Fungal Pathogen Entomophaga Maimaiga Research Articles

Further spread of the gypsy moth fungal pathogen, Entomophaga maimaiga, to the west and north in Central Europe

The gypsy moth, Lymantria dispar, is one of the most important pests of deciduous trees in Europe, occurring in all environmental zones of Europe except the Alpine North and part of the Boreal zone. The gypsy moth fungal pathogen Entomophaga maimaiga, originally from Asia, was imported from the USA and released in Bulgaria, in 1999–2000. Spread of E. maimaiga in Central Europe was documented from 2011 to 2013, but this pathogen was only detected as far north as Slovakia. Collections of dead L. dispar larvae in 2019 (but not 2018), in areas of both sporadic and cyclic L. dispar outbreaks, resulted in the first detections of E. maimaiga in the Czech Republic. Lymantria dispar larvae were exposed to soil from oak forests in 2014–2018, but only infection by the gypsy moth nucleopolyhedrovirus (LdNPV) and the microsporidium Vairimorpha (= Nosema) lymantriae was detected. At most localities in the cyclic outbreak area, in 2017 the first defoliations of a new outbreak cycle occurred and the numbers of gypsy moth egg masses began to increase, so the calculated duration of approximately 13 years from outbreak to outbreak in the Czech Republic was verified.

Replacement of a dominant viral pathogen by a fungal pathogen does not alter the collapse of a regional forest insect outbreak.

Natural enemies and environmental factors likely both influence the population cycles of many forest-defoliating insect species. Previous work suggests precipitation influences the spatiotemporal patterns of gypsy moth outbreaks in North America, and it has been hypothesized that precipitation could act indirectly through effects on pathogens. We investigated the potential role of climatic and environmental factors in driving pathogen epizootics and parasitism at 57 sites over an area of ≈72,300 km(2) in four US mid-Atlantic states during the final year (2009) of a gypsy moth outbreak. Prior work has largely reported that the Lymantria dispar nucleopolyhedrovirus (LdNPV) was the principal mortality agent responsible for regional collapses of gypsy moth outbreaks. However, in the gypsy moth outbreak-prone US mid-Atlantic region, the fungal pathogen Entomophaga maimaiga has replaced the virus as the dominant source of mortality in dense host populations. The severity of the gypsy moth population crash, measured as the decline in egg mass densities from 2009 to 2010, tended to increase with the prevalence of E. maimaiga and larval parasitoids, but not LdNPV. A significantly negative spatial association was detected between rates of fungal mortality and parasitism, potentially indicating displacement of parasitoids by E. maimaiga. Fungal, viral, and parasitoid mortality agents differed in their associations with local abiotic and biotic conditions, but precipitation significantly influenced both fungal and viral prevalence. This study provides the first spatially robust evidence of the dominance of E. maimaiga during the collapse of a gypsy moth outbreak and highlights the important role played by microclimatic conditions.

Detection of presumptive mycoparasites associated with Entomophaga maimaiga resting spores in forest soils

The fungal pathogen Entomophaga maimaiga can provide high levels of control of the gypsy moth, Lymantria dispar, an important forest defoliator. This fungus persists in the soil as resting spores and occurs naturally throughout many areas where gypsy moth is established. Studies on the spatial dynamics of gypsy moth population have shown high variability in infection levels, and one possible biological factor could be the variable persistence of E. maimaiga resting spores in the soil due to attacks by mycoparasites. We surveyed presumptive mycoparasites associated with parasitized E. maimaiga resting spores using baiting and molecular techniques and identified an ascomycete (Pochonia sp.) and oomycetes (Pythium spp.).

Impact of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) on outbreak gypsy moth populations (Lepidoptera: Erebidae): the role of weather.

