Imidacloprid disrupts larval molting regulation and nutrient energy metabolism, causing developmental delay in honey bee Apis mellifera.

Imidacloprid is a global health threat that severely poisons the economically and ecologically important honeybee pollinator, Apis mellifera. However, its effects on developing bee larvae remain largely unexplored. Our pilot study showed that imidacloprid causes developmental delay in bee larvae, but the underlying toxicological mechanisms remain incompletely understood. In this study, we exposed bee larvae to imidacloprid at environmentally relevant concentrations of 0.7, 1.2, 3.1, and 377 ppb. There was a marked dose-dependent delay in larval development, characterized by reductions in body mass, width, and growth index. However, imidacloprid did not affect on larval survival and food consumption. The primary toxicological effects induced by elevated concentrations of imidacloprid (377 ppb) included inhibition of neural transmission gene expression, induction of oxidative stress, gut structural damage, and apoptosis, inhibition of developmental regulatory hormones and genes, suppression of gene expression levels involved in proteolysis, amino acid transport, protein synthesis, carbohydrate catabolism, oxidative phosphorylation, and glycolysis energy production. In addition, we found that the larvae may use antioxidant defenses and P450 detoxification mechanisms to mitigate the effects of imidacloprid. Ultimately, this study provides the first evidence that environmentally exposed imidacloprid can affect the growth and development of bee larvae by disrupting molting regulation and limiting the metabolism and utilization of dietary nutrients and energy. These findings have broader implications for studies assessing pesticide hazards in other juvenile animals.

Maturation of subtilisin-like protease NbSLP1 from microsporidia Nosema bombycis.

Microsporidia are obligate intracellular parasites and possess a unique way of invading hosts, namely germination. Microsporidia are able to infect almost all animal cells by germination. During the process, the polar tube extrudes from the spores within, thus injecting infectious sporoplasm into the host cells. Previous studies indicated that subtilisin-like protease 1 (NbSLP1) of microsporidia Nosema bombycis were located at the polar cap of germinated spores where the polar tube extrusion. We hypothesized that NbSLP1 is an essential player in the germination process. Normally, SLP need to be activated by autoproteolysis under conditions. In this study, we found that the signal peptide of NbSLP1 affected the activation of protease, two self-cleavage sites were involved in NbSLP1 maturation between Ala104Asp105 and Ala124Asp125 respectively. Mutants at catalytic triad of NbSLP1 confirmed the decreasing of autoproteolysis. This study demonstrates that intramolecular proteolysis is required for NbSLP1 maturation. The protease undergoes a series of sequential N-terminal cleavage events to generate the mature enzyme. Like other subtilisin-like enzymes, catalytic triad of NbSLP1 are significant for the self-activation of NbSLP1. In conclusion, clarifying the maturation of NbSLP1 will be valuable for understanding the polar tube ejection mechanism of germination.

Melatonin enhances the antioxidant capacity to rescue the honey bee Apis mellifera from the ecotoxicological effects caused by environmental imidacloprid.

Imidacloprid severely poisons the nontarget insect honey bee Apis mellifera. Few treatments are available to mitigate the adverse effects of imidacloprid. The primary concern is that the molecular understanding of imidacloprid toxicity is not comprehensive enough. Oxidative stress is the primary pathophysiological mechanism by which pesticides cause high mortality. Our pilot study found for the first time that imidacloprid stimulates bee brains to secrete melatonin, a free radical scavenger. However, the molecular basis for imidacloprid toxicity and the role of melatonin in coping with imidacloprid have not been systematically investigated in bees. This study administered an environmental dose of imidacloprid (36 ng/bee) orally to A. mellifera. The detoxification gene cytochrome P450 CYP4G11 was significantly induced. However, potent cytotoxicity of imidacloprid suppressed the expression of the antioxidants catalase (CAT) and thioredoxin reductase (TrxR), and the activity of guaiacol peroxidase (GPX), superoxide dismutase (SOD), and reduced glutathione (GSH) was not induced. The levels of reactive oxygen species (ROS) and the lipid peroxidation marker malondialdehyde (MDA) were increased. The expression of the apoptotic genes cysteinyl aspartate specific proteinase (Caspase-3) and apoptosis inducing factor (AIF) increased, and the apoptotic features of midgut cells were prominently apparent. These results suggest that imidacloprid disrupts the bee antioxidant system, causing severe oxidative stress and tissue damage and ultimately leading to apoptosis. Significantly, however, imidacloprid exposure also stimulated bee brains to continuously secrete melatonin. Further preadministration of exogenous melatonin (200 ng/bee) orally to bees significantly reversed and enhanced the activity of the imidacloprid-suppressed antioxidants CAT, SOD, and GSH, which allowed imidacloprid-induced ROS accumulation to be effectively alleviated. The MDA content, apoptotic genes Caspase-3 and AIF, and detoxification gene CYPG411 expression were restored to normalization; midgut cell damage, apoptosis, and mortality were significantly reduced. These findings strongly suggest that melatonin enhanced bee antioxidant capacity, thus attenuating oxidative stress and apoptosis to confer imidacloprid tolerance to honey bees. Melatonin secretion may be a defense mechanism to mitigate imidacloprid toxicity.

