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The microsporidian genera Nosema and Vairimorpha comprise a clade described from insects. Currently the genus Nosema is defined as having a dimorphic life cycle characterized by diplokaryotic stages and diplosporoblastic sporogony with two functionally and morphologically distinct spore types ("early" or "primary" and "environmental"). The Vairimorpha life cycle, in addition to a Nosema-type diplokaryotic sporogony, includes an octosporoblastic sporogony producing eight uninucleate spores (octospores) within a sporophorous vesicle. Molecular phylogeny, however, has clearly demonstrated that the genera Nosema and Vairimorpha, characterized by the absence or presence of uninucleate octospores, respectively, represent two polyphyletic taxa, and that octosporogony is turned on and off frequently within taxa, depending on environmental factors such as host species and rearing temperature. In addition, recent studies have shown that both branches of the Vairimorpha-Nosema clade contain species that are uninucleate throughout their life cycle. The SSU rRNA gene sequence data reveal two distinct clades, those closely related to Vairimorpha necatrix, the type species for the genus Vairimorpha, and those closely related to Nosema bombycis, the type species for the genus Nosema. Here, we redefine the two genera, giving priority to molecular character states over those observed at the developmental, structural or ultrastructural levels and present a list of revised species designations. Using this approach, a series of species are renamed (combination novum) and members of two genera, Rugispora and Oligosporidium, are reassigned to Vairimorpha because of their phylogenetic position. Moreover, the family Nosematidae is redefined and includes the genera Nosema and Vairimorpha comprising a monophyletic lineage of Microsporidia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We describe a unique microsporidian species that infects the green stink bug, Chinavia hilaris; the brown marmorated stink bug, Halyomorpha halys; the brown stink bug, Euschistus servus; and the dusky stink bug, Euschistus tristigmus. All life stages are unikaryotic, but analysis of the consensus small subunit region of the ribosomal gene places this microsporidium in the genus Nosema, which historically has been characterized by diplokaryotic life stages. It is also characterized by having the reversed arrangement of the ribosomal gene (LSU -ITS- SSU) found in species within the "true Nosema" clade. This microsporidium is apparently Holarctic in distribution. It is present in H. halys both where it is native in Asia and where it is invasive in North America, as well as in samples of North American native C. hilaris collected prior to the introduction of H. halys from Asia. Prevalence in H. halys from mid-Atlantic, North America in 2015-2016 ranged from 0.0% to 28.3%, while prevalence in C. hilaris collected in Illinois in 1970-1972 ranged from 14.3% to 58.8%. Oral infectivity and pathogenicity were confirmed in H. halys and C. hilaris. Morphological, ultrastructural, and ecological features of the microsporidium, together with a molecular phylogeny, establish a new species named Nosema maddoxi sp. nov., (© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.)

