The gut parasite Nosema ceranae impairs olfactory learning in bumblebees.

Pollinators are exposed to numerous parasites and pathogens when foraging on flowers. These biological stressors may affect critical cognitive abilities required for foraging. Here, we tested whether exposure to Nosema ceranae, one of the most widespread parasites of honey bees also found in wild pollinators, impacts cognition in bumblebees. We investigated different forms of olfactory learning and memory using conditioning of the proboscis extension reflex. Seven days after being exposed to parasite spores, bumblebees showed lower performance in absolute, differential and reversal learning than controls. The consistent observations across different types of olfactory learning indicate a general negative effect of N. ceranae exposure that did not specifically target particular brain areas or neural processes. We discuss the potential mechanisms by which N. ceranae impairs bumblebee cognition and the broader consequences for populations of pollinators.

Release of Mediator Enzyme ß-Hexosaminidase and Modulated Gene Expression Accompany Hemocyte Degranulation in Response to Parasitism in the Silkworm Bombyx mori.

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme ß-hexosaminidase showed increased activity and expression of ß-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R2 = 0.71) between LDH activity and ß-hexosaminidase released after the infection showed parasitic-protein-induced hemocyte damage and accompanied release of the enzymes. Expression of ß-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R2 = 0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45-60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

Targeting the honey bee gut parasite Nosema ceranae with siRNA positively affects gut bacteria.

BACKGROUND: Gut microbial communities can contribute positively and negatively to host health. So far, eight core bacterial taxonomic clusters have been reported in honey bees. These bacteria are involved in host metabolism and defenses. Nosema ceranae is a gut intracellular parasite of honey bees which destroys epithelial cells and gut tissue integrity. Studies have shown protective impacts of honey bee gut microbiota towards N. ceranae infection. However, the impacts of N. ceranae on the relative abundance of honey bee gut microbiota remains unclear, and has been confounded during prior infection assays which resulted in the co-inoculation of bacteria during Nosema challenges. We used a novel method, the suppression of N. ceranae with specific siRNAs, to measure the impacts of Nosema on the gut microbiome. RESULTS: Suppressing N. ceranae led to significant positive effects on microbial abundance. Nevertheless, 15 bacterial taxa, including three core taxa, were negatively correlated with N. ceranae levels. In particular, one co-regulated group of 7 bacteria was significantly negatively correlated with N. ceranae levels. CONCLUSIONS: N. ceranae are negatively correlated with the abundance of 15 identified bacteria. Our results provide insights into interactions between gut microbes and N. ceranae during infection.

Cross-infectivity of honey and bumble bee-associated parasites across three bee families.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Effects of the microsporidian pathogen, Nosema adaliae (Nosematidae) on the seven-spotted lady beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae).

Lady beetles are important predators in nature. Some species, including the two-spotted lady beetle, Adalia bipunctata L., are native to North America, whereas others, such as the seven-spotted lady beetle, Coccinella septempunctata L., have been introduced in North America for pest control on agriculture crops. Microsporidia are obligate pathogens that cause chronic disease, and these pathogens are known to infect several lady beetle species. Lady beetles are cannibalistic and, because many species share a given landscape, there is potential for microsporidia to infect susceptible coccinellids when infected eggs are eaten. The objective of this study was to examine the effects of the microsporidium Nosema adaliae isolated from A. bipunctata on C. septempunctata fitness (larval development and mortality, sex ratio, adult longevity and fecundity). Mortality was higher for C. septempunctata larvae that ate four A. bipunctata eggs (≥96% mortality) than for those that ate only one (<63.8%), suggesting that the mortality observed was influenced by the number of eggs eaten. A. bipunctata eggs contain adaline and adalinine, two species-specific alkaloids that have been shown to be detrimental to C. septempunctata larvae. Development of larvae that consumed one uninfected or one N. adaliae-infected A. bipunctata egg, did not differ significantly (20.5 ±â€¯0.2 d and 21.3 ±â€¯0.4 d, respectively) and, although mortality remained high for these larvae (53.5% and 65.6% mortality, respectively), these values also did not differ significantly (p = 0.05). Over a 60-d period, mean fecundity for C. septempunctata adults that ate one uninfected A. bipunctata egg as first-instar larvae was significantly greater (776.6 ±â€¯122.0 eggs) than those that ate one N. adaliae-infected egg (335.6 ±â€¯86.6 eggs, p = 0.005). Larvae from the former group also lived significantly longer (58.2 ±â€¯1.8 d) than did those from the latter group (38.4 ±â€¯6.4 d, p = 0.010). Sex ratios of adult beetles did not differ significantly. Because A. bipunctata and C. septempunctata share similar habitats, it is reasonable to expect these two coccinellids to encounter one another in nature. Results of this study show that the consumption of only one infected A. bipunctata egg by C. septempunctata larvae can result in high larval mortality and reduced fecundity.

