Identification and characterization of an ecdysiotropic peptide from brain extracts of the gypsy moth,Lymantria dispar

A peptide (Lymantria TE) was isolated from brains of the gypsy moth, Lymantria dispar, which stimulates synthesis of ecdysteroid in the testes of larval and pupal insects. This ecdysiotropic peptide was purified and its structure determined to be NH₂-IIe-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH using protein sequence analysis and electrospray mass spectrometry. The peptide was biphasic in activity, with maximal activity in the pupal testes at 10⁻¹³ M and 10⁻⁹ M, with a minimum at 10⁻¹⁰ M, and with maxima at 10⁻¹⁵ M and 10⁻¹⁰ M and minimum at 10⁻¹³ M for larval testes. Arch. Insect Biochem. Physiol. 34:175–189, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Structure–function analysis of Lymantria testis ecdysiotropin: A search for the active core

A structure–function study was performed on the synthetic 21 residue neuropeptide, Lymantria testis ecdysiotropin (LTE), originally isolated from brains of Lymantria dispar pupae. The peptide induces ecdysteroid synthesis by testis sheaths of various lepidopteran species. LTE, as well as synthetic LTE 1-11, 11-21, and 11-15, stimulated synthesis in larval and pupal testes of Lymantria dispar at concentrations of 10^–9 to 10^–15 M; LTE 16-21 was weakly active, and an elongated LEU-LTE was inhibitory to synthesis at all but extremely low concentrations (10^–15M). Since the sequence and polarity of residues in LTE 1-11, 11-15, and 11-21 are quite different, several parts of the molecule must activate receptors which initiate the cascade, resulting in ecdysiogenesis in Lepidopteran testes. Arch. Insect Biochem. Physiol. 38:11–18, 1998. © 1998 Wiley-Liss, Inc.     

Second messengers mediating the effects of testis ecdysiotropin in testes of the gypsy moth,Lymantria dispar

Exposure of larval and pupal testes of Lymantria dispar to diacyl glycerol mimics, phorbol, 12-myristate, 13-acetate, and 11, 12 dibutyryl phorbol ester, induced or augmented synthesis of immunodetectable ecdysteroids. The non-esterified analog, 4α-phorbol, had little effect. H-7, a protein kinase C inhibitor, inhibited ecdysteroid synthesis. When testis ecdysiotropin and phorbol esters were administered together at the maximum effective dose of each, steroidogenesis was further enhanced. Therefore, diacyl glycerol may be a second messenger for testis ecdysiotropin in testes. In addition, testis ecdysiotropin induced a rapid rise and fall in cAMP titers in both larval and pupal testes. The cyclic AMP analog, dibutyryl cyclic AMP, induced ecdysteroid synthesis in larval testes, but had little steroidogenic effect in pupal testis sheaths. However, dibutyryl cyclic AMP inhibited the steroidogenic effect of testis ecdysiotropin in larval as well as pupal testes. Cyclic AMP may act to modulate the effects of testis ecdysiotropin in inducing ecdysteroid synthesis by testes of L. dispar. © 1993 Wiley-Liss, Inc.     

Mating-induced loss of sex pheromone and sexual receptivity in insects with emphasis on Helicoverpa zea and Lymantria dispar

Mating in most species of insects leads to a transient or permanent loss in sexual receptivity of the females. Among moths, this loss of receptivity is often accompanied with a loss of the sex pheromone in the absence of calling, which also could be temporary or permanent. Most of the earlier work on changes in reproductive behavior after mating was done with Diptera in which sperm and/or male accessory gland secretions were shown to be responsible for termination of receptivity. In the corn earworm moth, Helicoverpa zea, mated females become depleted of pheromone and become nonreceptive to further mating attempts, but only for the remainder of the night of mating. A pheromonostatic peptide isolated from the accessory glands of males may be responsible for the depletion of pheromone, while the termination of receptivity is independently controlled. In the gypsy moth, Lymantria dispar, the changes in behavior following mating are permanent. In this species, the switch from virgin to mated behavior involves three steps: a physical stimulation associated with mating, transfer of viable sperm to the spermatheca, and commencement of oviposition. Signals generated by these factors operate through neural pathways and, unlike in H. zea, accessory gland factors seem not to be involved. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