We recently discovered infections by a microsporidium closely related to Nosema fumiferanae in field populations of the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in the San Francisco region of California. E. postvittana originates from Australia and was first detected in California in 2006; therefore, our aim was to identify and determine the origin of the Nosema isolate. We characterized the pathogenicity, transmission pathways, and ultrastructure of this new Nosema isolate. In addition, we sequenced fragments of commonly used genetic markers (ITS, SSU, and RPB1), and examined the phylogenetic relationships between the Nosema isolate and other microsporidian species commonly found in lepidopteran hosts. The pathogenicity of the Nosema isolate was investigated by infecting second instar larvae of E. postvittana. Larval and pupal survivorship were reduced by 7% and 13% respectively, and pupation occurred 1-2d later in infected individuals than in healthy individuals. Emerging infected females died 5d earlier than healthy females, and daily fecundity was 22% lower. Hatch rate also was 22% lower for eggs oviposited by infected females. Vertical transmission was confirmed; spores were present in 68% of egg masses and 100% of the surviving larvae from infected females. Ultrastructure images, together with sequences from selected genetic markers, confirmed the Nosema isolate to be a member of the Nosema fumiferanae species complex (Nosema fumiferanae postvittana subsp. n.). The association of this pathogen with E. postvittana contributes further to the biotic resistance that E. postvittana has experienced since its introduction to California., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In Brazil, the sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), is controlled with massive releases of the hymenopteran parasitoid Cotesia flavipes Cam. (Hymenoptera: Braconidae); over 3 million hectares of sugarcane are treated annually with 18 billion parasitoids. In order to meet this demand, parasitoids are produced in D. saccharalis under laboratory conditions where a Nosema sp. is reported to be an important problem in mass rearing of the host. The goals for this work were to study the pathogenicity of the Nosema sp. and the progression of the disease in the host under laboratory conditions. The average median lethal time (LT₅₀) of Nosema sp. in first instar D. saccharalis varied from 9 ± 0.3 to 42 ± 2.3 days at concentration of 5 × 10(5)-0.5 spores/mm(3) artificial diet (10(7)-10 spores/μl). For third instar, the average of LT50 ranged from 32 ± 0.7 to 37 ± 0.7 days at concentration of 5 × 10(5)-5 × 10(2) spores/mm(3) artificial diet (10(7)-10(4) spores/μl in saline). The concentration necessary to cause 50% mortality (LC₅₀) of first instar larvae was 5.6 (0.9-17.6) spores/μl and the estimated LC50 for third instar larvae was 1,200 (200-4700) spores/μl. The impacts of Nosema sp. on D. saccharalis were analyzed for first instar larvae fed 0.5 spores/mm(3) artificial diet. Duration and viability of the larval and pupal stages, adult longevity, pupal weight and fertility life table were measured for offspring of mating pairs composed of infected females and uninfected males or infected males and uninfected females and compared to offspring of uninfected pairs. Nosema sp. infection resulted in adverse effects on all biological parameters measured except for the duration of the larval and pupal stages and the weight of the male pupae, which did not differ statistically between infected and uninfected groups. The intrinsic rates of growth (rm) were greater for uninfected pairs compared to pairs with either male or female infected. The growth rate of individual larvae produced by uninfected adults was 48.2% faster than of larval offspring of infected females and it was negative (-0.003) when males were infected. Our study confirms the negative impact of the Nosema sp. in mass rearing of D. saccharalis for parasitoid production but shows potential for use as a microbial control agent of the sugarcane borer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Honey bees (Apis mellifera) are infected by two species of microsporidia: Nosema apis and Nosemaceranae. Epidemiological evidence indicates that N. ceranae may be replacing N. apis globally in A. mellifera populations, suggesting a potential competitive advantage of N. ceranae. Mixed infections of the two species occur, and little is known about the interactions among the host and the two pathogens that have allowed N. ceranae to become dominant in most geographical areas. We demonstrated that mixed Nosema species infections negatively affected honey bee survival (median survival=15-17days) more than single species infections (median survival=21days and 20days for N. apis and N. ceranae, respectively), with median survival of control bees of 27days. We found similar rates of infection (percentage of bees with active infections after inoculation) for both species in mixed infections, with N. apis having a slightly higher rate (91% compared to 86% for N. ceranae). We observed slightly higher spore counts in bees infected with N. ceranae than in bees infected with N. apis in single microsporidia infections, especially at the midpoint of infection (day 10). Bees with mixed infections of both species had higher spore counts than bees with single infections, but spore counts in mixed infections were highly variable. We did not see a competitive advantage for N. ceranae in mixed infections; N. apis spore counts were either higher or counts were similar for both species and more N. apis spores were produced in 62% of bees inoculated with equal dosages of the two microsporidian species. N. ceranae does not, therefore, appear to have a strong within-host advantage for either infectivity or spore growth, suggesting that direct competition in these worker bee mid-guts is not responsible for its apparent replacement of N. apis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The historic genus Pleistophora (Plistophora) is a highly polyphyletic clade with invertebrate Microsporidia reassigned to several new genera since the 1980s. Two genera, Endoreticulatus and Cystosporogenes, clearly separate into distinct but closely related clades based on small subunit ribosomal RNA analysis but are included in different families that are each polyphyletic. A microsporidium with morphology resembling the Endoreticulatus/Cystosporogenes clade was isolated from the grasshopper Poecilimon thoracicus from a site in Northwest Bulgaria. It produced intense infections in the digestive tract of the host but no behavioral changes were noted in infected individuals. Prevalence of the microsporidium increased over the active feeding season yearly. Mature spores were oval and measured 2.58±0.21 μm×1.34±0.24 μm, with 16 to approximately 32 spores in a parasitophorous vacuole. The spores were uninucleate and polar filament coils numbered 8-9 situated in a single row. The spore polaroplast consisted of an anterior lamellar section and a posterior vesicular section, and the posterior vacuole was reduced. Analyses of a 1221 bp partial SSU-rRNA sequence indicated that the isolate is more closely related to the Endoreticulatus clade than to Cystosporogenes, but shows earlier phylogenetic separation from species infecting Lepidoptera and represents a new species, Endoreticulatus poecilimonae. To compare sequences of Endoreticulatus spp. from Lepidoptera to those infecting other insect orders, an isolate, Microsporidium itiitiMalone (1985), described from the Argentine stem weevil, Listronotus bonariensis, was sequenced. Like the grasshopper isolate, the weevil isolate is closely related but basal to the lepidopteran Endoreticulatus clade. The original description combined with the new sequence data confirms species status and permits transfer of the isolate from Microsporidium, a genus erected for microsporidian species of uncertain taxonomic status, to Endoreticulatus., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Conclusions about the nontarget effects of putatively invasive pathogens should be based on biologically relevant data. We disagree that the research experiments on a microsporidium isolated from Harmonia axyridis conducted by Vilcinskas et al. (Reports, 17 May 2013, p. 862) can explain the decline of native coccinellid species in the absence of such data.

The two etiological agents of nosema disease in honey bees, Nosema apis and Nosema ceranae (Microsporidia: Nosematidae), reproduce in the midgut tissues of the host. N. apis is tissue specific but the development and tissue tropism of N. ceranae is not well understood. Our investigations compared development of the two phylogenetically related pathogens in all major host tissues. Using microscopy, PCR and qPCR quantification to evaluate tissue tropism of infected bees in communal cages and of individually restrained infected bees, we found no detectable spores in cephalic or other body tissues except midgut tissues. Nosema DNA was detected in Malpighian tubules but the tubules could not be separated from the alimentary tract without release of spores from the midgut. Nosema DNA was not detected in hemolymph sampled from the head capsule or the abdomen of infected bees. We confirmed that N. ceranae only develops in midgut tissues. Spores of both species released from host midgut cells accumulated in the hindgut lumen, and we noted differences in numbers and ratios of spore types and in growth curves between the two pathogens. N. apis reached a consistent level of spore production after 12 days post inoculation (dpi); N. ceranae spore production increased linearly from 12 to 20 dpi and the number of mature N. ceranae spores was consistently higher., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2) in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.