Imidacloprid activates ROS and causes mortality in honey bees (Apis mellifera) by inducing iron overload.

Imidacloprid, a neonicotinoid pesticide widely used for insect pest control, has become a potential pollutant to pollinators. Previous reports have demonstrated the toxicity of this drug in activating oxidative stress resulting in high mortality in the honey bee Apis mellifera. However, the mechanisms underlying the toxicity of imidacloprid have not been fully elucidated. In this study, sublethal (36 ng/bee) and median lethal (132 ng/bee) doses of imidacloprid were administered to bees. The results showed dose-dependent increases in reactive oxygen species (ROS), Fe2+, and mortality in bees. Notably, imidacloprid also induced upregulation of the gene encoding ferritin (AmFth), which plays a pivotal role in reducing Fe2+ overload. Upregulation of AmFth has been suggested to be closely related to ROS accumulation and high mortality in bees. To confirm the role played by AmFth in imidacloprid-activated ROS, dsAmFth double-strand was orally administered to bees after exposure to imidacloprid. The results revealed aggravated Fe2+ overload, higher ROS activation, and elevated mortality in the bees, indicating that imidacloprid activated ROS and caused mortality in the bees, probably by inducing iron overload. This study helps to elucidate the molecular mechanisms underlying the toxicity of imidacloprid from the perspective of iron metabolism.

Lipidomic Profiling Reveals Distinct Differences in Sphingolipids Metabolic Pathway between Healthy Apis cerana cerana larvae and Chinese Sacbrood Disease.

Chinese sacbrood disease (CSD), which is caused by Chinese sacbrood virus (CSBV), is a major viral disease in Apis cerana cerana larvae. Analysis of lipid composition is critical to the study of CSBV replication. The host lipidome profiling during CSBV infection has not been conducted. This paper identified the lipidome of the CSBV-larvae interaction through high-resolution mass spectrometry. A total of 2164 lipids were detected and divided into 20 categories. Comparison of lipidome between healthy and CSBV infected-larvae showed that 266 lipid species were altered by CSBV infection. Furthermore, qRT-PCR showed that various sphingolipid enzymes and the contents of sphingolipids in the larvae were increased, indicating that sphingolipids may be important for CSBV infection. Importantly, Cer (d14:1 + hO/21:0 + O), DG (41:0e), PE (18:0e/18:3), SM (d20:0/19:1), SM (d37:1), TG (16:0/18:1/18:3), TG (18:1/20:4/21:0) and TG (43:7) were significantly altered in both CSBV_24 h vs. CK_24 h and CSBV_48 h vs. CK_48 h. Moreover, TG (39:6), which was increased by more than 10-fold, could be used as a biomarker for the early detection of CSD. This study provides evidence that global lipidome homeostasis in A. c. cerana larvae is remodeled after CSBV infection. Detailed studies in the future may improve the understanding of the relationship between the sphingolipid pathway and CSBV replication.

Nbseptin2 Expression Pattern and Its Interaction with NbPTP1 during Microsporidia Nosema bombycis Polar Tube Extrusion.