Test of the invasive pathogen hypothesis of bumble bee decline in North America.

Emergent fungal diseases are critical factors in global biodiversity declines. The fungal pathogenNosema bombiwas recently found to be widespread in declining species of North American bumble bees (Bombus), with circumstantial evidence suggesting an exotic introduction from Europe. This interpretation has been hampered by a lack of knowledge of global genetic variation, geographic origin, and changing prevalence patterns ofN. bombiin declining North American populations. Thus, the temporal and spatial emergence ofN. bombiand its potential role in bumble bee decline remain speculative. We analyzeNosemaprevalence and genetic variation in the United States and Europe from 1980, before an alleged introduction in the early 1990s, to 2011, extractingNosemaDNA fromBombusnatural history collection specimens from across this time period.Nosema bombiprevalence increased significantly from low detectable frequency in the 1980s to significantly higher frequency in the mid- to late-1990s, corresponding to a period of reported massive infectious outbreak ofN. bombiin commercial bumble bee rearing stocks in North America. Despite the increased frequency, we find no conclusive evidence of an exoticN. bombiorigin based on genetic analysis of globalNosemapopulations; the widespreadNosemastrain found currently in declining United States bumble bees was present in the United States before commercial colony trade. Notably, the USN. bombiis not detectably different from that found predominantly throughout Western Europe, with both regions characterized by low genetic diversity compared with high levels of diversity found in Asia, where commercial bee breeding activities are low or nonexistent.

Transcriptional response of honey bee (Apis mellifera) to differential nutritional status and Nosema infection.

BACKGROUND: Bees are confronting several environmental challenges, including the intermingled effects of malnutrition and disease. Intuitively, pollen is the healthiest nutritional choice, however, commercial substitutes, such as Bee-Pro and MegaBee, are widely used. Herein we examined how feeding natural and artificial diets shapes transcription in the abdomen of the honey bee, and how transcription shifts in combination with Nosema parasitism. RESULTS: Gene ontology enrichment revealed that, compared with poor diet (carbohydrates [C]), bees fed pollen (P > C), Bee-Pro (B > C), and MegaBee (M > C) showed a broad upregulation of metabolic processes, especially lipids; however, pollen feeding promoted more functions, and superior proteolysis. The superiority of the pollen diet was also evident through the remarkable overexpression of vitellogenin in bees fed pollen instead of MegaBee or Bee-Pro. Upregulation of bioprocesses under carbohydrates feeding compared to pollen (C > P) provided a clear poor nutritional status, uncovering stark expression changes that were slight or absent relatively to Bee-Pro (C > B) or MegaBee (C > M). Poor diet feeding (C > P) induced starvation response genes and hippo signaling pathway, while it repressed growth through different mechanisms. Carbohydrate feeding (C > P) also elicited 'adult behavior', and developmental processes suggesting transition to foraging. Finally, it altered the 'circadian rhythm', reflecting the role of this mechanism in the adaptation to nutritional stress in mammals. Nosema-infected bees fed pollen compared to carbohydrates (PN > CN) upheld certain bioprocesses of uninfected bees (P > C). Poor nutritional status was more apparent against pollen (CN > PN) than Bee-Pro (CN > BN) or MegaBee (CN > MN). Nosema accentuated the effects of malnutrition since more starvation-response genes and stress response mechanisms were upregulated in CN > PN compared to C > P. The bioprocess 'Macromolecular complex assembly' was also enriched in CN > PN, and involved genes associated with human HIV and/or influenza, thus providing potential candidates for bee-Nosema interactions. Finally, the enzyme Duox emerged as essential for guts defense in bees, similarly to Drosophila. CONCLUSIONS: These results provide evidence of the superior nutritional status of bees fed pollen instead of artificial substitutes in terms of overall health, even in the presence of a pathogen.

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.

Honey Bee (Apis mellifera) Pollen Foraging Reflects Benefits Dependent on Individual Infection Status.

Parasites often modify host foraging behavior, for example, by spurring changes to nutrient intake ratios or triggering self-medication. The gut parasite, Nosema ceranae, increases energy needs of the European or Western honey bee (Apis mellifera), but little is known about how infection affects foraging behavior. We used a combination of experiments and observations of caged and free-flying individual bees and hives to determine how N. ceranae affects honey bee foraging behavior. In an experiment with caged bees, we found that infected bees with access to a high-quality pollen were more likely to survive than infected bees with access to a lower quality pollen or no pollen. Non-infected bees showed no difference in survival with pollen quality. We then tested free-flying bees in an arena of artificial flowers and found that pollen foraging bees chose pollen commensurate with their infection status; twice as many infected bees selected the higher quality pollen than the lower quality pollen, while healthy bees showed no preference between pollen types. However, healthy and infected bees visited sucrose and pollen flowers in the same proportions. Among hive-level observations, we found no significant correlations between N. ceranae infection intensity in the hive and the proportion of bees returning with pollen. Our results indicate that N. ceranae-infected bees benefit from increased pollen quality and will selectively forage for higher quality while foraging for pollen, but infection status does not lead to increased pollen foraging at either the individual or hive levels.