The distribution of PBAN (pheromone biosynthesis activating neuropeptide)-like immunoreactivity in the nervous system of the gypsy moth,Lymantria dispar

The production of sex pheromone in many moths is regulated by the neuropeptide PBAN (pheromone biosynthesis-activating neuropeptide). Studies in a number of species have shown that pheromone production can be linked to a hemolymph factor and that continuity in the ventral chain of ganglia is not required. However, it has recently been shown that production of pheromone in the gypsy moth, Lymantria dispar, is largely prevented in females with a transected ventral nerve cord (VNC). To begin to understand the cellular basis for this dependence on the VNC, we sought to determine the distribution of PBAN in the central nervous system and its neurohemal sites, including those associated with the VNC. Using an antiserum to L. dispar-PBAN in immunocytochemical methods, we have mapped the distribution of PBAN-like immunoreactivity (PLI). PLI is found in three clusters of ventral midline somata in the subesophageal ganglion (SEG), in three clusters of midline cells in each segmental ganglion, and in bilateral pairs of cells located posterolaterally in each abdominal ganglion. The SEG cells comprise both interneurons, with endings in the neuropil of each segmental ganglion, as well as neurosecretory cells, with endings in the retrocerebral complex and in an unusual neurohemal structure near the anterior aspect of the SEG. The latter structure, which we have named the corpus ventralis, receives axons from the two anterior clusters of cells in the SEG. In the abdominal ganglia, the posterolateral clusters of cells have immunoretroreactive axons exiting the ganglia via the ventral nerves. Endings of these axons reach the perivisceral organ in the next posterior ganglion and pass anteriorly into the median nerve, forming additional varicose endings. We did not detect PLI in the terminal nerve. Thus, our findings raise the possibility that the requirement for an intact VNC in pheromone production reflects a role for descending regulation of neurosecretory cells in the segmental ganglia. Arch. Insect Biochem. Physiol. 34:391–408, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Testis ecdysiotropin, an insect gonadotropin that induces synthesis of ecdysteroid.

Testes of lepidoptera synthesized ecdysteroid in a somewhat different temporal pattern than the prothoracic glands that release ecdysteroid to the hemolymph. Brain extracts from Heliothis virescens and Lymantria dispar induced testes to synthesize ecdysteroid, but did not affect prothoracic glands. The testis ecdysiotropin (LTE) was isolated from L. dispar pupal brains by a series of high-pressure chromatography steps. Its sequence was Ile-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH, of molecular mass 2,473 Daltons. The predominant signaling pathway for LTE was via G(i) protein, IP3, diacylglycerol and PKC; a modulating pathway, apparently mediated by an angiotensin II-like peptide, was controlled via G(s) protein, cAMP, and PKA. Testis ecdysteroid caused isolated testis sheaths to also synthesize a growth factor that induced development of the male genital tract. The growth factor appeared to be a glycoprotein similar to vertebrate alpha-1-glycoprotein. A polyclonal antibody to LTE indicated LTE-like peptide in L. dispar brain medial neurosecretory cells, the suboesophageal, and other ganglia, and also in its target organ, the testis sheath. LTE immunoreactivity was also seen in testis sheaths of Rhodnius prolixus. LTE-like immunoactivity was also detected in developing optic lobes, antennae, frontal ganglia, and elongating spermatids of developing L. dispar pupae. This may indicate that LTE has a role in development as well as stimulation of testis ecdysteroid synthesis. Published 2001 Wiley-Liss, Inc.     