Nosema bombycis (Nb) is a deadly species of microsporidia capable of causing pébrine, leading to heavy losses in sericulture. Germination is an important biological event in the invasion process of microsporidia. Septins, a family of membrane-associated proteins, play a critical role in tissue invasion and have been recognized as a virulence factor in numerous pathogens. Previous work in our laboratory has shown that Nosema bombycis septin2 (Nbseptin2) interacts with subtilisin-like protease 2 (NbSLP2). Herein, we found that Nbseptin2 was mainly associated with the plasma membrane in spores. Following spore germination, Nbseptin2 was found to co-localize with polar tube protein 1 (NbPTP1) at the polar cap and proximal zone of the polar tube. Co-immunoprecipitation and yeast two-hybrid analysis further confirmed that Nbseptin2 interacted with NbPTP1. The translocation and interaction of Nbseptin2 in the spores suggest that Nbseptin2 may play a significant role in microsporidia polar tube extrusion process. Our findings improve understanding of the mechanisms underlying microsporidia germination.

A Domain of Herpes Simplex Virus pUL33 Required To Release Monomeric Viral Genomes from Cleaved Concatemeric DNA.

Monomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33-encoded protein (pUL33) interacts with pUL28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pUL33 C terminus did not affect viral replication or the interaction of pUL33 with pUL28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pUL33 mutant interacted with pUL28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA.IMPORTANCE This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.

Identification of a new subtilisin-like protease NbSLP2 interacting with cytoskeletal protein septin in Microsporidia Nosema bombycis.

Nosema bombycis is the pathogen of pébrine which brings heavy losses to sericulture every year. As a member of serine proteases, subtilisin-like protease (SLP) is related to the pathogenicity in fungi. In this study, we characterized a novel 63.8kDa subtilisin-like protease NbSLP2 with a predicted transmembrane domain from Microsporidia, N. bombycis. RT-PCR showed that the transcript of NbSLP2 was detected from third day post infection. Immunofluorescence assay (IFA) indicated that NbSLP2 mainly scattered around the spore wall of N. bombycis. Co-immunoprecipitation data and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed that NbSLP2 directly interacts with septin2 of N. bombycis, which is a cytoskeletal protein. IFA showed that NbSLP2 and Nbseptin2 co-localized beneath the spore wall. NbSLP2 can be pulled down by Nbseptin2, further confirming the interaction between NbSLP2 and Nbseptin2. As an important serine protease with a transmembrane domain, NbSLP2 interacting with Nbseptin2, a scaffold protein adjacent to the membrane may provide advantages to stabilize the NbSLP2 for its hydrolysis function.

Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells.

Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative and sporogonic phases of microsporidia Nosema bombycis in host cells. Based on the presence of chitin, Calcofluor White M2R was used to label the sporogonic phase, while ß-tubulin antibody coupled with fluorescence secondary antibody were used to label the proliferative phase by immunofluorescence. This method is simple, efficient and can be used on both infected cells and tissue slices, providing a great potential application in microsporidia research.

Comparative Analysis of the Proteins with Tandem Repeats from 8 Microsporidia and Characterization of a Novel Endospore Wall Protein Colocalizing with Polar Tube from Nosema bombycis.

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal-infecting microsporidia (Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect-infecting microsporidia (Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect-infecting microsporidia was larger than that of mammal-infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC-rich codons. The tandem repeat position within proteins of insect-infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal-infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite.

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.

Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall.

All microsporidia possess a unique, highly specialized invasion mechanism that involves the polar tube and spore wall. The interaction between spore wall proteins (SWPs) and polar tube proteins (PTPs) in the formation, arrangement, orderly orientation, and function of the polar tube and spore wall remains to be determined. This study was undertaken to examine the protein interactions of Nosema bombycis SWP7 (NbSWP7), NbSWP9, and PTPs. Coimmunoprecipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and yeast two-hybrid data demonstrated that NbSWP9, but not NbSWP7, interacts with NbPTP1 and NbPTP2. Furthermore, immunoelectron microscopy (IEM) showed that NbSWP9 was localized mainly in the developing polar tube of sporoblasts, while NbSWP7 was found randomly in the cytoplasm. However, both NbSWP9 and NbSWP7 were located in the polar tube and spore wall of N. bombycis mature spores. The reason why NbSWP7 was localized to the polar tube may be due to the interaction between NbSWP9 and NbSWP7. Interestingly, the majority of NbSWP9, but not NbSWP7, accumulated in the beginning part of the extruded polar tube and the ruptured spore wall called the anchoring disk (AD) when the mature spores germinated under weak-alkaline environmental stimulation. Additionally, anti-NbSWP9 antibody reduced spore germination in a dose-dependent manner. In conclusion, our study further confirmed that NbSWP9 is a scaffolding protein that not only anchors and holds the polar tube but also tethers the polar tube to the spore wall.

Characterization of a novel otubain-like protease with deubiquitination activity from Nosema bombycis (Microsporidia).

Otubains are a recently identified family of deubiquitinating enzymes (DUBs). They are involved in diverse biological processes including protein degradation, signal transduction, and cell immune response. Several microsporidian genomes have been published in the last decade; however, little is known about the otubain-like protease in these widely-spread obligate intracellular parasites. Here, we characterized a 25 kDa otubain-like protease (NbOTU1) from the microsporidian Nosema bombycis, the pathogen causing pebrine disease in the economically important insect Bombyx mori. Sequence analysis showed that this protein contained a conserved catalytic triad of otubains composed of aspartate, cysteine, and histidine residues. The expression of Nbotu1 began on day 3 postinfection as determined by the RT-PCR method. Immunofluorescence analysis indicated that NbOTU1 is localized on the spore wall of N. bombycis. The subcellular localization of the NbOTU1 was further detected with immunoelectron microscopy, which showed that NbOTU1 is localized at the regions around endospore wall and plasma membrane. Deubiquitination analysis confirmed that the recombinant NbOTU1 possessed deubiquitination activity in vitro. Taken together, a novel microsporidian otubain-like protease NbOTU1 was partially characterized in N. bombycis, demonstrating its subcellular location and deubiquitination activity. This study provided a basic reference for further dissecting the function of otubains in microsporidia.

Identification, Diversity and Evolution of MITEs in the Genomes of Microsporidian Nosema Parasites.

Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous DNA transposons, which are widespread in most eukaryotic genomes. However, genome-wide identification, origin and evolution of MITEs remain largely obscure in microsporidia. In this study, we investigated structural features for de novo identification of MITEs in genomes of silkworm microsporidia Nosema bombycis and Nosema antheraeae, as well as a honeybee microsporidia Nosema ceranae. A total of 1490, 149 and 83 MITE-related sequences from 89, 17 and five families, respectively, were found in the genomes of the above-mentioned species. Species-specific MITEs are predominant in each genome of microsporidian Nosema, with the exception of three MITE families that were shared by N. bombycis and N. antheraeae. One or multiple rounds of amplification occurred for MITEs in N. bombycis after divergence between N. bombycis and the other two species, suggesting that the more abundant families in N. bombycis could be attributed to the recent amplification of new MITEs. Significantly, some MITEs that inserted into the homologous protein-coding region of N. bombycis were recruited as introns, indicating that gene expansion occurred during the evolution of microsporidia. NbS31 and NbS24 had polymorphisms in different geographical strains of N. bombycis, indicating that they could still be active. In addition, several small RNAs in the MITEs in N. bombycis are mainly produced from both ends of the MITEs sequence.

Interaction and assembly of two novel proteins in the spore wall of the microsporidian species Nosema bombycis and their roles in adherence to and infection of host cells.

Microsporidia are obligate intracellular parasites with rigid spore walls that protect against various environmental pressures. Despite an extensive description of the spore wall, little is known regarding the mechanism by which it is deposited or the role it plays in cell adhesion and infection. In this study, we report the identification and characterization of two novel spore wall proteins, SWP7 and SWP9, in the microsporidian species Nosema bombycis. SWP7 and SWP9 are mainly localized to the exospore and endospore of mature spores and the cytoplasm of sporonts, respectively. In addition, a portion of SWP9 is targeted to the spore wall of sporoblasts earlier than SWP7 is. Both SWP7 and SWP9 are specifically colocalized to the spore wall in mature spores. Furthermore, immunoprecipitation, far-Western blotting, unreduced SDS-PAGE, and yeast two-hybrid data demonstrated that SWP7 interacted with SWP9. The chitin binding assay showed that, within the total spore protein, SWP9 and SWP7 can bind to the deproteinated chitin spore coats (DCSCs) of N. bombycis. However, binding of the recombinant protein rSWP7-His to the DCSCs is dependent on the combination of rSWP9-glutathione S-transferase (GST) with the DCSCs. Finally, rSWP9-GST, anti-SWP9, and anti-SWP7 antibodies decreased spore adhesion and infection of the host cell. In conclusion, SWP7 and SWP9 may have important structural capacities and play significant roles in modulating host cell adherence and infection in vitro. A possible major function of SWP9 is as a scaffolding protein that supports other proteins (such as SWP7) that form the integrated spore wall of N. bombycis.