Pathological analysis of silkworm infected by two microsporidia Nosema bombycis CQ1 and Vairimorpha necatrix BM.

Microsporidia Nosema bombycis CQ1 can be vertically transmitted in silkworm Bombyx mori but Vairimorpha necatrix BM cannot. Therefore, the pathological differences in silkworm infected with these two microsporidia required clarification. Here, we compared the virulence of N. bombycis CQ1 and V. necatrix BM against silkworm. The pathological characteristics in intestine, testis and ovary were surveyed using paraffin sections, scanning electron microscopy and transmission electron microscopy. Our data firstly showed that the virulence of V. necatrix BM was weaker than that of N. bombycis CQ1. Secondly, the typical symptom of V. necatrix BM infection is making xenomas, which are full of pathogens in different stages, at the posterior of intestine. However, no xenomas were formed surrounding intestines infected with N. bombycis CQ1. Thirdly, N. bombycis CQ1 can cluster spores near the trachea while infecting ovaries. It is worth noting that N. bombycis CQ1 infected epithelial cells and connective tissues of ovaries, while V. necatrix BM did not. Although silkworm ovaries can not be infected by V. necatrix BM in vivo, it can infect embryonic and ovarian cell lines in vitro. This study is the first report about comparing infection features of N. bombycis CQ1 and V. necatrix BM in silkworm tissues and it provided elaborate and visual information of pathological characteristics which can help to explain the different transmission strategies of these two microsporidia.

Greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) as a resistant model host for Nosema pyrausta (Microsporidia: Nosematidae).

Galleria mellonella fed 3 million Nosema pyrausta spores per larva showed 0 and 5% infestation rate at 30 °C and 24 °C, respectively. N. pyrausta virulence did not increase after passage through G. mellonella for three generations. When larvae were pretreated with phenylthiourea, Bacillus thuringiensis or combination of both, infection rates were 11%, 15% and 22%, respectively. Injection of untreated and potassium hydroxide-primed spores resulted in approximately 10% and 50% infection, respectively. G. mellonella is resistant to high dosages of N. pyrausta spores, serving as a prospective model of insect resistance to microsporidia, while host immunosuppression and/or spore activation increases success of the pathogen.

Infection dynamics of Nosema ceranae in honey bee midgut and host cell apoptosis.

Nosema ceranae is an intracellular microsporidian parasite that infects epithelial cells of the honey bee (Apis mellifera) midgut. Previous studies have shown that Nosema may alter cell renewal and apoptosis in honey bees. We found that the amount of apoptotic cells progressively declines from the anterior towards posterior regions of the midgut in Nosema-infected sensitive bees. There was no such pattern in the infected Nosema tolerant honey bees and controls. These data provide additional evidence that N. ceranae appears to alter apoptosis in its host cells for its own advantage.

Global decline of bumblebees is phylogenetically structured and inversely related to species range size and pathogen incidence.

Conservation biology can profit greatly from incorporating a phylogenetic perspective into analyses of patterns and drivers of species extinction risk. We applied such an approach to analyse patterns of bumblebee (Bombus) decline. We assembled a database representing approximately 43% of the circa 260 globally known species, which included species extinction risk assessments following the International Union fo Conservation of Nature Red List categories and criteria, and information on species traits presumably associated with bumblebee decline. We quantified the strength of phylogenetic signal in decline, range size, tongue length and parasite presence. Overall, about one-third of the assessed bumblebees are declining and declining species are not randomly distributed across the Bombus phylogeny. Susceptible species were over-represented in the subgenus Thoracobombus (approx. 64%) and under-represented in the subgenus Pyrobombus (approx. 6%). Phylogenetic logistic regressions revealed that species with small geographical ranges and those in which none of three internal parasites were reported (i.e. Crithidia bombi, Nosema spp. or Locustacarus buchneri) were particularly vulnerable. Bumblebee evolutionary history will be deeply eroded if most species from threatened clades, particularly those stemming from basal nodes, become finally extinct. The habitat of species with restricted distribution should be protected and the importance of pathogen tolerance/resistance as mechanisms to deal with pathogens needs urgent research.

Bacterium-Expressed dsRNA Downregulates Microsporidia Nosema bombycis Gene Expression.

The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain (DE3) to express double-strand RNAs targeting the gene encoding ADP/ATP protein in N. bombycis. The results showed that dsRNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the dsRNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm.

Changes in the Bacteriome of Honey Bees Associated with the Parasite Varroa destructor, and Pathogens Nosema and Lotmaria passim.

