of offspring, nor did it have a significant effect on the levels of Vt, GRP or egg weight. Levels of hemolymph proteins arylphorin and vitellogenin in the maternal parent during the prepupal stage had no influence on levels of yolk proteins, larval longevity, or tendency to balloon.     

The effect of varying alkaloid concentrations on the feeding behavior of gypsy moth larvae, Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine, and strychnine) were evaluated as feeding deterrents for gypsy moth larvae (Lymantria dispar (L.); Lepidoptera: Lymantriidae). Our aim was to determine and compare the taste threshold concentrations, as well as the ED₅₀ values, of the nine alkaloids to determine their potency as feeding deterrents. The alkaloids were applied to disks cut from red oak leaves (Quercus rubra) (L.), a plant species highly favored by larvae of this polyphagous insect species. We used two-choice feeding bioassays to test a broad range of biologically relevant alkaloid concentrations spanning five logarithmic steps. We observed increasing feeding deterrent responses for all the alkaloids tested and found that the alkaloids tested exhibited different deterrency threshold concentrations ranging from 0.1 to 10 mM. In conclusion, it appears that this generalist insect species bears a relatively high sensitivity to these alkaloids, which confirms behavioral observations that it avoids foliage containing alkaloids. Berberine and aristolochic acid were found to have the lowest ED₅₀ values and were the most potent antifeedants. Handling Editor: Joseph Dickens.     

Exploration into a mechanism of transgenerational transmission of nucleopolyhedrovirus in Lymantria dispar L. in Western Siberia

The mechanisms of nucleopolyhedrovirus (NPV)transmission through gypsy moth (Lymantriadispar L., Lepidoptera: Lymantriidae)populations in Western Siberia wereinvestigated. Field and laboratory studies werecarried out during 1992–1998 in a gypsy mothoutbreak in the Novosibirsk Area (WesternSiberia), Russia. The damaged trees were primarilytwo species of birch, Betula pendula Roth. and B. pubescensEhrh. Investigations were performed at threestages of the outbreak: population increase(1992–1993), population peak (1994–1995) andpopulation decline (1996–1998). Experiments onactivation of occult virus by different stressfactors were carried out in order to clarifythe mechanism of transgenerational transmissionof NPV. In both laboratory and fieldexperiments NPV-caused mortality levels werelow; most insects did not acquire a lethal doseof virus. Occult virus can provide an importantroute of transgenerational NPV transmission,particularly in Western Siberia where gypsymoths migrate by female flight and can moveaway from trees contaminated by virus fromprevious larval infections.     

Starvation resistance of gypsy moth,Lymantria dispar (L.) (Lepidoptera: Lymantriidae): tradeoffs among growth,body size,and survival

Summary Survival and body composition of starving gypsy moth larvae initially reared on aspen foliage or artificial diet differeing in nitrogen (N) and carbohydrate concentration were examined under laboratory conditions. Diet nitrogen concentration strongly affected starvation resistance and body composition, but diet carbohydrate content had no effects on these. Within any single diet treatment, greater body mass afforded greater resistance to starvation. However, starving larvae reared on 1.5% N diet survived nearly three days longer than larvae reared on 3.5% N diet. Larvae reared on artificial diet survived longer than larvae reared on aspen. Differences in survival of larvae reared on artificial diet with low and high nitrogen concentrations could not be attributed to variation in respiration rates, but were associated with differences in body composition. Although percentage lipid in larvae was unaffected by diet nitrogen concentration, larvae reared on 1.5% N diet had a higher percentage carbohydrate and lower percentage protein in their bodies prior to starvation than larvae reared on 3.5% N diet. Hence, larger energy reserves of larvae reared on low nitrogen diet may have contributed to their greater starvation resistance. Whereas survival under food stress was lower for larvae reared on high N diets, growth rates and pupal weights were higher, suggesting a tradeoff between rapid growth and survival. Larger body size does not necessarily reflect larger energy reserves, and, in fact, larger body size accured via greater protein accumulation may be at the expense of energy reserves. Large, fast-growing larvae may be more fit when food is abundant, but this advantage may be severely diminished under food stress. The potential ecological and evolutionary implications of a growth/survival tradeoff are discussed.     

