Molecular basis of host plant recognition by silkworm larvae.

Herbivorous insects can identify their host plants by sensing plant secondary metabolites as chemical cues. We previously reported the two-factor host acceptance system of the silkworm Bombyx mori larvae. The chemosensory neurons in the maxillary palp (MP) of the larvae detect mulberry secondary metabolites, chlorogenic acid (CGA), and isoquercetin (ISQ), with ultrahigh sensitivity, for host plant recognition and feeding initiation. Nevertheless, the molecular basis for the ultrasensitive sensing of these compounds remains unknown. In this study, we demonstrated that two gustatory receptors (Grs), BmGr6 and BmGr9, are responsible for sensing the mulberry compounds with attomolar sensitivity for host plant recognition by silkworm larvae. Calcium imaging assay using cultured cells expressing the silkworm putative sugar receptors (BmGr4-10) revealed that BmGr6 and BmGr9 serve as receptors for CGA and ISQ with attomolar sensitivity in human embryonic kidney 293T cells. CRISPR/Cas9-mediated knockout (KO) of BmGr6 and BmGr9 resulted in a low probability of making a test bite of the mulberry leaves, suggesting that they lost the ability to recognize host leaves. Electrophysiological recordings showed that the loss of host recognition ability in the Gr-KO strains was due to a drastic decrease in MP sensitivity toward ISQ in BmGr6-KO larvae and toward CGA and ISQ in BmGr9-KO larvae. Our findings have revealed that the two Grs, previously considered to be sugar receptors, are molecules responsible for detecting plant phenolics in host plant recognition.

Exercise training affects hemodynamics and exercise capacity in cases of heart failure with preserved ejection fraction: a non-randomized controlled trial in individuals aged 65-80 years.

Introduction: Exercise training is an established intervention method for improving exercise capacity and survival rates in patients with heart failure with preserved ejection fraction (HFpEF). However, most reports have focused on European and American patients, with limited data regarding the effects of exercise training on cardiac function, hemodynamics, and exercise capacity in East Asian patients. This study investigated the effects of exercise training on cardiac function, hemodynamics, and exercise capacity in Japanese patients aged 65-80 years with HFpEF. Methods: This single-center, open-label, non-randomized, controlled trial prospectively enrolled 99 outpatients. Eligibility criteria for HFpEF patients were an HFA score ≥5 in addition to clinical symptoms of heart failure and left ventricular diastolic dysfunction. Exercise training in the intervention group consisted of aerobic exercise and strength training thrice weekly for 5 months. Patients in the control group continued the usual treatment for 5 months. Resting cardiac function was evaluated using echocardiography. Peak oxygen uptake (peakVO2), ventilatory equivalent (VE) vs. carbon dioxide output (VCO2) slope, peak cardiac output index, and arteriovenous oxygen difference were calculated using cardiopulmonary exercise testing combined with impedance cardiography. Results: After 5 months of exercise training, remarkable interactions were observed, with peakVO2 as the primary outcome. Additionally, significant interactions were observed between hemodynamic indices and some echocardiographic parameters. The mean percentage change in peakVO2 from baseline was 8.3% in the intervention group. Fifteen study participants (30.1%) in the intervention group achieved a clinically meaningful change of 3.0 ml/min/kg (10% improvement) in peakVO2 from baseline. The group with 3.0 ml/min/kg or 10% improvement in peakVO2 from baseline had a considerably lower prevalence of diabetes mellitus and VE vs. VCO2 slope and considerably higher left atrial-global longitudinal strain values than the group without any notable improvements. Conclusions: Although exercise training can help improve exercise intolerance in Japanese patients aged 65-80 years with HFpEF, its benefits are limited. Our results suggest that HFpEF, complicated by diabetes mellitus and decreased ventilatory efficiency during exercise, may require reconsideration of intervention strategies. This trial was registered with the University Hospital Medical Information Network, a trial registry in Japan (registration number: UMIN000045474).

Diversity of Antimicrobial Peptides in Silkworm.