The honey bee, Apis mellifera, is a globally important species that suffers from a variety of pathogens and parasites. These parasites and pathogens may have sublethal effects on their bee hosts via an array of mechanisms, including through a change in symbiotic bacterial taxa. Our aim was to assess the influence of four globally widespread parasites and pathogens on the honey bee bacteriome. We examined the effects of the ectoparasitic mite Varroa destructor, the fungal pathogens Nosema apis and Nosema ceranae, and the trypanosome Lotmaria passim. Varroa was detected by acaricidal treatment, Nosema and L. passim by PCR, and the bacteriome using MiSeq 16S rRNA gene sequencing. Overall, the 1,858,850 obtained sequences formed 86 operational taxonomic units (OTUs) at 3 % dissimilarity. Location, time of year, and degree of infestation by Varroa had significant effects on the composition of the bacteriome of honey bee workers. Based on statistical correlations, we found varroosis more important factor than N. ceranae, N. apis, and L. passim infestation influencing the honey bee bacteriome and contributing to the changes in the composition of the bacterial community in adult bees. At the population level, Varroa appeared to modify 20 OTUs. In the colonies with high Varroa infestation levels (varroosis), the relative abundance of the bacteria Bartonella apis and Lactobacillus apis decreased. In contrast, an increase in relative abundance was observed for several taxa including Lactobacillus helsingborgensis, Lactobacillus mellis, Commensalibacter intestini, and Snodgrassella alvi. The results showed that the "normal" bacterial community is altered by eukaryotic parasites as well as displaying temporal changes and changes associated with the geographical origin of the beehive.

Characterization of Nosema ceranae Genetic Variants from Different Geographic Origins.

In recent years, large-scale colony losses of honey bees (Apis mellifera) have been reported and the infection with the microsporidia Nosema ceranae has been involved. However, the effect of N. ceranae at the colony level and its role in colony losses vary in different geographic areas. This difference may be related to the presence of multiple N. ceranae genetic variants resulting in different biological consequences. In this study, we analyzed the genetic diversity of 75 N. ceranae samples obtained from 13 countries and Hawaii through inter-sequence single repetition (ISSR) and evaluated if two of these genetic variants triggered different immune responses when infecting Apis mellifera iberiensis. The genetic diversity analysis showed that 41% of the samples had the same DNA amplification pattern, including samples from most European countries except Spain, while the remaining samples showed high variability. Infection assays were performed to analyze the infection levels and the immune response of bees infected with N. ceranae from Spain and Uruguay. The infected bees presented similar infection levels, and both isolates downregulated the expression of abaecin, confirming the ability of the microsporidia to depress the immune response. Only N. ceranae from Uruguay downregulated the expression level of imd compared to control bees. On the other hand, both genetic variants triggered different expression levels of lysozyme. As imd and lysozyme play important roles in the response to pathogens, these results could reflect differences in the biological consequences of N. ceranae variants in A. mellifera infection.

Pathogenicity, prevalence and intensity of a microsporidian infection by Nosema fumiferanae postvittana in the light brown apple moth, Epiphyas postvittana, in California.

While biological invasions are increasing, in some cases exotic species exhibit an initial phase of population growth and spread, followed by a subsequent phase of natural decline. The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), provides a unique opportunity to examine potential mechanisms for the natural suppression of an exotic insect species that has become established in coastal California. We recently discovered a microsporidian pathogen, Nosema fumiferanae postvittana, from E. postvittana in its novel range. In the laboratory, we examined the pathogenicity and latent period of this microsporidium, and in the field we determined its prevalence and intensity in five locations using quantitative real-time PCR (qPCR). In the laboratory, when comparing healthy larvae to larvae infected with up to 10(5) spores, we found a reduction in juvenile survivorship (from 100% to 26%), a prolongation of juvenile development time (of up to 9-10 days), a reduction in viable lifetime fecundity (from 788 to 1) and a reduction in the intrinsic rate of increase (from 0.18 to 0.008). The median lethal dose (LD50) was estimated to be 1.8 × 10(4) spores, and the mean latent period for infections with 10(3) spores was 12.67 days. Our field sampling revealed that E. postvittana populations have further declined from previously reported densities in San Francisco and Santa Cruz. We detected N. fumiferanae postvittana in all five locations with an overall prevalence of 5%, which did not appear to be influenced consistently by either host density or season. Mean microsporidian intensity in field-infected individuals was 226 spores. Although the laboratory results demonstrated the potential for host suppression, the field sampling indicated that the prevalence and intensity of microsporidian infection were too low to account for the continued decline in population densities of E. postvittana in coastal California.

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.

Ricin-B-lectin enhances microsporidia Nosema bombycis infection in BmN cells from silkworm Bombyx mori.

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion.