Utilizing Genomics to Study Entomopathogenicity in the Fungal Phylum Entomophthoromycota: A Review of Current Genetic Resources.

The order Entomophthorales, which formerly contained c.280 species, has recently been recognized as a separate phylum, Entomophthoromycota, consisting of three recognized classes and six families. Many genera in this group contain obligate insect-pathogenic species with narrow host ranges, capable of producing epizootics in natural insect populations. Available sequence information from the phylum Entomophthoromycota can be classified into three main categories: first, partial gene regions (exons+introns) used for phylogenetic inference; second, protein coding gene regions obtained using degenerate primers, expressed sequence tag methodology or de novo transcriptome sequencing with molecular function inferred by homology analysis; and third, primarily forthcoming whole-genome sequencing data sets. Here we summarize the current genetic resources for Entomophthoromycota and identify research areas that are likely to be significantly advanced from the availability of new whole-genome resources.

Thermoregulatory behavior and fungal infection of Anoplophora glabripennis (Coleoptera: Cerambycidae).

Asian longhorned beetles, Anoplophora glabripennis (Motschulsky), are invasive wood borers that are native to China and Korea but have been introduced to North America and Europe. These beetles have great potential to negatively impact economic and environmental interests in hardwood and urban forests if they become established. The entomopathogenic fungus Metarhizium brunneum Petch (previously Metarhizium anisopliae (Metschnikoff) Sorokin) is under development for control of A. glabripennis. Some insect species eliminate pathogens or delay disease progression through thermoregulation. Because Asian longhorned beetles had been observed occupying sunlit areas of the tree canopy, we hypothesized that behavioral fevering could be used to delay mortality of fungal-infected beetles. M. brunneum cultures incubated at 34°C for 5 h/d grew significantly slower compared with cultures incubated at lower temperatures. Holding M. brunneum-infected A. glabripennis at 34°C for 5 h/d significantly delayed mortality by 2 d compared with infected beetles held at ≤31°C. Adult A. glabripennis did not exhibit behavioral fever when infected. Uninfected males, when provided with food, and both uninfected males and females when deprived of food, slightly increased their preferred temperature over time. When held at 15°C before being placed into temperature gradients, uninfected beetles did not increase their temperatures above ambient. Results demonstrate that M. brunneum-infected A. glabripennis do not exhibit behaviors necessary to elevate their body temperatures enough to combat M. brunneum infections through thermoregulation.

Bizarre interactions and endgames: entomopathogenic fungi and their arthropod hosts.

Invertebrate pathogens and their hosts are taxonomically diverse. Despite this, there is one unifying concept relevant to all such parasitic associations: Both pathogen and host adapt to maximize their own reproductive output and ultimate fitness. The strategies adopted by pathogens and hosts to achieve this goal are almost as diverse as the organisms themselves, but studies examining such relationships have traditionally concentrated only on aspects of host physiology. Here we review examples of host-altered behavior and consider these within a broad ecological and evolutionary context. Research on pathogen-induced and host-mediated behavioral changes demonstrates the range of altered behaviors exhibited by invertebrates including behaviorally induced fever, elevation seeking, reduced or increased activity, reduced response to semiochemicals, and changes in reproductive behavior. These interactions are sometimes quite bizarre, intricate, and of great scientific interest.

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.

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.

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.

A disjunct Californian strain of Entomophaga aulicae infecting Orgyia vetusta.

Fungal epizootics occurred in abundant Orgyia vetusta (western tussock moth; Lepidoptera: Lymantriidae) populations on Lupinus arboreus bushes growing on the Pacific coast north of San Francisco, California. The causative pathogen was isolated and identified as Entomophaga aulicae, Group II, based on RFLPs using rDNA and PCR-amplified rDNA products. Inability of this fungus to infect the lymantriid Lymantria dispar (gypsy moth) confirmed its distinction from Entomophaga maimaiga, the only other member of this species complex which predominantly infects lymantriids. Later instar wandering by O. vetusta in outbreak populations and close proximity of larvae in dense populations are characteristics most probably promoting development of E. aulicae epizootics; these life history patterns are also typical of Lymantria dispar populations experiencing epizootics of E. maimaiga.

Allozyme and restriction fragment length polymorphism analyses confirm Entomophaga maimaiga responsible for 1989 epizootics in North American gypsy moth populations.

In 1989, populations of North American gypsy moth, Lymantria dispar, in seven contiguous northeastern states were severely reduced by a fungal pathogen. Based on morphology, development, and pathology, this organism appeared to be Entomophaga maimaiga. We have now used allozyme and restriction fragment length polymorphism analyses to confirm this identification. Previously, this mycopathogen had been reported only from gypsy moth populations in Japan. During 1989, E. maimaiga occurred only in areas that had been initially defoliated by gypsy moth >10 years ago. E. maimaiga caused 60-88% mortality in late instar larvae on research sites in central Massachusetts.