Survival and differential development of Entomophaga maimaiga and Entomophaga aulicae (Zygomycetes: Entomophthorales) in Lymantria dispar hemolymph

The closely related entomophthoralean fungi Entomophaga aulicae and E. maimaiga are both host-specific pathogens of lepidopteran larvae. However, these fungi do not have the same host range. The first objective of this study was to compare the fate of E. aulicae in the nonpermissive host Lymantria dispar with the fate of the successful pathogen E. maimaiga over the same time period. In the hemolymph of L. dispar injected with E. maimaiga protoplasts, the number of hemocytes demonstrated a decreasing trend after the first day postinjection and hemocytes completely disappeared by day 5, with the majority of larvae dying in 5.6 +/- 0.1 days. In L. dispar larvae, E. maimaiga infections developed successfully, evidenced by increasing numbers of protoplasts and hyphal bodies prior to host mortality. In contrast, at day 5 hemocytes were readily visible in hemolymph of E. aulicae-injected larvae, but E. aulicae cells did not increase in numbers, although persisting in the hemolymph for at least 16 days postinjection. For both fungal species, when hemolymph samples from injected insects were introduced to culture media viable fungal cultures were always produced. Both E. aulicae and E. maimaiga occurred in hemolymph initially after injection as protoplasts. For E. maimaiga, after day 3, <50% of fungal cells were hyphal bodies until insect death when most cells regenerated cell walls. For E. aulicae, from day 2 equal numbers of fungal cells in the hemolymph occurred as protoplasts and hyphal bodies. To investigate the cause of fungistasis in E. aulicae-injected larvae, E. aulicae cell cultures exposed to partially purified protein fractions from hemolymph of larvae infected with either fungus displayed increased lysis and decreased viability at lower concentrations of protein fractions compared with E. maimaiga cell cultures. These studies demonstrate that E. aulicae does not increase in L. dispar hemolymph, although it persists and results suggest that proteinaceous factors induced within the hemolymph may limit the capacity of E. aulicae to develop successful infections.     

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.     

Introduced pathogens follow the invasion front of a spreading alien host

1.?When an invasive species first colonizes an area, there is an interval before any host-specific natural enemies arrive at the new location. Population densities of newly invading species are low, and the spatial and temporal interactions between spreading invasive species and specific natural enemies that follow are poorly understood. 2.?We measured infection rates of two introduced host-specific pathogens, the entomophthoralean fungus Entomophaga maimaiga and the baculovirus Lymantria dispar nucleopolyhedrovirus (LdNPV), occurring in spreading populations of an invasive forest defoliator, L. dispar (gypsy moth), in central Wisconsin. 3.?Over 3 years, we found that host density was closely associated with the presence and prevalence of both pathogens. The fungal and viral pathogens differed in the sensitivity of their response as E. maimaiga was present in lower-density host population than LdNPV. 4.?We examined the relationship between weather conditions and infection prevalence and found that activity of both the fungus and virus was strongly seasonally influenced by temperature and rainfall or temperature alone, respectively. 5.?Purposeful releases of pathogens (median distances of study sites from release sites were 65·2 km for E. maimaiga and 25·6 km for LdNPV) were not associated with pathogen prevalence. 6.?A generalist fly parasitoid, Compsilura concinnata, also killed L. dispar larvae collected from the study sites, and parasitism was greater when infection by pathogens was lower. 7.?Our results demonstrated that although infection levels were low in newly established host populations, host-specific pathogens had already moved into host populations close behind advancing populations of an invasive host; thus, spreading hosts were released from these enemies for only a relatively short time.     

Diurnal pattern of death and sporulation in Entomophaga maimaiga-infected Lymantria dispar

Entomophaga maimaiga Humber, Shimazu, et Soper (Zygomycotina: Entomophthoraceae) is a naturally occurring obligate fungal pathogen specific to gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae) larvae. This fungus is considered the most important natural enemy of this pest insect in North America and Asia. A critically important step for the development of E. maimaiga epizootics is the transmission of propagules to healthy larvae, a process known to require high humidity. Some pathogens are known to manipulate the time of day that hosts die so that propagules are produced to maximize chances of survival and thus enhance transmission. The objective of this study was to assess whether E. maimaiga manipulates L. dispar to die at a certain time of day. Laboratory bioassays were conducted at 15 and 20 °C to record the 24‐h activity pattern of death and sporulation exhibited under an L14:D10 photoperiod and 100% r.h. by four isolates of E. maimaiga in its host L. dispar. Events were recorded every 4 h. Our results clearly demonstrate that E. maimaiga‐infected L. dispar larvae die mainly in the afternoon and that the fungus sporulates during the night. The rhythm was independent of the fungal isolate tested and type of spores produced after larval death. By raising the temperature from 15 to 20 °C, the peak death time narrowed and sporulation was initiated earlier at night.     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.     