A new microsporidium was isolated from the endemic, Taiwanese shrimp, Caridina formosae (Decapoda, Atyidae) from northern Taiwan. A conspicuous symptom of infection was presence of opaque white xenomas located in the proximity of the alimentary tract, the surface of the hepatopancreas, and the gills. A fully developed xenoma consisted of a hard, thick capsule filled with sporophorous vesicles containing multiple spores. Microsporidia developed synchronously within the same sporophorous vesicle, although the stage of parasite development differed among the vesicles. Fresh spores were pyriform, mononucleated and measured 6.53 × 4.38 μm. The polar filament was anisofilar with 9-11 coils. Phylogenetic analysis based on the small subunit ribosomal DNA sequence showed that the isolate is most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 80%, was with Spraguea spp. Based on pathogenesis, life cycle and phylogenetic analysis, we erect a new genus and species, Triwangia caridinae for the novel microsporidium., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nosema ceranae, a microsporidian entomopathogen, was first reported from honey bees, Apis mellifera, in 2005 in Taiwan (Huang et al., 2007) and has become a major concern in apiculture worldwide. In Taiwan, we found one infection peak for N. ceranae during the winter months, compared to two peaks in spring and fall reported in 1980 for Nosema apis. N. ceranae infection intensity in apiaries reached a high level earlier than N. apis, a possible factor in replacement. We found a significant negative correlation of N. ceranae pathogen load with temperature; the highest spore counts were recorded at an average temperature of approximately 15 °C and infection intensity equaled the annual average at 23.8 °C. This model corresponds with published results but is most reliable for subtropical to tropical climates., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to exploit their hosts more fully. Here, we describe the complete sequences of two intermediate-sized genomes (2.5 Mbp), from Encephalitozoon hellem and Encephalitozoon romaleae. Overall, the E. hellem and E. romaleae genomes are strikingly similar to those of Encephalitozoon cuniculi and Encephalitozoon intestinalis in both form and content. However, in addition to the expected expansions and contractions of known gene families in subtelomeric regions, both species also were found to harbor a number of protein-coding genes that are not found in any other microsporidian. All these genes are functionally related to the metabolism of folate and purines but appear to have originated by several independent HGT events from different eukaryotic and prokaryotic donors. Surprisingly, the genes are all intact in E. hellem, but in E. romaleae those involved in de novo synthesis of folate are all pseudogenes. Overall, these data suggest that a recent common ancestor of E. hellem and E. romaleae assembled a complete metabolic pathway from multiple independent HGT events and that one descendent already is dispensing with much of this new functionality, highlighting the transient nature of transferred genes.

Several bumble bee (Bombus) species in North America have undergone range reductions and rapid declines in relative abundance. Pathogens have been suggested as causal factors, however, baseline data on pathogen distributions in a large number of bumble bee species have not been available to test this hypothesis. In a nationwide survey of the US, nearly 10,000 specimens of 36 bumble bee species collected at 284 sites were evaluated for the presence and prevalence of two known Bombus pathogens, the microsporidium Nosema bombi and trypanosomes in the genus Crithidia. Prevalence of Crithidia was ≤10% for all host species examined but was recorded from 21% of surveyed sites. Crithidia was isolated from 15 of the 36 Bombus species screened, and were most commonly recovered from Bombus bifarius, Bombus bimaculatus, Bombus impatiens and Bombus mixtus. Nosema bombi was isolated from 22 of the 36 US Bombus species collected. Only one species with more than 50 sampled bees, Bombus appositus, was free of the pathogen; whereas, prevalence was highest in Bombus occidentalis and Bombus pensylvanicus, two species that are reportedly undergoing population declines in North America. A variant of a tetranucleotide repeat in the internal transcribed spacer (ITS) of the N. bombi rRNA gene, thus far not reported from European isolates, was isolated from ten US Bombus hosts, appearing in varying ratios in different host species. Given the genetic similarity of the rRNA gene of N. bombi sampled in Europe and North America to date, the presence of a unique isolate in US bumble could reveal one or more native North American strains and indicate that N. bombi is enzootic across the Holarctic Region, exhibiting some genetic isolation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pathogens have been implicated as potential factors in the recent decline of some North American bumble bee (Bombus) species, but little information has been reported about the natural enemy complex of bumble bees in the United States. We targeted bumble bee populations in a state-wide survey in Illinois and several sites in California and Oregon where declines have been reported to determine presence and prevalence of natural enemies. Based on our observations, most parasites and pathogens appear to be widespread generalists among bumble bee species, but susceptibility to some natural enemies appeared to vary., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bumble bees (Bombus) are vitally important pollinators of wild plants and agricultural crops worldwide. Fragmentary observations, however, have suggested population declines in several North American species. Despite rising concern over these observations in the United States, highlighted in a recent National Academy of Sciences report, a national assessment of the geographic scope and possible causal factors of bumble bee decline is lacking. Here, we report results of a 3-y interdisciplinary study of changing distributions, population genetic structure, and levels of pathogen infection in bumble bee populations across the United States. We compare current and historical distributions of eight species, compiling a database of >73,000 museum records for comparison with data from intensive nationwide surveys of >16,000 specimens. We show that the relative abundances of four species have declined by up to 96% and that their surveyed geographic ranges have contracted by 23-87%, some within the last 20 y. We also show that declining populations have significantly higher infection levels of the microsporidian pathogen Nosema bombi and lower genetic diversity compared with co-occurring populations of the stable (nondeclining) species. Higher pathogen prevalence and reduced genetic diversity are, thus, realistic predictors of these alarming patterns of decline in North America, although cause and effect remain uncertain.

Several species of microsporidia are important chronic pathogens of Lymantria dispar in Europe but have never been recovered from North American gypsy moth populations. The major issue for their introduction into North American L. dispar populations is concern about their safety to native non-target insects. In this study, we evaluated the susceptibility of sympatric non-target Lepidoptera to two species of microsporidia, Nosema lymantriae and Vairimorpha disparis, isolated from European populations of L. dispar and applied in field plots in Slovakia. Application of ultra low volume sprays of the microsporidia maximized coverage of infective spores in a complex natural environment and, thus, exposure of non-target species to the pathogens. Of 653 non-target larvae collected from plots treated with V. disparis in 2002, 18 individual larvae representing nine species in four families were infected. These plots were monitored for two subsequent seasons and V. disparis was not recovered from non-target species. Of 2571 non-target larvae collected in N. lymantriae-treated sites, one larva was found to be infected. Both species of microsporidia, particularly N. lymantriae, appear to have a very narrow host range in the field, even when an inundative technique is used for their introduction. V. disparis infections in L. dispar exceeded 40% of recovered larvae in the treated study sites; infection rates were lower in sites sprayed with N. lymantriae. Several naturally-occurring pathogens were recorded from the non-target species. The most common pathogen, isolated from 21 species in eight families, was a microsporidium in the genus Cystosporogenes., (Copyright 2010 Elsevier Inc. All rights reserved.)

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 26.87h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3weeks post inoculation. The highest level of spore production was at 4weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34+/-0.9x10(6)spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.

We describe a new microsporidian species, Encephalitozoon romaleae n. sp., isolated from an invertebrate host, the grasshopper Romalea microptera, collected near Weeks Island, Louisiana, and Jacksonville, Florida. This microsporidian is characterized by specificity to the gastric caecae and midgut tissues of the host and a life cycle that is nearly identical to that of Encephalitozoon hellem and Encephalitozoon cuniculi. Mature spores are larger (3.97 x 1.95 microm) than those of other Encephalitozoon species. Polar filament coils number 7 to 8 in a single row. Analysis of the small subunit (SSU) rDNA shows that E. romaleae fits well into the Encephalitozoon group and is a sister taxon to E. hellem. This is the first Encephalitozoon species that has been shown to complete its life cycle in an invertebrate host.

Encephalitozoon species are the most common microsporidian pathogens of humans and domesticated animals. We recently discovered a new microsporidium, Encephalitozoon romaleae, infecting the eastern lubber grasshopper Romalea microptera. To understand its evolutionary relationships, we compared partial gene sequences of alpha- and beta-tubulin and methionine aminopeptidase 2 enzyme from this and related species. We also analyzed the rRNA internal transcribed spacer. Based on tubulin and MetAP-2 gene phylogenetic analysis, E. romaleae clustered with the Encephalitzoon group with strong bootstrap support (>99%). Within the Encephalitozoon clade, E. romaleae clustered with Encephalitozoon hellem for both the beta-tubulin and MetAP-2 phylogenies based on ML tree. The alpha-tubulin based ML tree, however, placed the new microsporidium closer to Encephalitozoon cuniculi. The rRNA internal transcribed spacer region of E. romaleae has 91% homology with E. hellem.

A microsporidium possessing molecular and morphological characteristics of the genus Nosema was isolated from larvae of the thee-spot grass yellow butterfly, Eurema blanda arsakia. The complete rRNA gene sequences of the E. blanda isolate contained 4,428 base pairs (GenBank Accession No. EU338534). The organization of the rRNA genes is LSU rRNA-ITS-SSU rRNA-IGS-5S, which corresponds with that of Nosema species closely related to Nosema bombycis. Phylogenetic analysis based on rRNA gene sequences show that this isolate is closely related to Nosema bombycis, Nosema plutellae, Nosema spodopterae, and Nosema antheraeae. The ultrastructure of all developmental stages of this microsporidium confirmed its placement in the genus Nosema. The isolate was successfully propagated in cell lines IPLB-LD652Y (Lymantria dispar) and NTU-LY (Lymantria xylina) and, in the in vitro system, it was frequently found to develop in the nuclei of the host cells, a circumstance that seldom occurs in other Nosema species. An extra-cellular vegetative stage of this microsporidium was also observed in the culture medium after 14 days of infection. The ECMDFs might be released from disrupted host cells.