The fungal pathogen Entomophaga maimaiga Humber, Shimazu, and Soper is prevalent in gypsy moth [Lymantria dispar (L.)] populations throughout North America. To understand how weather-related variables influence gypsy moth-E. maimaiga interactions in the field, we measured fungal infection rates at 12 sites in central Pennsylvania over 3 yr, concurrently measuring rainfall, soil moisture, humidity, and temperature. Fungal mortality was assessed using both field-collected larvae and laboratory-reared larvae caged on the forest floor. We found significant positive effects of moisture-related variables (rainfall, soil moisture, and relative humidity) on mortality due to fungal infection in both data sets, and significant negative effects of temperature on the mortality of field-collected larvae. Lack of a clear temperature relationship with the mortality of caged larvae may be attributable to differential initiation of infection by resting spores and conidia or to microclimate effects. These relationships may be helpful in understanding how gypsy moth dynamics vary across space and time, and in forecasting how the gypsy moth and fungus will interact as they move into warmer or drier areas, or new weather conditions occur due to climate change.

Interactions between the introduced fungal pathogen Entomophaga maimaiga and indigenous tachinid parasitoids of gypsy moth Lymantria dispar in Bulgaria

Interactions between the gypsy moth, Lymantria dispar L. (Lepidoptera: Erebidae), pathogenic fungus Entomophaga maimaiga, and the tachinid parasitoid (Diptera: Tachinidae) complex in gypsy moth larvae were investigated in Bulgaria, where E. maimaiga was recently released as a biological control agent. Gypsy moth larvae were collected in oak stands where E. maimaiga was originally introduced (nine sites) and in sites where the pathogen has invaded by natural extension of the range (ten sites). In total, 4,375 host larvae were examined and 401 tachinid larvae emerged from parasitized hosts. Host mortality caused by tachinids varied from 0 to 48.5% among sites, with an overall average of 9.2%. Emerging adult tachinid parasitoids included 54 individuals belonging to six species: Compsilura concinnata, Exorista larvarum, Senometopia separata, Senometopia excisa, Drino incospiqua and Zenilia libatrix; the remaining parasitoids (86.5%) died in the pupal stage. E. maimaiga azygospores were observed on puparia surfaces, an indication that the gypsy moth host larvae were infected with the fungus. No azygospores were observed in parasitoid tissues. The high parasitoid mortality may be the result of the competition with E. maimaiga during development in the same host.

Release, establishment, and initial spread of the fungal pathogen Entomophaga maimaiga in island populations of Lymantria dispar

Biological invasions represent a major threat to the function and composition of ecosystems. Although the degree of invasion success of a non-native species and the consequent damage it causes can vary among and within invading species, the absence or presence of natural enemies associated with the invader can also play roles in the invasion dynamics. We used newly established, spatially isolated populations of the gypsy moth, Lymantria dispar (L.), on some of the islands within the archipelago of the Apostle Islands National Lakeshore in northwestern Wisconsin, USA, to study the establishment and initial spread of two releases of the entomophthoralean fungus Entomophaga maimaiga. We also explored patterns in rates of infection by the L. dispar nucleopolyhedrovirus, which was also released on three islands, and in the rates of parasitism by generalist parasitoids. The mean initial rate of spread by E. maimaiga following its successful establishment was 0.8kmyear−1, although it was detected as far as 6.1km from a release site after one year. Infection rates by both entomopathogens were highest on those islands where they were released; however, rates of parasitism by larval parasitoids were highest where neither pathogen had been released, suggesting that pathogens outcompete larval parasitoids. Understanding the intricate relationship between an invading host species and their associated pathogens during the early stages of invasion could enhance the use of biological control in invasive species management.

Assessing the climatic potential for epizootics of the gypsy moth fungal pathogen Entomophaga maimaiga in the North Central United States

The fungal pathogen Entomophaga maimaiga Humber, Shimazu et Soper has become an important biocontrol for gypsy moth ( Lymantria dispar (L.)) in the northeastern United States and is commonly introduced into new areas with established gypsy moth populations. Germination of the fungus is dependent on spring temperature and moisture, but specific conditions associated with epizootics have not been determined. Whether E. maimaiga will be as effective in other regions that experience different weather conditions is not yet known. We examined similarity of weather conditions associated with 16 documented E. maimaiga epizootics with conditions at 1351 North American locations using the climate-matching software CLIMEX. Based on CLIMEX’s overall index of climatic similarity, long-term annual climatic patterns across much of the eastern United States were 60%–80% similar to the conditions associated with epizootics. Monthly weather records from 1971 to 2000 in nine North Central states were examined to compare precipitation and temperature with conditions observed during epizootics. Based on climatic averages identified with the documented epizootics, temperature and precipitation conditions in Illinois, Indiana, Iowa, Kentucky, Missouri, and Ohio were more conducive for epizootics than conditions in Minnesota, Wisconsin, and Michigan, which were likely to support E. maimaiga epizootics in fewer than 6 of the 30 years considered.

Nondormancy in Entomophaga maimaiga azygospores: effects of isolate and cold exposure

Azygospores (resting spores) of the fungal pathogen Entomophaga maimaiga are produced in later larval instars of the gypsy moth Lymantria dispar and normally enter constitutive dormancy. In the laboratory cadavers of recently dead larvae containing immature azygospores were placed on 1.0% water agar at 20 C for 2 wk after host death, allowing time for azygospore maturation. We found that some azygospores produced in this way did not enter dormancy. To investigate nondormancy, suspensions of azygospores from individual cadavers were transferred to moist, sterile soil at 15 C. Groups of gypsy moth larvae were exposed sequentially to azygospores for consecutive 4 d periods for 196 d. Infections first were seen among larvae exposed 24–28 d after bioassays began, and infection potential continued 196 d. Germination tests confirmed azygospore germination. Additional cadavers containing azygospores produced under the same conditions were maintained at 4 C for 1–8 mo, and each month new sequential bioassays were initiated. There was a general trend of earlier initiation of infection with longer durations of 4 C exposure; after 6–8 mo at 4 C first infections occurred 6–10 d after bioassays began. With 5–8 mo at 4 C infection levels declined after 96 d of sequential bioassays. Activity of azygospores differed by the individual larval cadaver in which they were produced; azygospores from 29.2% of cadavers yielded only 0–0.3% infection. Infection from non-dormant azygospores did not differ among three Japanese isolates and two North American isolates, although azygospores from a third North American isolate caused no infections in 84 d, suggesting that dormancy had not been prevented.

Genetic diversity in the gypsy moth fungal pathogen Entomophaga maimaiga from founder populations in North America and source populations in Asia

Entomophaga maimaiga is a naturally occurring fungal pathogen specific to larvae of the gypsy moth, Lymantria dispar. E. maimaiga is thought to be native to Asia where its epizootics can suppress gypsy moth outbreaks. However, in the USA this beneficial fungal pathogen was not observed until 1989, although an isolate of E. maimaiga from Tokyo was released in Massachusetts to control gypsy moths as early as in 1910-1911, and another isolate from Ishikawa Prefecture in Japan was later released in 1985 and 1986 in New York and Virginia. Our objectives were to: (1) test the hypothesis that E. maimaiga populations in the USA have reduced genetic variability due to founder effects compared to the putative ancestral populations in Asia; (2) track the origin of the North American populations of this fungus; and (3) assess whether genetic differences among E. maimaiga isolates are correlated to morphological differences. We compared genetic diversity among 30 E. maimaiga isolates originating from seven states in the USA, five prefectures in Japan, one province of China and one region of far eastern Russia by AFLPs. Among 14 USA isolates, only ten polymorphic AFLP loci were found, whereas 56 polymorphic loci were found among 16 Asian isolates; 29 loci were polymorphic among 12 isolates from Japan alone. Average gene diversity (h) for the polymorphic loci was 0.223 +/- 0.005 for Asia (including Japan), 0.131 +/- 0.006 for Japan only, and 0.041 +/- 0.004 for the USA. Thus, native populations from Asia were more diverse than the USA populations. These results are consistent with the expectation of a population founded from a source population by a small number of individuals. Distance and parsimony analyses of AFLP data showed that the isolates from the USA formed one distinct clade that was most closely related to Japanese isolates collected outside the Tokyo area. No morphological variation of E. maimaiga from different geographical locations was detected.