Effect of food plant switching by a herbivore on its parasitoid: Cotesia melanoscela development in Lymantria dispar exposed to reciprocal dietary crosses

1. Host plant switching by dispersing early instar lepidopterans could have implications for parasitoid performance, but this possibility has not been evaluated thoroughly. 2. The relative growth rates of Lymantria dispar parasitized by Cotesia melanoscela, and the weight of larvae at the time of parasitoid emergence, were affected most by the second larval food plant consumed. 3. The relative growth rates, pupal weights, weight of larva at the time of parasitoid emergence, and development times of L. dispar were affected significantly by the second larval food plant consumed. 4. Development time and size of Cotesia melanoscela were affected most by the second larval food plant consumed. 5. Parasitoid performance was affected most by the larval host’s relative growth rate and the final weight of the host larva at the time of parasitoid emergence. 6. Host plant switching affected the weight of L. dispar larvae at the time of parasitoid emergence, but the effect of switching per se was not a significant factor in C. melanoscela size or development. 7. Lymantria dispar larvae that fed on Populus as their second host outperformed larvae that fed ultimately on Acer. 8. Parasitoids yielded from L. dispar larvae that fed ultimately on Populus outperformed parasitoids yielded from larvae that fed ultimately on Acer. 9. Per cent mortality of L. dispar due to parasitism and percentage adult C. melanoscela emergence were highest in parasitized larvae fed Populus, poor in hosts fed Acer, and intermediate in switching larvae.     

Underestimated mitochondrial diversity in gypsy moth Lymantria dispar from Asia

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Characterization of hemolymph juvenile hormone esterase from Lymantria dispar

A major peak of juvenile hormone esterase (JHE) activity approaching 330 nmol JH III hydrolyzed/min/ml of hemolymph was observed during the last larval growth stage in Lymantria dispar. A smaller peak of JHE occurred 3–5 days after pupation. The gypsy moth JHE was purified from larval hemolymph using a classical approach. A specific activity of 766 units per mg of protein and a K_m of 3.6 × 10⁻⁷ M for racemic JH III and the (10R, 11S) enantiomer of JH II was determined for the purified enzyme. The 62 kDa esterase was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate (DFP), or by phenylmethylsulfonyl fluoride (PMSF). Two forms of JHE isolated by RP-HPLC were indistinguishable by HPLC tryptic peptide mapping and share an identical N-terminal amino acid sequence. Polyclonal antisera raised against gypsy moth enzyme cross-reacted with JHE from Trichoplusia ni but not with JHE from Manduca sexta. A weak cross-reactivity was observed with JHE from Heliothis virescens. Forty amino acid residues of the N-terminus were placed in sequence. The N-terminal sequence of JHE from L. dispar showed little homology to the sequence of JHE from H. virescens. The immunological and structural data support the conclusion that markedly different esterases, which catalyze the hydrolysis of juvenile hormone, are present in the hemolymph of different Lepidoptera.     

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.     

Phenology of Lymantria dispar (Lepidoptera: Lymantriidae), male flight and the effect of moth dispersal in heterogeneous landscapes

 A model of Lymantria dispar development was assembled from the published literature and used to predict the period of male moth flight in the United States. Model predictions were compared with observations made with pheromone traps in several locations throughout the United States but especially in Virginia, West Virginia and North Carolina between 1995 and 1996. The model was found to provide accurate and unbiased forecasts of the dates of 5%, 50% and 95% cumulative trap catch, particularly at lower elevations. In areas of high topographic diversity (such as West Virginia), deviations between model output and observations were minimized by basing predictions of 5% and 50% cumulative catch on minimum elevation within neighborhoods of 25–81 km². This model of L. dispar male flight phenology can be used to time the deployment and retrieval of pheromone traps in intensive or extensive monitoring programs. However, a better understanding of moth movement is needed to fully explain the patterns of local trap catch. Received: 9 October 1997/Accepted: 8 December 1997     

Juvenile hormone biosynthesis by corpus cardiacum-corpus allatum complexes of larval Lymantria dispar

• 1.1. A radiochemical assay was used to examine juvenile hormone (JH) synthesis and secretion in vitro by incubating two pairs of larval corpus cardiacum-corpus allatum complexes (CC-CA) from, Lymantria dispar, in 50 μl of osmotically balanced Grace's medium containing 1 μC1 [³H-methyl]-methionine for 6 hr.
• 2.2. For CC-CA of fourth instar female larvae, maximal incorporation of ³H-methyl was 0.15 pmol/pr/hr between days 2 and 3. High pressure liquid chromatographic (HPLC) analysis suggested that the biosynthetic products are mainly JH III with a little JH II at times.
• 3.3. For CC-CA of last instar female larvae, incorporation of ³H-methyl was 0.48 pmol/pr/hr at the beginning of the stadium and decreased to negligible levels by day 10. HPLC analysis suggested that CC-CA of last instar larvae produced only JH III. Volume increases in CA during the last instar were associated with declining activities of JH secretion.
• 4.4. Comparisons of maximal rates of ³ H-methyl incorporation by each unit volume of CA revealed that in the last instar each unit volume (μm³) of glandular tissue secreted 50% more JH than in the fourth instar.

     

Testis ecdysiotropic peptides in Rhodnius prolixus: biological activity and distribution in the nervous system and testis

In Rhodnius prolixus, testes from both pharate adult and adult males are shown to produce and release ecdysteroids in vitro. Proteinaceous brain extracts from these stages caused stimulation of ecdysteroid production by testes of unfed adults. Therefore, the brain of Rhodnius contains peptides with testis ecdysiotropic activity. The Lymantria testis ecdysiotropin (LTE) also stimulated the in vitro production of ecdysteroids by unfed adult testis but had no stimulatory effect on prothoracic glands. Western blot analysis of brain peptides using anti-LTE revealed the presence of several medium to small size immunoreactive peptides. Two of these peptides with sizes of 16.8 and 11.0 kDa were present only during pharate adult development and the adult stage. Immunohistochemical analysis using confocal laser scanning microscopy revealed abundant LTE-immunoreactive material in cytoplasmic granules of specific neurosecretory cells in the brain and suboesophageal ganglion and the epithelium of the testis sheath. Clusters of two cytologically distinct cell types were seen within the medial neurosecretory cells (MNC) and also a pair of neurons in the posterior protocerebrum. Feeding in both larvae and adult males resulted in massive release of LTE-immunoreactive material from the MNC cells, suggesting a role of LTE-related peptides in both larval-adult development and in male reproductive development. Release from the MNC cells of LTE-immunoreactive material exhibited a clear daily cycling during larval-adult development, which was synchronous with the rhythms of release of prothoracicotropic hormone and bombyxin reported previously. The testis sheath exhibited intense immunofluorescence in pharate adults and unfed adults, which disappeared following a blood meal. It is concluded that LTE-related peptides are developmentally regulated in several locations and may act as ecdysiotropins in Rhodnius. Those in the MNC cells are very probably classical hormones, i.e. are transported to their target sites via the insect haemolymph.     

Soluble cuticular phenoloxidase in the gypsy moth Lymantria dispar

The soluble enzyme phenoloxidase (tyrosinase) from the larval cuticle of Lymantria dispar has been partially purified using Ultrogel ACA 34, and the activity has been determined using phenolic substrates. The enzyme exhibited more activity toward O-diphenolic substrates and monophenolic substrates. The enzyme is inhibited by diethyl dithiocarbamate, phenylthiourea, and thiourea. The enzyme has been localized in the 7% slab and disc PAGE as an intense band. The enzyme is suggested to be involved in wound healing. © 1992 Wiley-Liss, Inc.     