Decline in gypsy moth (Lymantria dispar) performance in an elevated CO2 atmosphere depends upon host plant species

Plant species differ broadly in their responses to an elevated CO₂ atmosphere, particularly in the extent of nitrogen dilution of leaf tissue. Insect herbivores are often limited by the availability of nutrients, such as nitrogen, in their host plant tissue and may therefore respond differentially on different plant species grown in CO₂-enriched environments. We reared gyspy moth larvae (Lymantria dispar) in situ on seedlings of yellow birch (Betula allegheniensis) and gray birch (B. populifolia) grown in an ambient (350 ppm) or elevated (700 ppm) CO₂ atmosphere to test whether larval responses in the elevated CO₂ atmosphere were species-dependent. We report that female gypsy moths (Lymantria dispar) reared on gray birch (Betula populifolia) achieved similar pupal masses on plants grown at an ambient or an elevated CO₂ concentration. However, on yellow birch (B. allegheniensis), female pupal mass was 38% smaller on plants in the elevated-CO₂ atmosphere. Larval mortality was significantly higher on yellow birch than gray birch, but did not differ between the CO₂ treatments. Relative growth rate declined more in the elevated CO₂ atmosphere for larvae on yellow birch than for those on gray birch. In preference tests, larvae preferred ambient over elevated CO₂-grown leaves of yellow birch, but showed no preference between gray birch leaves from the two CO₂ atmospheres. This differential response of gypsy moths to their host species corresponded to a greater decline in leaf nutritional quality in the elevated CO₂ atmosphere in yellow birch than in gray birch. Leaf nitrogen content of yellow birch dropped from 2.68% to 1.99% while that of gray birch leaves only declined from 3.23% to 2.63%. Meanwhile, leaf condensed tannin concentration increased from 8.92% to 11.45% in yellow birch leaves while gray birch leaves only increased from 10.72% to 12.34%. Thus the declines in larval performance in a future atmosphere may be substantial and host-species-specific.     

Male death resulting from hybridization between subspecies of the gypsy moth,Lymantria dispar

We explored the origin of all-female broods resulting from male death in a Hokkaido population of Lymantria dispar through genetic crosses based on the earlier experiments done by Goldschmidt and by testing for the presence of endosymbionts that are known to cause male killing in some insect species. The mitochondrial DNA haplotypes of the all-female broods in Hokkaido were different from those of normal Hokkaido females and were the same as those widely distributed in Asia, including Tokyo (TK). Goldschmidt obtained all-female broods through backcrossing, that is, F1 females obtained by a cross between TK females (L. dispar japonica) and Hokkaido males (L. dispar praeterea) mated with Hokkaido males. He also obtained all-male broods by mating Hokkaido females with TK males. Goldschmidt inferred that female- and male-determining factors were weakest in the Hokkaido subspecies and stronger in the Honshu (TK) subspecies. According to his theory, the females of all-female broods mated with Honshu males should produce normal sex-ratio broods, whereas weaker Hokkaido sexes would be expected to disappear in F1 or F2 generations after crossing with the Honshu subspecies. We confirmed both of Goldschmidt''s results: in the case of all-female broods mated with Honshu males, normal sex-ratio broods were produced, but we obtained only all-female broods in the Goldschmidt backcross and obtained an all-male brood in the F1 generation of a Hokkaido female crossed with a TK male. We found no endosymbionts in all-female broods by 4,′6-diamidino-2-phenylindole (DAPI) staining. Therefore, the all-female broods observed in L. dispar are caused by some incompatibilities between Honshu and Hokkaido subspecies.     

Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth,Lymantria dispar (L.) following studies on identification,titers and synthesis of ecdysteroids in last-instar females

Summary Hemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Grace's medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Grace's medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.Abbreviations A _r activation ratio - HPLC high performance liquid chromatography - HPSEC high performance size-exclusion chromatography - PG prothoracic gland - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

Sexual dimorphism in neuronal projections from the antennae of silk moths (Bombyx mori,Antheraea polyphemus) and the gypsy moth (Lymantria dispar)

Summary The antennal lobe of both sexes of the silk moth Bombyx mori contains 55–60 ventrally located antennal glomeruli; in addition, that of the male contains a dorsal macroglomerular complex (MGC). A group of identifiable glomeruli consisting of two lateral large glomeruli (LLG) and four medial small glomeruli (MSG) is present in both sexes, but the LLG are greatly enlarged in the female. A MGC is also present in the male gypsy moth Lymantria dispar and male giant silk moth Antheraea polyphemus. The MGC in all of these species is organized into 3–4 distinct levels of glomeruli. Antennal sensory fibers were stained by cobalt backfills in B. mori, A. polyphemus, and L. dispar. Most fibers stained from cut long hairs (sensilla trichodea) projected to MGC in males and LLG in both sexes of B. mori. The distribution of fibers in the MGC of B. mori was topographically biased in that a majority of fibers from anterior branches projected medially in MGC while most fibers from posterior branches projected laterally or anteriorly. Terminal arborizations of single fibers were each restricted to a single glomerular level of the MGC. Fibers projecting to the posterior antennal center were frequently stained in cut-hair and control preparations, apparently by uptake of cobalt through intact sensilla on flagellar branches.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.