Virulence and fitness of the fungal pathogen Entomophaga maimaiga in its host Lymantria dispar, for pathogen and host strains originating from Asia, Europe, and North America

In this study, we tested (1) whether non-North American gypsy moth strains are susceptible to North American isolates of Entomophaga maimaiga and (2) the potential for erosion in the efficacy of E. maimaiga in controlling gypsy moth. We used bioassays to assess the variability in virulence (measured as time to death) as well as fitness of the pathogen (measured as spore production) in four gypsy strains challenged with six E. maimaiga isolates, using host and pathogen strains originating from Asia, Europe, and North America. We found that all E. maimaiga isolates tested were pathogenic to all strains of Lymantria dispar, regardless of the geographical origin of the fungal isolate, with at least 86% mortality for all combinations of fungal isolate and gypsy moth strain. We therefore conclude that Asian gypsy moths are susceptible to North American strains of E. maimaiga. No significant interactions between fungal isolates and gypsy moth strains with regard to time to death were found, indicating that each fungal isolate had the same overall effect on all the gypsy moth strains tested. However, fungal isolates differed significantly with regard to virulence, with a Russian isolate being the slowest to kill gypsy moth (5.1 ± 0.1 days) and a Japanese isolate being the overall fastest to kill its host (4.0 ± 0.1 days). Fungal isolates also differed in fitness, with variability in types of spores produced. These differences in virulence and fitness were, however, not correlated with geographical origin of the fungal isolate. Gypsy moth strains had no or only little effect on fungal virulence and fitness. Based on our studies with laboratory-reared gypsy moth strains, erosion of successful control of gypsy moth by E. maimaiga seems unlikely.

Epizootic occurrence of Entomophaga maimaiga at the leading edge of an expanding population of the gypsy moth (Lepidoptera: Lymantriidae) in north-central Ontario

Ever since its entry from New York State in the late 1960s, the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), has continued to expand its distribution in Ontario to the north and west (Nealis and Erb 1993). Outbreaks were recorded for the first time in the Sudbury – North Bay region in the early 1990s, by which time there was evidence of resident populations extending along the north shore of Lake Huron as far west as Lake Superior. The population expansion along the north shore is characterized by a patchy occurrence of small outbreaks, which typically last for a few years and then disappear (Annual Forest Health Reports, Great Lakes Forestry Centre, http://www.glfc.cfs.nrcan.gc.ca/foresthealth/index_e.html). Nealis et al. (1999) found that winter weather, parasitoids, and the gypsy-moth-specific fungal pathogen Entomophaga maimaiga Humber, Shimazu et Soper (Zygomycetes: Entomophthorales) were the most prominent sources of mortality in those transient outbreaks.

Persistence of the fungal pathogen Entomophaga maimaiga and its impact on native Lymantriidae

The entomopathogenic fungus Entomophaga maimaiga has been reported infecting gypsy moth, Lymantria dispar , larvae in the United States since 1989. Laboratory bioassays demonstrated that larvae of numerous species of native tussock moths (Lymantriidae) are susceptible to E. maimaiga . From 1997 to 2001, larvae of gypsy moth and native lymantriids were collected in long-term plots in national forests in Virginia and West Virginia to evaluate infection levels. Throughout this time, gypsy moth populations were low although increasing the last 2 years, whereas native lymantriids remained uncommon throughout. Among gypsy moth larvae, infection by E. maimaiga first occurred during 2000 and increased in both areas during 2001 as gypsy moth populations increased. Seven species of native lymantriids were collected but only three were infected: Dasychira obliquata , Dasychira vagans , and Orgyia leucostigma . Infection of these native lymantriid species did not occur in 1997–1999 and was always <50% per species during 2000 and 2001. Ecological studies were conducted to evaluate when and where native lymantriids could be exposed to E. maimaiga inoculum. Field bioassays demonstrated that E. maimaiga resting spores were active in soil at bases of trees beginning in April, especially when soil was moist, but few gypsy moth larvae were infected by airborne conidia from resting spores early in the season. In 2000 and 2001, cadavers that produced conidia were collected throughout the season and conidia were the predominant spore form produced for at least the first month of the field season. Resting spore densities in soil were high during 1997 but titers declined through the study, with the exception of an increase following E. maimaiga infections in increasing gypsy moth populations in West Virginia during 2000.

Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle.

The lepidopteran-specific fungal pathogen Entomophaga maimaiga is highly virulent against Lymantria dispar (gypsy moth) larvae, and other members of the family Lymantriidae. Numerous species in the subfamily Cuculliinae (Family Noctuidae) are not susceptible to E. maimaiga due to the inability of this fungus to penetrate the larval cuticle. Conidial attachment and germination were compared among five cuculliine species and L. dispar using bioassays and scanning electron microscopy. Although conidia were showered evenly across larvae during bioassays, on L. dispar conidia were most abundant on segments, where they adhered well to the cuticle and germinated at high percentages. Conidia on cuculliine cuticles were predominantly found in large, loose aggregations in intersegmental areas. Few conidia on cuculliine cuticle germinated and scanning electron microscopy revealed a thick film of mucous enveloping conidia. We hypothesize that the conidia on cuculliines become coated by this film and were only loosely attached to the larval cuticle. No such film was seen on L. dispar larvae where individual conidia appeared well attached. On L. dispar larvae many conidia also adhered to setae. To determine if hydrophobicity affected the ability of E. maimaiga conidia to attach and germinate on a substrate, a goniometer was used to determine relative hydrophobicity of larval cuticles. L. dispar cuticle was more hydrophobic than cuculliine cuticle, suggesting that a high level of hydrophobicity could be a required characteristic for hosts. Cuticles from four cuculliine species and L. dispar were sequentially extracted using hexane, chloroform, and methanol. Conidia were showered onto glass slides coated with the different extracts and germination was quantified. Methanol extracts of cuculliine cuticle consistently decreased germination, compared to all extracts of L. dispar cuticle. For all L. dispar extracts, the majority of conidia produced germ tubes, which is a normal prerequisite for cuticular penetration. For the cuculliines, conidia exposed to hexane and chloroform extracts produced secondary conidia as did all controls, but the conidia exposed to cuculliine methanol extracts that germinated produced germ tubes. These studies demonstrated that a range of factors act in concert to prevent E. maimaiga infection of the cuculliine species investigated.

Modeling the Impact of the FungusEntomophaga maimaiga(Zygomycetes: Entomophthorales) on Gypsy Moth (Lepidoptera: Lymantriidae): Incorporating Infection by Conidia

A computer model of the interaction between the fungal pathogen Entomophaga maimaiga Humber, Shimazu & Soper and its host, the gypsy moth, Lymantria dispar (L.), had previously been developed to simulate infection by fungus resting spores (azygospores) that are present in soil and germinate in spring if moisture is adequate. This model is now expanded to include infection caused by conidia that are produced on dead, infected caterpillars when relative humidity is near 100%. As inputs, the model uses temperature, relative humidity, rainfall, abundance of resting spores in the soil, and gypsy moth population density. From plots in 1999, 2000, and 2001, data on weather conditions (the first year from daily weather station data and the last two from in-plot data loggers) were obtained, and gypsy moth density and resting spore abundance were determined. Also, collections of gypsy moth larvae were taken weekly to determine proportion of infection by the fungus. These data were used to find best-fitting parameters for the model. Using the same parameter values, model output was close to data for all years. When conidial dispersal was incorporated in the model, output and data were even closer. A validated model should be useful for evaluating and predicting gypsy moth-fungus interactions in forests.

Larval behavior in Lymantria dispar increases risk of fungal infection.