Effects of pathogen exposure on life‐history variation in the gypsy moth (Lymantria dispar)

Investment in host defences against pathogens may lead to trade‐offs with host fecundity. When such trade‐offs arise from genetic correlations, rates of phenotypic change by natural selection may be affected. However, genetic correlations between host survival and fecundity are rarely quantified. To understand trade‐offs between immune responses to baculovirus exposure and fecundity in the gypsy moth (Lymantria dispar), we estimated genetic correlations between survival probability and traits related to fecundity, such as pupal weight. In addition, we tested whether different virus isolates have different effects on male and female pupal weight. To estimate genetic correlations, we exposed individuals of known relatedness to a single baculovirus isolate. To then evaluate the effect of virus isolate on pupal weight, we exposed a single gypsy moth strain to 16 baculovirus isolates. We found a negative genetic correlation between survival and pupal weight. In addition, virus exposure caused late‐pupating females to be identical in weight to males, whereas unexposed females were 2–3 times as large as unexposed males. Finally, we found that female pupal weight is a quadratic function of host mortality across virus isolates, which is likely due to trade‐offs and compensatory growth processes acting at high and low mortality levels, respectively. Overall, our results suggest that fecundity costs may strongly affect the response to selection for disease resistance. In nature, baculoviruses contribute to the regulation of gypsy moth outbreaks, as pathogens often do in forest‐defoliating insects. We therefore argue that trade‐offs between host life‐history traits may help explain outbreak dynamics.     

Effects of phytochemical variation in quaking aspen Populus tremuloides clones on gypsy moth Lymantria dispar performance in the field and laboratory

1. This study investigated how phytochemical variation among clones of quaking aspen Populus tremuloides, growing in a common habitat, affects the growth and fecundity of a model herbivore. 2. Gypsy moth Lymantria dispar larvae were reared from egg hatch to pupation on 10 aspen clones in the field or on excised foliage in the laboratory. Foliage was collected from each clone, and concentrations of phenolic glycosides, condensed tannins, nitrogen, and water were determined. 3. Herbivore fitness parameters and aspen phytochemical concentrations varied significantly among clones. In both the field and laboratory, larvae reared on clones containing high concentrations of phenolic glycosides exhibited prolonged developmental times and reduced pupal weights and fecundity. Herbivore performance parameters were also related positively to foliar nitrogen concentrations in the laboratory. Food consumption, but neither growth nor reproductive parameters, were related positively to condensed tannin concentrations. 4. In this study, foliar concentrations of phenolic glycosides were implicated as a significant determinant of food quality for gypsy moths, consistent with results of previous laboratory experiments. Additionally, this study documents a case in which host plant variation at a local level influences the performance and possibly the distribution and abundance of an important herbivore.     

Two different and sublethal isolates of Nosema lymantriae (Microsporidia) reduce the reproductive success of their host,Lymantria dispar

We examined the effects of two microsporidian isolates of Nosema lymantriae (Germany isolate; Schweinfurt isolate) on the reproductive success of Lymantria dispar L. All possible mating combinations were tested. Both isolates affected the fecundity of infected females and the hatch of neonates. The infection of female L. dispar with either isolate resulted in a higher proportion of non-viable eggs; the survival of neonates during egg stage was not affected. When L. dispar larvae were infected with N. lymantriae [Germany] the number of eggs per egg mass decreased between 24 and 61%. When both adults were infected, the hatch rate decreased to 26%. While the infection of the male or the female host with the Germany isolate affected the number of eggs per egg mass and the hatch of progeny, we did not find a significant effect when male hosts were infected with the Schweinfurt isolate; only infection of the female L. dispar resulted in a reduction of the number of eggs per egg mass between 26 and 37%.     