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.     

New Tachinidae (Diptera) host records of eastern North American forest canopy Lepidoptera: baseline data in a Bacillus thuriengiensis variety kurstaki nontarget study.

Macrolepidopteran caterpillars collected in 1995 and 1996 in the Monongahela National forest, Pocahontas County, WV, and the George Washington National forest, Augusta County, VA, yielded 60 previously unreported tachinid host associations. Most associations were between native species, but the introduced polyphagous tachinid Compsilura concinnata (Meigen) produced eight new associations with native hosts. The tachinids collected were slightly broader in their host preferences than associated Braconidae and Ichneumonidae, illustrating their potential importance in regulating foliage-feeding macrolepidopteran populations in the region studied. The sample years reported herein are the pretreatment baseline portion of a long-term study on the effects of Lymantria dispar (L.) defoliation, efficacy of applied Bacillus thuringiensis variety kurstaki Berliner, and the natural occurrence of the L. dispar pathogen Entomophaga maimaiga Humber, Shimazu & Soper on nontarget organisms.     

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.     

Storage of Resting Spores of the Gypsy Moth Fungal Pathogen, Entomophaga maimaiga

The fungal pathogen, Entomophaga maimaiga causes epizootics in populations of the important North American forest defoliator gypsy moth ( Lymantria dispar ). Increasing use of this fungus for biological control is dependent on our ability to produce and manipulate the long-lived overwintering resting spores (azygospores). E. maimaiga resting spores undergo obligate dormancy before germination so we investigated conditions required for survival during dormancy as well as the dynamics of subsequent germination. After formation in the field during summer, resting spores were stored under various moisture levels, temperatures, and with and without soil in the laboratory and field. The following spring, for samples maintained in the field, germination was greatest among resting spores stored in plastic bags containing either moistened paper towels or sterile soil. Resting spores did not require light during storage to subsequently germinate. In the laboratory, only resting spores maintained with either sterile or unsterilized soil at 4°C (but not at 20 or -20°C) germinated the following spring, but at a much lower percentage than most field treatments. To further investigate the effects of relative humidity (RH) during storage, field-collected resting spores were placed at a range of humidities at 4°C. After 9.5 months, resting spore germination was highest at 58% RH and no resting spores stored at 88 or 100% RH germinated. To evaluate the dynamics of infections initiated by resting spores after storage, gypsy moth larvae were exposed to soil containing resting spores that had been collected in the field and stored at 4°C for varying lengths of time. No differences in infection occurred among larvae exposed to fall-collected soil samples stored at 4oC over the winter, versus soil samples collected from the same location the following spring. Springcollected resting spores stored at 4°C did not go into secondary dormancy. At the time that cold storage of soil containing resting spores began in spring, infection among exposed larvae was initiated within a few days after bringing the soil to 15°C. This same pattern was also found for spring-collected resting spore-bearing soil that was assayed after cold storage for 2-7 months. However, after 31-32 months in cold storage, infections started 14-18 days after soil was brought to 15°C, indicating a delay in resting spore activity after prolonged cold storage.     

Colonization History,Host Distribution,Anthropogenic Influence and Landscape Features Shape Populations of White Pine Blister Rust,an Invasive Alien Tree Pathogen

White pine blister rust is caused by the fungal pathogen Cronartium ribicola J.C. Fisch (Basidiomycota, Pucciniales). This invasive alien pathogen was introduced into North America at the beginning of the 20th century on pine seedlings imported from Europe and has caused serious economic and ecological impacts. In this study, we applied a population and landscape genetics approach to understand the patterns of introduction and colonization as well as population structure and migration of C. ribicola. We characterized 1,292 samples of C. ribicola from 66 geographic locations in North America using single nucleotide polymorphisms (SNPs) and evaluated the effect of landscape features, host distribution, and colonization history on the structure of these pathogen populations. We identified eastern and western genetic populations in North America that are strongly differentiated. Genetic diversity is two to five times higher in eastern populations than in western ones, which can be explained by the repeated accidental introductions of the pathogen into northeastern North America compared with a single documented introduction into western North America. These distinct genetic populations are maintained by a barrier to gene flow that corresponds to a region where host connectivity is interrupted. Furthermore, additional cryptic spatial differentiation was identified in western populations. This differentiation corresponds to landscape features, such as mountain ranges, and also to host connectivity. We also detected genetic differentiation between the pathogen populations in natural stands and plantations, an indication that anthropogenic movement of this pathogen still takes place. These results highlight the importance of monitoring this invasive alien tree pathogen to prevent admixture of eastern and western populations where different pathogen races occur.     