Late-instar larvae of the forest defoliator Lymantria dispar display relatively unusual behavior for Lepidoptera. Late instars move down from the tree canopy and wander and rest in dark locations during daylight hours. When we sampled the area extending from below 3m and within 200cm of tree trunks during daylight hours, 71% of L. dispar late instar larvae were found at ground level. Providing dark resting locations on the soil surface where there was no litter resulted in rapid location and colonization of these sites by late instar larvae. L. dispar larvae were always more prevalent in leaf litter 0-50cm from tree trunks compared with 50-200cm away. In an area where the fungal insect pathogen Entomophaga maimaiga is established, larvae were caged on tree trunks, in the foliage, or on top of soil during photophase or scotophase to determine in which locations risk of infection was greatest. At both times of day, highest infection levels always occurred on the soil, with least infection among larvae caged in the foliage. Infection levels were greater during photophase than scotophase. When larvae were exposed to soil for shorter periods during daylight hours to mimic wandering, 4.7 and 6.1% became infected after 30- and 60-min exposure intervals, respectively, with increasing infection associated with longer exposure times. The high levels of infection by E. maimaiga that have been documented in L. dispar populations since this pathogen was first found in North America are consistent with the strong litter-dwelling behavior of late-instar L. dispar larvae. Rarity of other lepidopteran larvae at ground level could help to explain the host specificity of this pathogen in the field.

Entomophaga maimaiga(Zygomycete: Entomophthorales) Resting Spores and Biological Control of the Gypsy Moth (Lepidoptera: Lymantriidae)

Neonate and larger gypsy moth, Lymantria dispar (L.), larvae were collected in a forest and exposed in the leaf litter and trees to determine proportions infected by the fungal pathogen Entomophaga maimaiga Humber, Shimazu & Soper. Results were used to refine a previously developed model of fungus infection of gypsy moths. The revised model only allowed infection of 4th- and larger instar gypsy moths through the germination of resting spores. Infection of neonates via germinating resting spores and of larger gypsy moths through release of conidia from sporulating cadavers was not included because adequate information about transmission rates were not available. This model produced a generally good fit to weekly infection data obtained from collected early to midsized gypsy moth larvae in 1990, 1991, and 1998, so long as a multiplying parameter that did not change the shape of the curve was permitted to vary. This parameter was correlated with a measure of infection caused by resting spores. The intensity of spore germination and resulting infection in the forest is an important predictor of infection by this fungus of early to midsized gypsy moth larvae, and ways to measure the infection potential of such spores before gypsy moth larvae hatch in the spring would greatly aid efforts to predict the impact of this biological control agent.

Pathology and epizootiology of Entomophaga maimaiga infections in forest Lepidoptera.

The insect-pathogenic fungal pathogen Entomophaga maimaiga is endemic to northeastern Asia and was first found in North America in 1989. Due to repeated epizootics and spread within populations of the major forest defoliator in northeastern North America, the gypsy moth (Lymantria dispar), this pathogen has gained much notoriety. Although this pathogen was purposely introduced to North America for biological control of L. dispar in 1910 to 1911, it is questionable whether it became established at the time of release and then remained at innocuous levels until relatively recently. Alternatively, the fungal strain present in North America today could be a more recent accidental introduction. DNA analysis demonstrates that this pathogen differs significantly from North American members of the same species complex (the Lepidoptera-specific Entomophaga aulicae species complex), and, to date, isolates of this introduced pathogen display little heterogeneity in North America. Nonsusceptible lepidopteran larvae have been identified, and either E. maimaiga is unable to penetrate the cuticle or the fungus cannot survive within the hemocoel. In the latter case, although E. maimaiga grows as protoplasts lacking cell walls in the host hemolymph, glycoproteins on plasma membranes of the protoplasts could lead to host recognition. Epizootiological studies demonstrate a clear association between fungal activity and environmental moisture but little association with host density under hypothesized conditions of high fungal density. Prediction of the occurrence of epizootics is not yet possible. E. maimaiga is easily established in new areas by releasing azygospores, but the ability to use this pathogen further for biological control will depend, in large part, on the development of mass production systems.