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2+/-3.5% at density of 10 inoculated per 100 larvae to 36.7+/-5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7+/-5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (<3.0%). Laboratory studies were conducted by orally exposing both larvae and adults of O. sulcatus to varying concentrations of Canningia sp. spores. Larval bioassays at a variety of dosages (0, 10, etc.) were performed to evaluate pathogen infectivity, larval survival and growth. Adult bioassays (dosages: 0, 10, etc.) were performed to evaluate longevity, fecundity and mechanisms of vertical pathogen transmission. Larvae and adults were infected in all spore treatments. Larval growth was significantly reduced at dosages above 10 spores/larva. Adults infected at all dosages experienced high levels of mortality and fecundity was reduced to zero. Greenhouse trials were performed to determine if larvae feeding in soil acquired infections when spores were topically applied as a drench application (0, 10(5), 10(6), 10(7) spores/pot). Established larvae feeding on plant roots in pots developed infections when exposed to drench treatments of 10(6) and 10(7) spores/pot after 14-21 days. Canningia sp. is an acute pathogen of O. sulcatus infective to both larvae and adults. Topically applied spores also infected larvae feeding on roots in soilless potting media, suggesting the possibility of using this pathogen in a microbial control program.

New and efficient methods to screen antibiotics are needed to counter increased antibiotic resistance in pathogens and the emergence of new diseases. Here we report a new insect model for screening antibiotics in vivo using the grasshopper Romalea microptera. The system is inexpensive, efficient, and flexible, avoids animal-welfare problems, and can be used to test against most major pathogenic groups. We employed this system to test 11 commercial antibiotics against a pathogenic Encephalitozoon species (Microsporidia). Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts, whereas spore counts of grasshoppers fed with albendazole, ampicillin, chloramphenicol, griseofulvin, metronidazole, sulfadimethoxine, or tetracycline were not significantly different from the infected controls. Quinine produced a distinct, but nonsignificant, reduction in spores, and streptomycin a nonsignificant increase in spores. Although 2 antibiotics significantly reduced spore counts, in no case was the pathogen totally eliminated. This study demonstrates the validity of this system as a method to screen antibiotics. It also corroborates the difficulty researchers and physicians have had in treating microsporidia infections, and suggests that quinine and related alkaloid compounds should be further examined as possible therapeutic agents against this group of ubiquitous pathogens. In addition, streptomycin and related compounds should be tested to determine if this widely used antibiotic enhances microsporidiosis.

The gypsy moth, Lymantria dispar L. (Lepidoptera, Lymantriidae), a serious defoliator of deciduous trees, is an economically important pest when population densities are high. Outbreaking populations are, however, subject to some moderating influences in the form of entomopathogens, including several species of microsporidia. In this study, we conducted laboratory experiments to investigate the transmission of an unusual Nosema sp. isolated from L. dispar in Schweinfurt, Germany; this isolate infects only the silk glands and, to a lesser extent, Malpighian tubules of the larval host. The latent period ended between 8 and 15 days after oral inoculation and spores were continuously released in the feces of infected larvae until pupation. Exclusion of feces from the rearing cages resulted in a 58% decrease in horizontal transmission. The silk of only 2 of 25 infected larvae contained microsporidian spores. When larvae were exposed to silk that was artificially contaminated with Nosema sp., 5% became infected. No evidence was found for venereal or transovum (including transovarial) transmission of this parasite.

The interactions in multiple species infections and effects on the horizontal transmission of three microsporidian species, Vairimorpha disparis, Nosema lymantriae and Endoreticulatus schubergi, infecting Lymantria dispar were evaluated in the laboratory. Simultaneous and sequential inoculations of host larvae were performed and the resulting infections were evaluated. Test larvae were exposed to the inoculated larvae to measure horizontal transmission. Dual species infections demonstrated interspecific competition between Nosema and Vairimorpha in the host larvae, but no observable competition occurred between Endoreticulatus and either of the other microsporidian species. Timing of inoculation was an important factor determining the outcome of competition between Nosema and Vairimorpha. The species inoculated first showed a higher rate of successful establishment; a time lag of 7 days between inoculations allowed the first species to essentially exclude the second. The microsporidia differed in efficiency of horizontal transmission. Nosema and Endoreticulatus were transmitted at very high rates, close to 100%. Horizontal transmission of Vairimorpha was less efficient, ranging from 25% to a maximum of 75%. The patterns of infection observed in inoculated larvae were reflected in the test larvae that acquired infections in the horizontal transmission experiments. Competition with Vairimorpha suppressed horizontal transmission of Nosema after simultaneous and sequential inoculation. In simultaneous inoculation experiments Endoreticulatus had no effect on transmission of Nosema and Vairimorpha.

Consideration of vertical transmission is particularly important for understanding the life cycles of entomopathogens that are naturally occurring in invertebrate populations, are a problem in beneficial insect colonies, or are under consideration as classical biological control agents. Empirical studies generally corroborate the evolutionary hypothesis that virulence should be relatively low for pathogen species that utilize vertical transmission as one mechanism for maintenance in the host population. Nevertheless, many entomopathogens with significant effects on host populations are vertically as well as horizontally transmitted. In addition to gaining a better understanding of pathogen-host interactions and population dynamics, studies of the host range and specificity of putative biological control agents can benefit by using transmission studies to better predict ecological host specificity from physiological data. Horizontal transmission requires a tightly organized host-pathogen relationship to succeed, but still involves, albeit restricted by host behavior and pathogen dosage, the physiological susceptibility of the nontarget host. Vertical transmission studies can provide increased stringency for determining the ecological host specificity of a species and may be one very accurate predictor of the ability of a pathogen to successfully host-switch when introduced into a naïve population.