Effects of heavy metal stress on free amino acids in the haemolymph and proteins in haemolymph and total body tissue of Lymantria dispar larvae parasitized by Glyptapanteles liparidis

Heavy metal contamination of the forest pest insect Lymantria dispar (L.) (Lepidoptera; Lymantriidae), the gypsy moth, can alter its haemolymph composition, as has already been shown for carbohydrates and lipids in recent studies. L. dispar larvae are frequently parasitized by Glyptapanteles liparidis (Bouché) (Hymenoptera; Braconidae) larvae, which can—to some extent—regulate the population size of the pest insect. The parasitoids feed on the haemolymph of L. dispar larvae; hence, a different haemolymph composition of the host alters the trophic situation of the parasitoids. The aim of the present study was to investigate whether metal contamination also affects the concentrations of free amino acids in L. dispar haemolymph, and protein concentrations in their haemolymph and tissue. L. dispar larvae were parasitized on the first day of the second instar and then reared on diets contaminated with Cd, Pb, Cu or Zn at two concentrations each. Haemolymph and total body tissue of the larvae (fourth instar/third day) were analyzed. The concentrations of the free amino acids were elevated in five out of the eight contamination groups (Cd6, Pb4, Cu6, Cu10, Zn60), whereas haemolymph protein concentrations were significantly reduced in all contaminated individuals. The haemolymph protein concentration was 18 mg/ml in the control group and decreased to less than 10 mg/ml due to cadmium and zinc contamination at both concentrations and in the low copper contamination group. In contrast, total body proteins (136 g/mg dry weight in the control group) were elevated due to heavy metal stress. Analyses of haemolymph protein concentrations during the fourth instar demonstrated an increase of the proteins from day one to day four (followed by a decrease on the fifth day) in the control group and the cadmium contamination group. A steady increase of proteins from the first to the fifth day in the copper and zinc contaminated larvae indicated a retarded development in these groups. Thus, the present study along with other recent studies demonstrated, that heavy metal stress changes the concentrations of all main haemolymph compounds of L. dispar larvae.     

Suitability,digestibility and assimilation of various host plants of the gypsy moth Lymantria dispar L.

Summary The development and survival of gypsy moth (Lymantria dispar) larvae is strongly influenced by the host plant upon which they feed. The most rapid development and largest pupae were produced from grey birch fed larvae. Beech and maple-fed larvae produced the smallest pupae while maple-fed larvae exhibited prolonged development. White and red oak-fed larvae exhibited development and pupal weights intermediate between the above two groups. The approximate digestibility (AD) and efficiencies of conversion of food (ECD and ECI) were generally highest among grey birchfed individuals. The utilization of the relatively closely related oak species, as reflected in AD and ECD values, differed.Leaves were examined for 14 elements. The content of each element varied among host plant species and over time. For example, nitrogen levels were highest in grey-birch and dropped over time in all host plants.Lepidoptera: LymantriidaePaper No. 2277 Massachusetts Agricultural Experiment Station, University of Massachusetts at Amherst, MA U.S.A. This research supported (in part) from Experiment Station Project No. 437     

Vertical transmission and overwintering of microsporidia in the gypsy moth, Lymantria dispar

Vertical transmission and the overwintering success of three different microsporidia infecting Lymantria dispar (Lepidoptera: Lymantriidae) larvae were investigated. Endoreticulatus schubergi, a midgut pathogen, was transmitted to offspring via female and male via the egg chorion (transovum transmission). Between 8% and 29% of the emerging larvae became infected. No spores of E. schubergi were found in surface-washed eggs. Nosema lymantriae, a microsporidium that causes systemic infections, was transovarially transmitted. Between 35% and 72% of the progeny were infected. Vairimorpha disparis, a fat body pathogen, was not vertically transmitted. The infectivity of spores that overwintered in cadavers of infected L. dispar varied by species, placement in the environment, and weather conditions. Spores of E. schubergi were still infective after an eight month exposure period of cadavers on the ground. Spores of N. lymantriae and V. disparis remained highly infective only when cadavers overwintered under a more or less continuous snow cover for four months.