Characterization of a novel spore wall protein NbSWP16 with proline-rich tandem repeats from Nosema bombycis (microsporidia).

Nosema bombycis, a pathogen of silkworm pebrine, is an obligate unicellular eukaryotic parasite. It is reported that the spore wall proteins have essential functions in the adherence and infection process of microsporidia. To date, the information related to spore wall proteins from microsporidia is still limited. Here, a 44 kDa spore wall protein NbSWP16 was characterized in N. bombycis. In NbSWP16, a 25 amino acids signal peptide and 3 heparin binding motifs were predicted. Interestingly, a region that contains 3 proline-rich tandem repeats lacking homology to any known protein was also present in this protein. The immunofluorescence analysis (IFA) demonstrated that distinct fluorescent signals were detected both on the surface of mature spores and the germinated spore coats. Immunolocation by electron microscopy revealed that NbSWP16 localized on the exospore regions. Finally, spore adherence analysis indicated that spore adherence to host cell was decreased more than 20% by anti-NbSWP16 blocking compared with the negative control in vitro. In contrast with anti-NbSWP16, no remarkable decrement inhibition was detected when antibodies of NbSWP16 and NbSWP5 were used simultaneously. Collectively, these results suggest that NbSWP16 is a new exospore protein and probably be involved in spore adherence of N. bombycis.

NbHSWP11, a microsporidia Nosema bombycis protein, localizing in the spore wall and membranes, reduces spore adherence to host cell BME.

Microsporidia are obligate intracellular parasites, and a derivative of fungi, which harbor a rigid spore wall to resist adverse environmental pressures. The spore wall protein, which is thought to be the first and direct protein interacting with the host cell, may play a key role in the process of microsporidia infection. In this study, we report a protein, NbHSWP11, with a dnaJ domain. The protein also has 6 heparin-binding motifs which are known to interact with extracellular glycosaminoglycans. Syntenic analysis indicated that gene loci of Nbhswp11 are conserved and syntenic between Nosema bombycis and Nosema ceranae. Phylogenetic tree analysis showed that Nbhswp11 clusters with fungal dnaJ proteins and has 98% identity with an N. bombycis dnaJ protein. Nbhswp11 was transcribed throughout the entire life stages, and gradually increased during 1-7 days, in a silkworm that was infected by N. bombycis, as determined by reverse-transcription PCR (RT-PCR). The recombinant protein NbHSWP11 (rSWP11-HIS) was obtained and purified using gene cloning and prokaryotic expression. Western blotting analysis displayed NbHSWP11 expressed in the total mature spore proteins and spore coat proteins. Indirect immunofluorescence assay revealed NbHSWP11 located at the spore wall of mature spores and the spore coats. Furthermore, immune electron microscopy showed that NbHSWP11 localized in the cytoplasm of the sporont. Within the developmental process of N. bombycis, a portion of NbHSWP11 is targeted to the spore wall of sporoblasts and mature spores. However, most of NbHSWP11 distributes on the membraneous structures of the sporoblast and mature spore. In addition, using a host cell binding assay, native protein NbHSWP11 in the supernatant of total soluble mature spore proteins is shown to bind to the host cell BmE surface. Finally, an antibody blocking assay showed that purified rabbit antibody of NbHSWP11 inhibits spore adherence and decreases the adherence rate of spores by 20% compared to untreated spores. Collectively, the present results suggest that NbHSWP11 is involved in host cell adherence in vitro. Therefore NbHSWP11, which has a dnaJ domain, may modulate protein assembly, disassembly, and translocation in N. bombycis.

Morphological and molecular studies of Vairimorpha necatrix BM, a new strain of the microsporidium V. necatrix (Microsporidia, Burenellidae) recorded in the silkworm, Bombyx mori.