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.     

Population genetic structure of a common host predicts the spread of white‐nose syndrome,an emerging infectious disease in bats

Landscape complexity influences patterns of animal dispersal, which in turn may affect both gene flow and the spread of pathogens. White‐nose syndrome (WNS) is an introduced fungal disease that has spread rapidly throughout eastern North America, causing massive mortality in bat populations. We tested for a relationship between the population genetic structure of the most common host, the little brown myotis (Myotis lucifugus), and the geographic spread of WNS to date by evaluating logistic regression models of WNS risk among hibernating colonies in eastern North America. We hypothesized that risk of WNS to susceptible host colonies should increase with both geographic proximity and genetic similarity, reflecting historical connectivity, to infected colonies. Consistent with this hypothesis, inclusion of genetic distance between infected and susceptible colonies significantly improved models of disease spread, capturing heterogeneity in the spatial expansion of WNS despite low levels of genetic differentiation among eastern populations. Expanding our genetic analysis to the continental range of little brown myotis reveals strongly contrasting patterns of population structure between eastern and western North America. Genetic structure increases markedly moving westward into the northern Great Plains, beyond the current distribution of WNS. In western North America, genetic differentiation of geographically proximate populations often exceeds levels observed across the entire eastern region, suggesting infrequent and/or locally restricted dispersal, and thus relatively limited opportunities for pathogen introduction in western North America. Taken together, our analyses suggest a possibly slower future rate of spread of the WNS pathogen, at least as mediated by little brown myotis.     

First record of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) in Georgia

In 2005, high levels of mortality occurred in an outbreak of the gypsy moth population in Georgia. Resting spores typical of entomophthoralean fungi were found within larval cadavers and molecular analyses confirmed that the pathogen was Entomophaga maimaiga. This is the first record of this entomopathogen in Georgia and in this part of Europe.     

Host persistence or extinction from emerging infectious disease: insights from white-nose syndrome in endemic and invading regions

Predicting species'' fates following the introduction of a novel pathogen is a significant and growing problem in conservation. Comparing disease dynamics between introduced and endemic regions can offer insight into which naive hosts will persist or go extinct, with disease acting as a filter on host communities. We examined four hypothesized mechanisms for host–pathogen persistence by comparing host infection patterns and environmental reservoirs for Pseudogymnoascus destructans (the causative agent of white-nose syndrome) in Asia, an endemic region, and North America, where the pathogen has recently invaded. Although colony sizes of bats and hibernacula temperatures were very similar, both infection prevalence and fungal loads were much lower on bats and in the environment in Asia than North America. These results indicate that transmission intensity and pathogen growth are lower in Asia, likely due to higher host resistance to pathogen growth in this endemic region, and not due to host tolerance, lower transmission due to smaller populations, or lower environmentally driven pathogen growth rate. Disease filtering also appears to be favouring initially resistant species in North America. More broadly, determining the mechanisms allowing species persistence in endemic regions can help identify species at greater risk of extinction in introduced regions, and determine the consequences for disease dynamics and host–pathogen coevolution.     

A PCR-based method to identify Entomophaga spp. infections in North American grasshoppers

A PCR-based method was developed for the detection and identification of two species of grasshopper-specific pathogens belonging to the genus Entomophaga in North America, Entomophaga calopteni and Entomophaga macleodii. Two separate sets of primers specific for amplification of a DNA product from each species of Entomophaga as well as a positive control were utilized. Grasshoppers were collected from two sites in Mexico during an epizootic with grasshoppers found in "summit disease", typical of Entomophaga infections. There was a preponderance of Melanopline grasshoppers infected by E. calopteni. The described method is an accurate tool for identification of North American grasshopper infections by Entomophaga species.     