Antimicrobial resistance is a phenomenon that the present-day world is witnessing that poses a serious threat to global health. The decline in the development of novel therapeutics over the last couple of decades has exacerbated the situation further. In this scenario, the pursuit of new alternative therapeutics to commonly used antibiotics has gained predominance amongst researchers across the world. Antimicrobial peptides (AMPs) from natural sources have drawn significant interest in the recent years as promising pharmacological substitutes over the conventional antibiotics. The most notable advantage of AMPs is that microorganisms cannot develop resistance to them. Insects represent one of the potential sources of AMPs, which are synthesized as part of an innate immune defence against invading pathogens. AMPs from different insects have been extensively studied, and silkworm is one of them. Diverse classes of AMPs (including attacins, cecropins, defensins, enbocins, gloverins, lebocins and moricins) were identified from silkworm that exhibit antimicrobial property against bacteria, fungi and viruses, indicating their potential therapeutic benefits. This review briefs about the immune responses of silkworm to invading pathogens, the isolation of AMPs from silkworms, AMPs reported in silkworms and their activity against various microorganisms.

Association of complication of type 2 diabetes mellitus with hemodynamics and exercise capacity in patients with heart failure with preserved ejection fraction: a case-control study in individuals aged 65-80 years.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a frequently observed complication in patients with heart failure with preserved ejection fraction (HFpEF). Although a characteristic finding in such patients is a decrease in objective exercise capacity represented by peak oxygen uptake (peakVO2), exercise capacity and its predictors in HFpEF with T2DM remain not clearly understood. This case-control study aimed to investigate the association between exercise capacity and hemodynamics indicators and T2DM comorbidity in patients with HFpEF aged 65-80 years. METHODS: Ninety-nine stable outpatients with HFpEF and 50 age-and-sex-matched controls were enrolled. Patients with HFpEF were classified as HFpEF with T2DM (n = 51, median age, 76 years) or without T2DM (n = 48, median age, 76 years). The peakVO2 and ventilatory equivalent versus carbon dioxide output slope (VE vs VCO2 slope) were measured by cardiopulmonary exercise testing. The peak heart rate (HR) and peak stroke volume index (SI) were measured using impedance cardiography, and the estimated arteriovenous oxygen difference (peak a-vO2 diff) was calculated with Fick's equation. The obtained data were compared among the three groups using analysis of covariance adjusted for the ß-blocker medication, presence or absence of sarcopenia, and hemoglobin levels in order to determine the T2DM effects on exercise capacity and hemodynamics in patients with HFpEF. RESULTS: In HFpEF with T2DM compared with HFpEF without T2DM and the controls, the prevalence of sarcopenia, chronotropic incompetence, and anemia were significantly higher (p < 0.001). The peakVO2 (Controls 23.5 vs. without T2DM 15.1 vs. with T2DM 11.6 mL/min/kg), peak HR (Controls 164 vs. without T2DM 132 vs. with T2DM 120 bpm/min), peak a-vO2 (Controls 13.1 vs without T2DM 10.6 vs with T2DM 8.9 mL/100 mL), and VE vs VCO2 slope (Controls 33.2 vs without T2DM 35.0 vs with T2DM 38.2) were significantly worsened in patients with HFpEF with T2DM (median, p < 0.001). There was no significant difference in peak SI among the three groups. CONCLUSIONS: Our results suggested that comorbid T2DM in patients with HFpEF may reduce exercise capacity, HR response, peripheral oxygen extraction, and ventilation efficiency. These results may help identify cardiovascular phenotypes of HFpEF complicated with T2DM and intervention targets for improving exercise intolerance.

Dietary compounds activate an insect gustatory receptor on enteroendocrine cells to elicit myosuppressin secretion.

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae.

[Perioperative Outcome of Laparoscopic Surgery after Colonic Stent Placement for Obstructive Colorectal Cancer].

In recent years, bridge to surgery(BTS), in which surgery is performed after colorectal stenting for obstructive colorectal cancer, has gradually become popular, and laparoscopic surgery is also a treatment option. From January 2020 to December 2022, we retrospectively evaluated clinicopathological factors in 18 colorectal cancer cases who underwent radical resection after colorectal stenting. We found no difference in patient background, histopathological factors, primary anastomosis rate, stoma creation rate, operative time, postoperative complication rate and length of hospital stay between the laparoscopic surgery(L)and open surgery(O)groups. Blood loss was significantly lower in group L. In T4 patients, laparoscopic surgery after colorectal stenting can be safely performed, but conversion to open surgery may be necessary. Surgery after colorectal stenting should be performed based on preoperative accurate imaging and sufficient experience.

Nodule formation in Bombyx mori larvae is regulated by BmToll10-3.