Vairimorpha sp. BM (2012) is a recent isolate of the microsporidia from the silkworm in Shandong, China. The ultrastructure, tissue pathology and molecular characterization of this isolate is described in this study. This pathogenic fungus causes pebrine disease in silkworms which manifests as a systemic infection. Meanwhile, the silkworm eggs produced by the infected moths were examined using a microscope and PCR amplification. Neither spores nor the expected PCR band were observed, suggesting that no vertical transmission occurred in Bombyx mori. In addition, the ultrastructure of the isolate was studied by light microscopy and transmission electron microscopy. Two types of spores were observed: diplokaroytic spores with 13-17 coils of polar tubes and monokaryotic spores with less coils of polar tubes which could form octospores; however, no sporophorous vesicles were observed. Finally, phylogenetic analysis of the small subunit rRNA genes of Vairimorpha species showed that this isolate has a closer relationship to Vairimorpha necatrix than the other species studied. This result also is supported by phylogenetic analysis based on their actin genes, heat shock protein 70 (HSP70) and RNA polymerase II (RPB1). Based on the information gained during this study, we propose that this microsporidian species infecting B. mori should be given the name V. necatrix BM.

Development of an approach to analyze the interaction between Nosema bombycis (microsporidia) deproteinated chitin spore coats and spore wall proteins.

Nosema bombycis is an obligate intracellular parasite of the Bombyx mori insect. The spore wall of N. bombycis is composed of an electron-dense proteinaceous outer layer and an electron-transparent chitinous inner layer, and the spore wall is connected to the plasma membrane. In this study, the deproteinated chitin spore coats (DCSCs) were acquired by boiling N. bombycis in 1M NaOH. Under a transmission electron microscope, the chitin spore coat resembles a loosely curled ring with strong refractivity; organelles and nuclei were not observed inside the spore. The anti-SWP25, 26, 30 and 32 antibodies were used to detect whether spore wall proteins within the total soluble and mature spore proteins could bind to the DCSCs. Furthermore, a chitin binding assay showed that within the total soluble and mature spore proteins, the SWP26, SWP30 and SWP32 spore wall proteins, bound to the deproteinated chitin spore coats, although SWP25 was incapable of this interaction. Moreover, after the DCSCs were incubated with the alkali-soluble proteins, the latter were obtained by treating N. bombycis with 0.1M NaOH. Following this treatment, SWP32 was still capable of binding the DCSCs, while SWP26 and SWP30 were unable to bind. Collectively, the DCSCs are useful for investigating the arrangement of spore wall proteins, and they shed light on how the microsporidia spore wall is self-assembled.

Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation.

BACKGROUND: Microsporidian Nosema bombycis has received much attention because the pébrine disease of domesticated silkworms results in great economic losses in the silkworm industry. So far, no effective treatment could be found for pébrine. Compared to other known Nosema parasites, N. bombycis can unusually parasitize a broad range of hosts. To gain some insights into the underlying genetic mechanism of pathological ability and host range expansion in this parasite, a comparative genomic approach is conducted. The genome of two Nosema parasites, N. bombycis and N. antheraeae (an obligatory parasite to undomesticated silkworms Antheraea pernyi), were sequenced and compared with their distantly related species, N. ceranae (an obligatory parasite to honey bees). RESULTS: Our comparative genomics analysis show that the N. bombycis genome has greatly expanded due to the following three molecular mechanisms: 1) the proliferation of host-derived transposable elements, 2) the acquisition of many horizontally transferred genes from bacteria, and 3) the production of abundnant gene duplications. To our knowledge, duplicated genes derived not only from small-scale events (e.g., tandem duplications) but also from large-scale events (e.g., segmental duplications) have never been seen so abundant in any reported microsporidia genomes. Our relative dating analysis further indicated that these duplication events have arisen recently over very short evolutionary time. Furthermore, several duplicated genes involving in the cytotoxic metabolic pathway were found to undergo positive selection, suggestive of the role of duplicated genes on the adaptive evolution of pathogenic ability. CONCLUSIONS: Genome expansion is rarely considered as the evolutionary outcome acting on those highly reduced and compact parasitic microsporidian genomes. This study, for the first time, demonstrates that the parasitic genomes can expand, instead of shrink, through several common molecular mechanisms such as gene duplication, horizontal gene transfer, and transposable element expansion. We also showed that the duplicated genes can serve as raw materials for evolutionary innovations possibly contributing to the increase of pathologenic ability. Based on our research, we propose that duplicated genes of N. bombycis should be treated as primary targets for treatment designs against pébrine. The genome data and annotation information of N. bombycis and N.antheraeae were submitted to GenBank (Accession numbers ACJZ01000001 -ACJZ01003558).