Investigation of pathogens of populations of the gypsy moth, Lymantria dispar (L.) in Central and Eastern Europe revealed the existence of a microsporidium (Fungi: Microsporidia) of the genus Vairimorpha. The parasite produced three spore morphotypes. Internally infective spores are formed in the gut and adjacent muscle and connective tissue; single diplokaryotic spores and monokaryotic spores grouped by eight in sporophorous vesicles develop in the fat body tissues. The small subunit rDNA gene sequences of various isolates of the Vairimorpha microsporidia, obtained from L. dispar in various habitats in the investigated region, revealed their mutual identity. In phylogenetic analyses, the organism clustered with other L. dispar microsporidia that form only diplokaryotic spores in the sporogony cycle. The octospores of certain microsporidia infecting Lepidoptera that were previously described as Thelohania spp., have recently been shown to be one of the several spore morphotypes produced by species in the genus Vairimorpha. Because the description and drawings of a parasite described as Thelohania disparis by Timofejeva fit the characteristics of Vairimorpha, and all octospore-producing microsporidia collected from L. dispar since 1985 are genetically identical Vairimorpha species, it is believed that the parasite characterized here is identical to T. disparis Timofejeva 1956, and is herein redescribed, characterized, and transferred to the genus Vairimorpha as the new combination Vairimorpha disparis n. comb.

Entomopathogenic nematodes were screened for efficacy against the cottonwood borer, Plectrodera scalator (Fabricius). Steinernema feltiae SN and S. carpocapsae All killed 58 and 50% of larvae, respectively, in filter paper bioassays but less than 10% in diet cup bioassays. S. glaseri NJ, S. riobrave TX, and H. indica MG-13 killed less than 10% of larvae in both assays. H. marelata IN was ineffective in the diet cup bioassay and killed 12.9% of larvae in a filter paper bioassay. The nematode isolates we tested are not suitable for use as biological control agents against P. scalator.

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth (Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Schädlingskunde, Pflanzen-Umweltschutz, 48, 11-12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.

We investigated vertical and horizontal transmission as means by which entomopathogenic microsporidia may be isolated in their hosts. Ostrinia nubilalis larvae were challenged with microsporidia isolated from other stalk-boring and row crop Lepidoptera and were susceptible to seven species. Two species were horizontally transmitted. A Nosema sp. from Eoreuma loftini was transmitted among O. nubilalis larvae but not among larvae of the E. loftini host. This species was also vertically transmitted to the offspring of infected O. nubilalis females. An rDNA sequence showed the E. loftini isolate to be Nosema pyrausta, a naturally occurring species in O. nubilalis. Our results suggest that both horizontal and vertical transmission provide physiological barriers to host switching in the microsporidia, thus restricting the natural host range.

A microsporidium from the Ficus pest, Ocinara lida, in Taiwan is characterized. The taxonomic position of this species was preliminarily determined by sequencing small subunit rRNA gene (SSUrRNA). Analysis of the SSUrRNA sequence indicated that this isolate from O. lida is a member of the genus Endoreticulatus and belongs to the genetic grouping containing other lepidopteran Endoreticulatus species we have analyzed phylogenetically. The taxonomic position of this isolate was also confirmed by the ultrastructural characteristics of this isolate. The congruence between SSUrRNA sequence analysis and ultrastructural characteristics shows that this isolate is more closely related to Endoreticulatus bombycis than to Endoreticulatus schubergi Zwölfer.

We investigated host-parasite interactions of two Nosema-type microsporidian isolates recovered from populations of Lymantria dispar L. in northwestern Bulgaria, one near Veslec and one near Levishte. Bioassay studies produced information on development, stage specific mortality, pupation, and adult eclosion of infected individuals. Horizontal transmission of the two isolates was investigated in a second set of experiments. At dosages ranging from 2 x 10(2) to 5 x 10(4) spores/microl, the infection rates varied between 77 and 100% for the isolate from Veslec and between 92 and 99% for the Levishte isolate. The Veslec isolate caused a slightly higher mortality rate and the median time to death was shorter compared to the isolate from Levishte. The total mortality for both isolates varied between 79 and 99%, independent of spore dosages. A lower relative growth rate was recorded for male and female L. dispar larvae infected with either isolate during the third larval instar and a higher relative growth rate during the fourth instar compared to the control groups. Pupal weight did not differ significantly among females, but male infected pupae were heavier than the controls. Nosema sp. [Veslec] was as efficiently transmitted as Nosema sp. [Levishte]; 42% of the susceptible larvae became infected with the Veslec isolate when uninfected larvae were exposed to infected larvae; 43% of larvae became infected with the Nosema sp. [Levishte]. The latency period varied between 7 and 8 days for both isolates.

The outcome of mixed infection by three species of microsporidia in the genera Endoreticulatus, Nosema, and Vairimorpha, isolated from different populations of Lymantria dispar in Bulgaria, was evaluated in the laboratory. All possible combinations of two species were administered either simultaneously or sequentially to larvae, and mortality, duration of development, and larval weight at 20 days post-infection (simultaneous inoculation) or 23 days post-infection (sequential inoculation) were chosen as the outcome variables. Larvae were also dissected and the presence of each species of microsporidia and the tissues infected were recorded for each treatment. Effects of infection were dependent on both host sex and the type of exposure. Infected larvae were more likely to die than uninfected larvae, but there were no differences in mortality between single and mixed infections. Addition of Endoreticulatus to infections of Nosema or Vairimorpha significantly increased duration of development to the fourth ecdysis; this effect was additive. Addition of Nosema or Vairimorpha to an existing infection had no such effect. When Nosema was administered simultaneously with Endoreticulatus or Vairimorpha, infected larvae weighed more than larvae that had single infections with either pathogen. Nosema was displaced from the silk glands by Vairimorpha and Nosema suppressed octospore formation by Vairimorpha in fat body. The histological evidence combined with the data on larval weight supports the hypothesis that competition occurred in mixed infections.

