Richard S. Soper

Isolation and characterization of Entomophaga maimaiga sp. nov., a fungal pathogen of gypsy moth, Lymantria dispar, from Japan.

Abstract A Japanese zygomycete pathogen from the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae), is described as Entomophaga maimaiga sp. nov. (Entomophthorales: Entomophthoraceae). This fungus belongs to the Entomophaga aulicae species complex. Isolates from the E. aulicae species complex were tested for infectivity and pathogenicity toward larvae of the gypsy moth, and only those isolates from gypsy moths were pathogenic to gypsy moth larvae. Electrophoretic studies of isoenzymes indicated that the Japanese gypsy moth isolates differed from other Japanese and North American strains of the E. aulicae species complex. Among the isolates of E. maimaiga from Japan, ARSEF 1400 appeared most promising for biological control use; it caused 90–95% mortality in third instar larvae after an average of 5.2 days and can be cultured in both protoplast and mycelial vegetative stages. In host range trials, ARSEF 1400 infected only a few of the lepidopteran species tested. Among these alternate hosts, mortality was the greatest (92.5%) for the Douglas-fir tussock moth, Orgyia pseudotsugata, another economically important lymantriid.

Germination and infection processes of the entomophthoralean fungus Erynia radicans on the potato leafhopper, Empoasca fabae

The germination and penetration processes of Erynia radicans on fifth instar Empoasca fabae nymphs at 20°C, 100% relative humidity, were investigated by scanning electron and fluorescence microscopy. Oval primary and secondary conidia attached to the host cuticle and germinated rapidly (60% within 2 hr). While percentage germination did not differ on the three body regions of the host, significant differences were observed in the modes of germination. The percentage of spores that produced germ tubes increased from 10% on the leafhopper head to 13% on the thorax and 22% on the abdomen, while capilliconidiophore production decreased from 88 to 84 to 71% on the respective body regions. Hyphae from spores deposited on sclerites displayed strong directional growth toward interscleral membranes, especially on the host abdomen. Penetration of the cuticle was effected by formation of appressoria. Small, globular appressoria were formed immediately adjacent to spores or on short, nonseptate germ tubes, while large, elongate appressoria were produced usually on long, but occasionally on short, septate hyphae. Approximately 3% of penetrations occurred on the leafhopper head, 16% on the thorax, and 74% on the abdomen; ca. 61% of all penetrations occurred through membranes, especially in the intersegmental folds of the abdomen. Earliest penetrations were observed 3–4 hr after inoculation, but significant numbers of penetrations were not observed until after 6 hr. The number of penetrations on individual leafhoppers was largely independent of dose (mean r2 = 0.174), indicating substantial variability in susceptibility of individual leafhoppers. The percentage of viable oval conidia which ultimately gave rise to penetrations (either directly or indirectly via secondary spore production) was ca. 5% after 10–12 hr and 20% after 48 hr. The percentage of leafhoppers infected (percentage penetrated by one or more hyphae from a mean dose of 41 spores per insect) increased rapidly from only 2.4% at 4 hr postinoculation to ca. 60% after 8 hr. The LD50 was estimated at 4.1 spores per leafhopper.

Humoral encapsulation of the fungus Erynia radicans (Entomophthorales) by the potato leafhopper, Empoasca fabae (Homoptera: Cicadellidae)

Abstract The immune response of the potato leafhopper, Empoasca fabae, to fungal infection is humoral encapsulation. Noncellular, melanotic capsules were formed around invading hyphae of Erynia radicans, Metarhizium anisopliae, and Hirsutella guyana. Melanization of E. radicans hyphae was observed in the cuticle, epidermis, and hemolymph of infected leafhoppers, in some cases within 4–6 hr post-inoculation. The encapsulation response was nearly always ineffective in preventing fungal infection. Rarely, hyphae in the hemolymph were completely encased in thick melanotic capsules. Thin sections of melanized hyphae showed the melanin to be a granular, electron-opaque substance. A few hyphae were observed coated with a filamentous, electron-opaque substance which also may be melanin.

Infection of grasshoppers (Orthoptera: Acrididae) by members of the Entomophaga grylli species complex (Zygomycetes: Entomophthorales)

Abstract Five pathotypes of the Entomophaga grylli species complex (Zygomycetes: Entomophthorales) were compared on the basis of infectivity to grasshoppers of the genera Melanoplus and Camnula . Intrahemocoelic and topical inoculations demonstrated that the pathotypes can be grouped into three infective range patterns. The Australian pathotype and North American pathotype 1 have the broadest spectrum of infectivity and may hold the greatest potential for future development as microbial agents against grasshoppers. Japanese and Indonesian pathotypes consistently caused disease by injection but were not consistently infective by topical inoculation. North American pathotype 2 was most variable in infectivity and did not infect Camnula pellucida through either infection route.

The effect of solar radiation on the survival of Entomophaga grylli (Entomophthorales: Entomophthoraceae) conidia

Abstract Laboratory studies on the effects of simulated solar radiation on Entomophaga grylli conidial viability showed definite interactions between the duration and intensity of exposure to the light source. When either variable was increased, the viability of E. grylli conidia was reduced. An exponential decay model was developed based on combining these two factors into a single predictive variable, exposure to cummulative solar radiation in Langleys. A minimum of approximately 3 Langleys was required before conidial viability decreased, with nearly 100% noted after exposure to 20 Langleys. The exponential decay model when applied to conidial viability data collected in the field provided an excellent fit; however, parameter estimates were significantly different from laboratory estimates as conidia were found to survive different periods of time in different habitats. The model predicted over 95% mortality in the most open habitats after exposure to ca. 50 Langleys. As expected, conidia exposed in lower canopy areas and in canopies with more dense foliage survived significantly longer (ca. 95% mortality after exposure to 700 Langleys).