Distribution of resting spores of the Lymantria dispar pathogen Entomophaga maimaiga in soil and on bark

Cadavers of late instar Lymantria dispar (gypsy moth) larvae killed by the fungal pathogen Entomophaga maimaiga predominantly contain resting spores (azygospores). These cadavers frequently remain attached to tree trunks for several weeks before they detach and fall to the ground. Density gradient centrifugation was used to quantify resting spores in the soil and on tree bark. Titers of resting spores were extremely high at 0–10 cm from the base of the tree and the number decreased with distance from the trunk of the tree. Titers were also highest in the organic layer of the soil with numbers decreasing precipitously with increasing depth in the soil. While resting spores were obtained from tree bark, densities per unit area were much lower than those found in the organic soil layer at the base of the tree. Field bioassays were conducted with caged L. dispar larvae to compare infection levels with distance from the tree trunk as well as on the trunk. Highest infection levels were found at 50cm from the tree base with lowest infection on the tree trunk at 0.5 m height, although we expected the highest infection levels among larvae caged at the bases of trees, where highest spore titers occurred. Laboratory experiments demonstrated that L. dispar larvae exposed to resting spore- bearing soil at the soil surface became infected while larvae exposed to soil with resting spores buried at least 1 cm below the surface did not become infected.

Formation and germination of Entomophaga maimaiga azygospores

Azygospores (resting spores) of the gypsy moth fungal pathogen Entomophaga maimaiga are produced in abundance during late spring and early summer in late-instar gypsy moth larvae (Lymantria dispar). Azygospores subsequently form, each from an individual hyphal body. Development of azygospores occurs asynchronously over several days; by 5 days after host death, greater than 60% of fungal cells had matured from hyphal bodies to the final double-walled resting state. Azygospores undergo constitutive dormancy and, under field conditions, will not germinate for approximately 9 months after production. Azygospores do not require nutrients to germinate. Germination of field-collected resting spores under laboratory conditions began more than 2 days after transfer from the field to the laboratory. Higher levels of germination occurred with a 14 h L: 10 h D cycle compared with 13 h L: 11 h D or 12 h L: 12 h D. Azygospores germinate relatively slowly and germination rates were greatest between 4 and 8 days, with a total of 71.8 or 72.5% germination by 16 days at 14 h L: 10 h D and 15 or 20 °C, respectively. During 1994 and 1995, resting spores began causing infections in experimental larvae in early May, about 1 – 2 weeks prior to gypsy moth egg hatch, and ceased causing infections in mid to late June, when late instars were present. This latter timing is a correction of previously reported information. Bioassays investigating resting spore activity determined that during 1994, once resting spores began germinating in the field, levels of infection were positively associated with soil moisture. Key words: azygospores, resting spores, entomopathogenic fungi, Entomophaga maimaiga, Lymantria dispar, biological control.

Fungal and Viral Epizootics in Gypsy Moth (Lepidoptera: Lymantriidae) Populations in Central New York

Abstract The activity of the gypsy moth fungal pathogen Entomophaga maimaiga was documented from 1991 to 1996 in central New York. This fungus had been introduced to the area in 1990 but also generally spread throughout the region in 1990–1992. In 1991, gypsy moth populations were abundant in 8 mixed hardwood plots (mean ± SE = 15,295 ± 4373 egg masses/ha), the gypsy moth nuclear polyhedrosis virus (LdNPV) was the most important cause of larval mortality by microorganisms, and by the end of the season, populations had decreased to 6875 ± 1345 egg masses/ha. During 1991, E. maimaiga -infected larvae were found at 7 of 8 plots causing 0–37% infection but this was not associated with 1990 E. maimaiga infection levels. In 1992, the major cause of larval mortality by microorganisms was E. maimaiga, with infection levels ranging from 64 to 98%; LdNPV was also present at all 11 plots sampled. At lower density plots in 1992, E. maimaiga infections only increased dramatically during 5th and later instars. In higher density plots, 16 and 24% of 2nd and 3rd instars per the two early June sample dates were infected by E. maimaiga but infection levels once again peaked during later instars, although earlier in higher than lower density sites. During 1992, more cadavers of 4th and 5th + instars produced resting spores in the higher density plots than in the lower density plots, suggesting increased secondary transmission by conidia actively ejected from cadavers. The amount of rain falling during the periods of time that larvae were active differed by year; LdNPV was the dominant pathogen during the dry 1991 spring, while E. maimaiga flourished during 1992, when rain fell at approximately normal levels. From 1993 to 1996, gypsy moth populations remained low, with almost no LdNPV infections recovered, and E. maimaiga infections increased only after larval densities increased.