Larval susceptibility to Bacillus thuringiensis was determined for Nosema pyrausta-infected and uninfected European corn borers, Ostrinia nubilalis (Hübner), in bioassays using a commercial formulation of B. thuringiensis subsp. kurstaki, Dipel ES, incorporated into diet. LC50 values for N. pyrausta-infected larvae were significantly lower (P<0.0001) than for uninfected larvae and declined with increasing levels of infection. LC50 values for a 15-d bioassay using field-colony first instars were 0.006 and 0.027 mg of Dipel ES/kg of diet for larvae moderately infected by N. pyrausta and uninfected larvae, respectively. Nosema pyrausta-infected larvae reared on Dipel ES-amended diets produced 70-fold fewer spores (P<0.0001) than larvae reared on standard diet. For example, 15 d after placement as first instars on standard diet, infected field-colony larvae produced 7.6-8.7 million N. pyrausta spores per larva; similar larvae placed on diet containing 0.09 mg of Dipel ES/kg of diet produced 85-103 thousand spores per larva. Infected larvae also weighed less and failed to mature on Dipel ES-amended diets. Increased susceptibility of N. pyrausta-infected larvae to Dipel ES and reduced N. pyrausta spore production in larvae feeding on diet containing Dipel ES suggest that Bt corn will have a direct adverse effect on the survival and continual impact of N, pyrausta as a regulating factor on European corn borer populations.

We employed a phylogenomic approach to study the evolution of alpha subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven alpha proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple alpha subunit sequences generated single alpha proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct alpha proteasome genes each. The presence of seven distinct alpha proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the alpha proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the alpha proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented.

Infection of the gypsy moth, Lymantria dispar, with the microsporidium Vairimorpha sp. strongly influences the development of the host in ways typical of many species of terrestrial entomopathogenic Microsporidia; growth is reduced while development time is extended in infected insects. The appearance of the different stages of the parasite in the host relative to the elapsed time after oral infection, as well as the influence of the parasite proliferation on food utilization of the host, were examined. At 3 days postinfection, midgut muscle cells were infected with primary spores, and the fat body tissues contained meronts, sporonts, and primary spores. Many more fat body cells contained vegetative stages and primary spores at 4 and 5 days postinfection, and diplokaryotic spores and immature octospores were also present. Approximate digestibility of infected larvae increased during this time period, whereas the conversion of ingested and digested food to body substance decreased. The relative growth rate of infected and uninfected groups did not differ significantly between 4 and 5 days postinfection, although the relative consumption rate in infected L. dispar larvae was higher. Between 8 and 10 days postinfection, the relative growth rate of uninfected larvae increased. The infected group did not demonstrate this increase at a time period characterized by maturation of diplokaryotic spores and octospores in larval fat body tissues. Total body weight of uninfected larvae remained higher than that of infected larvae after 8 days postinfection.

A microsporidium Nosema portugal n. sp. was isolated from gypsy moths, Lymantria dispar L, collected near Lisbon, Portugal, in 1985. The life cycle includes two sequential developmental cycles, a primary and a secondary cycle. The primary cycle occurs in midgut epithelial cells, where primary spores are produced within 48 h. The primary spores immediately extrude their polar filaments, presumably to infect other cells. In the target tissues (salivary glands and fat body) the secondary development cycle is followed by the formation of environmental spores. Primary spores were also sometimes present in target tissues. Fresh unfixed and unstained primary spores have a large posterior vacuole and measured 4.8 x 2.7 &mgr;m. Ultrastructurally, they have 5-8 polar filament coils, a large posterior vacuole, abundant endoplasmic reticulum, and were binucleate. Mature unfixed and unstained environmental spores were highly refractive and the posterior vacuole and nuclei could not be seen through the spore coat. Fresh environmental spores measured 4.5 x 1.9 &mgr;m. Ultrastructurally, environmental spores were binucleate, with a typical polaroplast, 10-11 isofilar polar filament coils, and a series of 4-6 thin polar filament-like tubules situated at the posterior end of the row of typical polar filament coils. The ssu rRNA sequences strongly suggest that this species is more closely related to the Vairimorpha subgroup within the Nosema/Vairimorpha clade than to the Nosema subgroup. Copyright 1999 Academic Press.

An early sporulation event in the host midgut tissues has been reported for several species of microsporidia infecting Diptera, Hymenoptera, and Lepidoptera. The role of these primary spores, formed between 35 and 96 h postinfection per os, has been suggested to be the cell to cell spread of infection within the host, but the sequence of events during the early sporulation stages has been reported for only a few species of microsporidia. We investigated these early life cycle events for two species of microsporidia, Vairimorpha necatrix and Vairimorpha sp. from Lymantria dispar, tested in the laboratory hosts Spodoptera exigua and L. dispar, respectively. We injected hemolymph drawn from orally infected host larvae into uninfected larvae at time periods from 2 to 96 h postinfection to determine the timing of infection of the hemolymph and target tissues. Our studies demonstrated that, for both Vairimorpha species, the early sporulation in the midgut tissues is a discrete first stage of infection. Invasion of the hemolymph and infection of the target tissues follows maturation of "primary spores" in the midgut and coincides with the germination of these spores beginning approximately 30 h postinfection for hosts held at a constant 24 degreesC. The developmental stages of the microsporidia observed in the target tissues at specific time periods postinfection corresponded to the stages observed in the hemocytes, suggesting that sporoplasms from the germinating primary spores are directly injected into the target tissues and the hemolymph. Copyright 1998 Academic Press.