Simulation of insect disease dynamics: an application of SERB to a rangeland ecosystem:

An object-oriented simulation program, SERB (Simulation En vironment for Research Biologists), has been used to simulate insect disease dynamics in a rangeland ecosystem. SERB was used to construct a mechanistic simulation model of a fungal pathogen of grasshoppers by synthesizing experimentally derived compo nents. Emphasis is placed on how SERB was used to construct and evaluate the model both in a piece-wise and combined manner to aid in understanding population interactions and dynamics. The interactive nature of SERB in the ZetaLisp environ ment was found to be highly conducive to model construction, manipulation and evaluation. Detailed examples from several phases of this process are presented from the perspective of a population bioligist. The orientation of SERB allows the research er to focus on a problem in an intuitive and systematic manner, in biological terms, unconstrained by details of an unfamiliar programming environment.

Simulation and object-oriented programming: the development of SERB:

An object-oriented programming language (OOPL), Flavors, was used to implement a modeling language and ancillary simula tion tools designed specifically to meet the needs of research biologists. A biological system is typically considered as a set of interacting entities, an abstraction which closely matches the pro gramming paradigm of Flavors. Some of the important charac teristics of Flavor programming are presented. Then SERB (the Simulation Environment for Research Biologists) is described. SERB uses Flavor objects to represent biological entities, resulting in a high degree of transparency between model and simuland. The simulationist creates model components from a library of component types and defines a model as some interacting set of these components. These models can then be used in simula tions or by higher level functions, such as sensitivity analysis. Flavors was found to be an ideal language for implementing modeling and simulation software for this domain.

Intraspecific genetic variability in laboratory strains of Entomophthora as determined by enzyme electrophoresis

Intraspecific variability of isoenzymes is described for approximately 20 isolates each of two species of Entomophthora. E. sphaerosperma was found to possess two electrophoretically distinct alleles for phosphoglucoisomerase (PGI), and E. virulenta was found to have three alleles for leucine aminopeptidase, two alleles for 6-phosphogluconate dehydrogenase, and five alleles for PGI. The data suggest that only a single locus codes for PGI in E. sphaerosperma , while two loci code for PGI in E. virulenta . Two strains of E. virulenta isolated in England suggest complete genome duplication. Difficulties inherent in interpreting electrophoretic data from fungi with complicated life histories are discussed.

A bioassay technique for Entomophthora sphaerosperma on the spruce budworm, Choristoneura fumiferana

Abstract A technique for conducting bioassays of Entomophthora sphaerosperma on sixth-instar larvae of the spruce budworm, Choristoneura fumiferana , was developed. Four assays were conducted by showering conidia on 10 larvae for each of 10 to 20 doses per assay. Dose was estimated by averaging estimates of the concentration of spores falling on water agar dishes before and after insect exposure. Maximum-likelihood probit analysis indicated significant regressions between log dose and probit mortality for all four assays. LC 50 values ranged from 11.21 to 18.77 spores/mm 2 with a weighted mean of 16.13 spores/mm 2 . Slope estimates ranged from 0.92 to 1.87 with a weighted mean of 1.13. These low slope values may have been indicative of a highly variable test insect population, but also suggested a nontoxic infection process by the pathogen.

OBSERVATION AND INTERPRETATION OF NUCLEAR REDUCTIONS DURING MATURATION AND GERMINATION OF ENTOMOPHTHORALEAN RESTING SPORES

tory ofthe pathogen. It is important to determine if karyogamy and meiosis occur in the life cycle of this fungus, and if so, at what site and time, before any strain improvement through genetic manipulation might be attempted. Conflicting data about the nuclear events associated with resting spore pro? duction and germination in the Entomophthorales have presented difficulty in the correct interpretation of entomophthoralean life cycles. Sawyer (1931) believed the resting spores of E. radicans {=Entomophthora sphaerosperma Fres.) re? mained multinucleate at maturity; Perry et al. (1982) studied the germination of resting spores of this species but did not note the nuclear condition at any time in mature, activated, or germinating spores. Latge (1976) reported a reduction in the number of nuclei to one during resting spore maturation in Conidiobolus thromboides Drechsler {=Entomophthora virulenta Hall & Dunn). Resting spores of E. radicans (strain RS-141, Insect Pathology Research Unit) were harvested from 3% yeast extract agar at various stages of development and stained with haematoxylin following the procedures of Lu and Raja (1970). Resting spores of Entomophaga grylli (Fres.) Batko were recovered from field-collected cadavers of Melanoplus bivittatus Say after several months of storage at 4 C in a sealed container. Confirmatory studies on the nuclear condition of resting spores of C thromboides (strain RS-40) were done with spores produced on egg yolk/ Sabouraud maltose agar (Soper et al, 1975). Nuclear conditions of germinating resting spores in this species were studied from spores stored over silica gel in a sealed container at 4 C for 3 yr; approximately 25% of these spores would ger? minate after being wetted. These 3-yr-old spores were used to determine nuclear events during the germination process. In the early formation of E. radicans resting spores, the prespores may contain as many as 25 nuclei (Fig. 1). The young resting spore forms in less than 24 h inside the parental prespore. During the deposition of the spore wall some nuclei may be trapped between the prespore (parental) wall and newly formed inner (sporangial) wall (Fig. 2). The excluded nuclei rapidly disintegrate leaving a more or less empty space between the two thickened wall layers. As the resting spore matures, many ofthe nuclei degenerate until, after two wk, only two nuclei remain in each spore (Fig. 3). No further reduction in the number of nuclei was observed,