Cloned DNA probes distinguish endemic and exotic Entomophaga grylli fungal pathotype infections in grasshopper life stages

Entomophaga grylli is a fungal pathogen of grasshoppers and at least three pathotypes are recognized world-wide. Pathotypes 1 and 2 are endemic to North America while the Australian pathotype 3 had been released into two field sites in North Dakota between 1989 and 1991. Grasshoppers were collected over the summer at the field sites in 1992 and assessed for pathotype infection by cloned DNA probe analysis. The three most predominant grasshopper species that were infected ( Melanoplus sanguinipes, M. bivittatus and Camnula pellucida ) were assessed for pathotype infection with respect to their life stages (nymphal instars and adult males and females). Pathotype 1 predominantly infected grasshoppers in the subfamilies Oedipodinae and Gomphocerinae and pathotype 2 predominantly infected grasshoppers in the subfamily Melanoplinae. Early-instar M. sanguinipes and M. bivittatus had higher pathotype 2 infection frequencies, while late-instar and adult C. pellucida had higher pathotype 1 infection frequencies. Cross-infection by the pathotypes did occur in up to 3% of the individuals, on a per species basis, and primarily in later instar and adult grasshoppers. Pathotype 3 infections occurred in later instar and adults of the three grasshopper species. Infection of grasshoppers by E. grylli pathotypes is discussed with reference to the fungal life cycles.     

The role of resting spores in the survival of the mite-pathogenic fungus Neozygites floridana from Mononychellus tanajoa during dry periods in Brazil

Survival of pathogens during long periods of unfavorable conditions can be critical to their ecology and to their use in biological control. In northeastern Brazil, the mite pathogen Neozygites floridana must survive hot and dry conditions between wet seasons when it infects the cassava green mite Mononychellus tanajoa. We report on large numbers of mite cadavers bearing resting spores towards the end of epizootics in mid-1995. High within-leaf variability indicated that local factors may be important in determining resting spore formation. These spores remain in the host cadaver on a leaf until the cadaver breaks up, whereupon the spores fall freely to the soil, there to remain dormant. Laboratory simulation of field conditions led to ca. 25% of mycosed individuals bearing resting spores. Mummies (without resting spores) kept in hot and dry conditions showed little or no viability within 2 months, implying no role for survival over extended dry periods. It is proposed that resting spores form the principal means by which this pathogen survives the dry season in the study area. This has implications for its introduction to new areas in classical biological control.     

Chytrid mycoparasitism of entomophthoralean azygospores

Mycoparasitism – when one fungus parasitizes another – has been reported to affect Beauveria bassiana and mycorrhizal fungi in the field. However, mycoparasitism of any fungi in the Order Entomophthorales has never been reported before now. The majority of entomophthoralean species persist as resting spores (either zygospores or azygospores) in the environment and dormant entomophthoralean resting spores (whether formed as zygospores or azygospores) are thought to be especially well adapted for survival over long periods due to their thick double walls. Entomophthoralean resting spores can accumulate in the soil as large reservoirs of inoculum which can facilitate the onset and development of epizootics. We report parasitism of azygospores of the gypsy moth pathogen Entomophaga maimaiga caged in soil from southern Ohio by the chytrid fungus Gaertneriomyces semiglobifer. G. semiglobifer had previously been isolated from soil samples from North America, Europe and Australia or horse manure from Virginia. After isolation and identification of G. semiglobifer, azygospores of E. maimaiga exposed to zoospores of G. semiglobifer exhibited high levels of mycoparasitism and G. semiglobifer was subsequently reisolated from mycoparasitized resting spores. We discuss the importance of this finding to the epizootiology of insect diseases caused by entomophthoralean fungi.     

Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes.

The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplification of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, originally from an Australian grasshopper (Praxibulus sp.), through a North America grasshopper resulted in no differences in the resultant RAPD fragment patterns. In the second method, polymorphic RAPD fragments were used to probe the genomic DNA from the three pathotypes, and pathotype-specific fragments were found. In the third method, restriction fragments from genomic DNA of the three pathotypes were cloned and screened for pathotype specificity. A genomic probe specific for each pathotype was isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers. To facilitate the use of RAPD analysis and other molecular tools to identify pathotypes, a method for extracting DNA from resting spores from infected grasshoppers was developed. The DNA from the fractured resting spores was of sufficient integrity to be blotted and probed with the pathotype-specific DNA probes, thus validating the use of these probes for pathotype identification in field-collected grasshoppers.