For most groups of biological control agents the relationship between laboratory (physiological) host range and the host range in the field (ecological host range) has not been explored empirically. The objective of our study was to investigate this relationship using the North America gypsy moth, Lymantria dispar, as a model nontarget host for microsporidia from native North American Lepidoptera. The gypsy moth, L. dispar, a native of Europe, has been established in North America for nearly 130 years and presumably exposed to many species of microsporidia from sympatric native Lepidoptera. Nevertheless, microsporidia have never been observed in North American populations of L. dispar. We conducted traditional laboratory feeding experiments using microsporidia from 20 lepidopteran host species and 1 coleopteran host species against L. dispar. Microsporidia from 18 native hosts infected L. dispar larvae. Although some of the infections were not typical of infections in the indigenous natural hosts, mature spores were produced in most of these infections. Horizontal transmission experiments, based on exposure of uninfected L. dispar larvae to infected L. dispar larvae, demonstrated that the microsporidia were far more host specific than the direct feeding experiments suggested. Of the three microsporidian biotypes that were horizontally transmitted between the nontarget L. dispar larvae, all were transmitted at very low levels. The results of our experiments provide additional evidence that the ecological host specificity of terrestrial microsporidia is much narrower than the physiological host specificity. Our studies establish the validity of using nonindigenous insect species with long-term data sets on natural enemies associated with them as a tool for testing hypotheses about host specificity., (Copyright 1998 Academic Press.)

Helicoverpa zea larvae were infected with Vairimorpha necatrix. The fat body was triturated and sporulation stages were fractionated according to buoyant density by Ludox density gradient centrifugation. Spores and sporulation stages formed two minor bands with buoyant densities of 1.072 and 1.121 g/ml, and two major bands with buoyant densities of 1.150 and 1.198 g/ml. The higher bands of less dense sporoblastic stages first appeared 96 hr following infection and the band containing the heaviest (1.198 g/ml) and most refringent mature spores appeared last, ca. 24 hr after the appearance of the other bands. Although the bands remained in the same relative positions regardless of the time after inoculation, the percentage of spores in Band 4 increased with time. The concentration of sugar in the spores from the 1.198-g/ml band was more than four times the concentration found in spores in the 1.150-g/ml band and accounted for more than 60% of the increase in spore weight between the two density classes. Similar tests with Nosema algerae yielded similar results, with sugars accounting for 88% of the increased spore weight. Sugar acquisition appears to occur during the attainment of the final spore density and perhaps signals spore maturation.

Results of traditional laboratory bioassays may not accurately represent ecological (field) host specificity of entomopathogens but, if carefully interpreted, may be used to predict the ecological host specificity of pathogens being considered for release as classical biological control agents. We conducted laboratory studies designed to evaluate the physiological host specificity of microsporidia, which are common protozoan pathogens of insects. In these studies, 49 nontarget lepidopteran species indigenous to North America were fed five biotypes of microsporidia that occur in European populations of Lymantria dispar but are not found in North American populations of L. dispar. These microsporidia, Microsporidium sp. from Portugal, Microsporidium sp. from Romania, Microsporidium sp. from Slovakia, Nosema lymantriae, and Endoreticulatus sp. from Portugal, are candidates for release as classical biological control agents into L. dispar populations in the United States. The microsporidia produced a variety of responses in the nontarget hosts and, based on these responses, the nontarget hosts were placed in the following categories: (1) no infection (refractory), (2) atypical infections, and (3) heavy infections. Endoreticulatus sp. produced patent, host-like infections in nearly two-thirds of the nontarget hosts to which it was fed. Such generalist species should not be recommended for release. Infections comparable to those produced in L. dispar were produced in 2% of the nontarget hosts fed Microsporidium sp. from Portugal, 19% of nontarget hosts fed Microsporidium sp. from Romania, 13% fed spores of Microsporidium sp. from Slovakia, and 11% of nontarget species fed N. lymantriae. The remaining nontarget species developed infections that, despite production of mature spores, were not typical of infection in L. dispar. We believe it is very unlikely that these atypical infections would be horizontally transmitted within nontarget insect populations in the United States.

Thirty-six carrion beetles (Silphidae: Nicrophorus tomentosus Weber, Oiceoptoma noveboracense (Forster), Necrophila americana (L.] collected in the Great Swamp National Wildlife Refuge, Basking Ridge, N.J., were dissected and the midgut, hindgut, and associated hemolymph were cultured for bacteria. Analytical profile index rapid biochemical systems were used for bacterial identifications. Nineteen bacteria were identified to species and four to the genus level. Although frank pathogens were not found, several of the identified coliform and staphylococci bacteria were known opportunistic pathogens. More than 20 additional morphologically distinct bacteria were cultured, but these could not be identified using the rapid biochemical test strips because of data base limitations.