A formal redefinition of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) and reassignment of species based on molecular phylogenetics.

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.

Nosema maddoxi sp. nov. (Microsporidia, Nosematidae), a Widespread Pathogen of the Green Stink Bug Chinavia hilaris (Say) and the Brown Marmorated Stink Bug Halyomorpha halys (Stål).

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.

Israeli acute paralysis virus: epidemiology, pathogenesis and implications for honey bee health.

Israeli acute paralysis virus (IAPV) is a widespread RNA virus of honey bees that has been linked with colony losses. Here we describe the transmission, prevalence, and genetic traits of this virus, along with host transcriptional responses to infections. Further, we present RNAi-based strategies for limiting an important mechanism used by IAPV to subvert host defenses. Our study shows that IAPV is established as a persistent infection in honey bee populations, likely enabled by both horizontal and vertical transmission pathways. The phenotypic differences in pathology among different strains of IAPV found globally may be due to high levels of standing genetic variation. Microarray profiles of host responses to IAPV infection revealed that mitochondrial function is the most significantly affected biological process, suggesting that viral infection causes significant disturbance in energy-related host processes. The expression of genes involved in immune pathways in adult bees indicates that IAPV infection triggers active immune responses. The evidence that silencing an IAPV-encoded putative suppressor of RNAi reduces IAPV replication suggests a functional assignment for a particular genomic region of IAPV and closely related viruses from the Family Dicistroviridae, and indicates a novel therapeutic strategy for limiting multiple honey bee viruses simultaneously and reducing colony losses due to viral diseases. We believe that the knowledge and insights gained from this study will provide a new platform for continuing studies of the IAPV-host interactions and have positive implications for disease management that will lead to mitigation of escalating honey bee colony losses worldwide.

Pathogenicity, morphology, and characterization of a Nosema fumiferanae isolate (Microsporidia: Nosematidae) from the light brown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae) in California.

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.

Nosema ceranae escapes fumagillin control in honey bees.

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.

Infectivity and virulence of Nosema ceranae and Nosema apis in commercially available North American honey bees.

Nosema ceranae infection is ubiquitous in western honey bees, Apis mellifera, in the United States and the pathogen has apparently replaced Nosema apis in colonies nationwide. Displacement of N. apis suggests that N. ceranae has competitive advantages but N. ceranae was significantly less infective and less virulent than N. apis in commercially available lineages of honey bees in studies conducted in Illinois and Texas. At 5 days post eclosion, the most susceptible age of adult bees tested, the mean ID50 for N. apis was 359 spores compared to 3217 N. ceranae spores, a nearly 9-fold difference. Infectivity of N. ceranae was also lower than N. apis for 24-h and 14-day worker bees. N. ceranae was less infective than reported in studies using European strains of honey bees, while N. apis infectivity, tested in the same cohort of honey bees, corresponded to results reported globally from 1972 to 2010. Mortality of worker bees was similar for both pathogens at a dosage of 50 spores and was not different from the uninfected controls, but was significantly higher for N. apis than N. ceranae at dosages ⩾500 spores. Our results provide comparisons for evaluating research using different ages of bees and pathogen dosages and clarify some controversies. In addition, comparisons among studies suggest that the mixed lineages of US honey bees may be less susceptible to N. ceranae infections than are European bees or that the US isolates of the pathogen are less infective and less virulent than European isolates.

Impacts of Nosema sp. (Microsporidia: Nosematidae) on the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae).

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.

Review of the genus Endoreticulatus (Microsporidia, Encephalitozoonidae) with description of a new species isolated from the grasshopper Poecilimon thoracicus (Orthoptera: Tettigoniidae) and transfer of Microsporidium itiiti Malone to the genus.

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.

Comparative virulence and competition between Nosema apis and Nosema ceranae in honey bees (Apis mellifera).

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.

Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites.

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.

Patterns of widespread decline in North American bumble bees.

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.

Specific detection and localization of microsporidian parasites in invertebrate hosts by using in situ hybridization.

We designed fluorescence in situ hybridization probes for two distinct microsporidian clades and demonstrated their application in detecting, respectively, Nosema/Vairimorpha and Dictyoceola species. We used them to study the vertical transmission of two microsporidia infecting the amphipod Gammarus duebeni.

Comparative development and tissue tropism of Nosema apis and Nosema ceranae.

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.

A new microsporidium, Triwangia caridinae gen. nov., sp. nov. parasitizing fresh water shrimp, Caridina formosae (Decapoda: Atyidae) in Taiwan.

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.

Nosema ceranae infection intensity highly correlates with temperature.

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.

Interspecific geographic distribution and variation of the pathogens Nosema bombi and Crithidia species in United States bumble bee populations.

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.

Survey of bumble bee (Bombus) pathogens and parasites in Illinois and selected areas of northern California and southern Oregon.

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.

Host specificity of microsporidia pathogenic to the gypsy moth, Lymantria dispar (L.): field studies in Slovakia.

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.