Nodule formation is a two-step cell-mediated immune response that is elicited by the cytokine spätzle1. Spätzle1 is activated within 30 s of invasion by microorganisms via an extracellular signaling pathway that consists of pathogen-associated molecular pattern recognition receptors, C-type lectins, and serine proteases. Here, we investigated a hemocyte molecule that is involved in eliciting the first step of nodule formation. BmToll10-3 was one of 14 Toll homologs identified in the silkworm Bombyx mori; it is an ortholog of Spodoptera exigua Toll. Previous research suggested that SeToll elicits nodule formation, but no evidence was presented to indicate whether SeToll elicited the first or second step of nodule formation. Reverse transcription-polymerase chain reaction and immunostaining confirmed that BmToll10-3 is expressed in granulocytes. To determine whether BmToll10-3 is involved in eliciting the first step of nodule formation, we tested an antiserum raised against BmToll10-3 in a nodule formation assay. The antiserum strongly inhibited the first step of nodule formation in B. mori larvae. Next, we tried to knock out BmToll10-3 using genome editing. Strains that were heterozygous for a truncated BmToll10-3 allele were generated, but no strain that was homozygous for truncated BmToll10-3 was generated. Nonetheless, several healthy homozygous larvae were identified before pupation, and we used these larvae in a nodule formation assay. The larvae that were homozygous for truncated BmToll10-3 did not form nodules. These results suggest that BmToll10-3 is involved in a cellular immunity, nodule formation.

Resistance mechanism of Nid-1, a dominant non-susceptibility gene, against Bombyx mori densovirus 1 infection.

Bombyx mori densovirus 1 (BmDV1) is a pathogen that causes flacherie disease in mulberry silkworms (B. mori). The absolute resistance (non-susceptibility) to BmDV1 of certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. Previously, we investigated the expression of viral transcript in virus-inoculated silkworms carrying different nsd-1 and Nid-1 genotypes, and observed that nsd-1 and Nid-1 expression blocked the early and late steps of BmDV1 infection, respectively. In addition, we found that nsd-1 encoded a Bombyx-specific mucin-like membrane protein only present on the surface of the midgut, where BmDV1 could infect. In this study, we dissected the resistance mechanism by Nid-1 against BmDV1 infection by investigating the sequential changes in the accumulation of viral DNA, transcripts, and proteins derived from BmDV1 in susceptible strain (pxj) and Nid-1-carrying resistant strain (No. 908) after inoculation with BmDV1. Genomic PCR results showed that the BmDV1 DNA was detected immediately after the infection in both strains but rapidly decreased in the Nid-1-carrying strain No. 908 compared with the susceptible strain pxj. RT-PCR results also showed that the BmDV1 transcripts of Nid-1-carrying strain No. 908 were rapidly decreased after the infection. Moreover, BmDV1-derived proteins were not detected in No. 908 throughout the infection. These results suggest that Nid-1 expression might inhibit the accumulation of viral DNA and transcripts. As Nid-1 has not been molecularly characterized, its identification will contribute to the elucidation of the interactions between the silkworm and BmDV1.

An investigation into the effects of infection and ORF expression patterns of the Indian bidensovirus isolate (BmBDV) infecting the silkworm Bombyx mori.

The Indian isolate of Bombyx mori bidensovirus (BmBDV) is a bipartite virus that comprises of a segmented, non-homologous, two linear single-strands of DNA molecules (VD1 and VD2). It is one of the causative agents of the fatal silkworm disease 'Flacherie' that causes severe crop loss for the sericulture farmers. Genome analyses of the Indian isolate of BmBDV revealed that it consists of 6 putative ORFs similar to the Japanese and Chinese isolates. VD1 consists of 4 ORFs while VD2 has 2 ORFs that code for 4 non- structural (NS) and 2 structural (VP) proteins, in total. In this study, we investigated, in detail, the impact of BmBDV pathogenesis on growth and development of the silkworm Bombyx mori, at different developmental stages. Mortality rate and weight uptake analyses were also performed on newly ecdysed 4th instar larvae. BmBDV infection was not found to be developmental stage specific and it occurred at all stages. Onset of mortality took place 8 days post infection (dpi) and 100% mortality occurred at 11 dpi. The infected larvae showed a significant difference in weight uptake wherein from 7 dpi the larvae stopped gaining weight and from 8th dpi started demonstrating the typical symptoms of flacherie. Further, the expression pattern of the 6 viral ORFs were also investigated in the newly ecdysed 4th instar BmBDV infected silkworms. Among all the six ORFs, VD2 ORF 1 and 2 revealed the highest transcript numbers, which was followed by VD1 ORF 4 that encodes for the viral DNA polymerase enzyme. This was the first ever attempt to understand the pathogenesis and the expression pattern of all the six ORF transcripts of the Indian isolate of BmBDV.

Genetic characterisation of an Iflavirus associated with a vomiting disease in the Indian tropical tasar silkworm, Antheraea mylitta.

Antheraea mylitta, the Tropical tasar silkworm, is frequently affected by a vomiting disease called Virosis by sericulturists although not confirmed being of viral origin. Based on the symptoms and the disease pattern, the causal agent is however suspected to be a virus. The condition involves a series of characteristic and progressive symptoms that generally culminates in the death of the larva. The disease is common in autumn season (Sep- Oct), with widespread distribution causing severe damage to the tasar silk industry. The leads for this study were obtained from a transcript identified in the EST database in a different study, which matched the positive strand of Iflavirus, an RNA virus known to infect insects. In the present study the genome of this novel Iflavirus was characterised and the full length of the genome was found to be 9728 nucleotides long encoding for a single large open reading frame (ORF) with flanking NTR regions at 5' and 3' ends and a natural poly A tail at the 3' end. The ORF encoded structural proteins at the N-terminal end and non-structural proteins at the C-terminal end with a predicted 2967 amino acid long polyprotein. The structural proteins consisted of 4 proteins (VP1-VP4) and the non-structural proteins consisted of helicase, RNA-dependent RNA polymerase and 3C-protease. The virus is found in various tissues (midgut, fatbody, trachea, Malpighian tubules and silk gland) and also has a vertical route of transmission, i.e., from gravid females to the offspring. Based on the available data, the virus is a new member of Iflaviridae for which we propose the name Antheraea mylitta Iflavirus (AmIV).

[A Case of Sigmoid Colon Cancer with Retroperitoneal Fibrosis Using Intraoperative Indocyanine Green Fluorescence Angiography for Colorectal Anastomosis].

A 72-year-old man was presented with anemia and diagnosed with sigmoid colon cancer by colonoscopy. CT showed a soft tissue density around the retroperitoneum, leading to the diagnosis of retroperitoneal fibrosis. Stenosis of left ureter, inferior mesenteric artery, and left colic artery due to the soft tissues were detected. Sigmoidectomy and retroperitoneal biopsy were performed, and colorectal anastomosis was completed after confirming the intestinal blood flow by ICG fluorescence angiography. In retroperitoneal fibrosis, identifying blood vessels intraoperatively can be difficult. ICG fluorescence angiography is useful for reliable anastomosis in colorectal cancer surgery with retroperitoneal fibrosis.

Epicardial adipose tissue is associated with cardiorespiratory fitness and hemodynamics among Japanese individuals of various ages and of both sexes.

Epicardial adipose tissue may affect hemodynamics and cardiorespiratory fitness as it is a metabolically active visceral adipose tissue and a source of inflammatory bioactive substances that can substantially modulate cardiovascular morphology and function. However, the associations between epicardial adipose tissue and hemodynamics and cardiorespiratory fitness remain unclear. This cross-sectional study aimed to examine the association between epicardial adipose tissue volume and hemodynamics, and cardiorespiratory fitness among Japanese individuals of various ages and of both sexes. Epicardial adipose tissue volume was measured in 120 participants (age, 21-85 years) by cardiac magnetic resonance imaging. To evaluate cardiorespiratory fitness, peak oxygen uptake was measured by cardiopulmonary exercise testing. Peak cardiac output and arteriovenous oxygen difference were calculated by impedance cardiography. The epicardial adipose tissue volume was significantly increased in middle-aged and older women. The epicardial adipose tissue volume was significantly and negatively correlated to peak cardiac output and peak oxygen uptake, regardless of age and sex; furthermore, epicardial adipose tissue showed a strong negative correlation with peak heart rate. Epicardial adipose tissue and peak cardiac output were significantly associated (ß = -0.359, 95% confidence interval, -0.119 to -0.049, p < 0.001), even after multivariate adjustment (R2 = 0.778). However, in the multiple regression analysis with peak oxygen uptake as a dependent variable, the epicardial adipose tissue volume was not an independent predictor. These data suggest that increased epicardial adipose tissue volume may be correlated with decreased peak oxygen uptake, which might have mediated the abnormal hemodynamics among Japanese people of various ages and of both sexes. Interventions targeting epicardial adipose tissue could potentially improve hemodynamics and cardiorespiratory fitness.

Host Response against Virus Infection in an Insect: Bidensovirus Infection Effect on Silkworm (Bombyx mori).

Silk cocoons obtained from silkworms are the primary source of commercial silk, making the silkworm an economically important insect. However, the silk industry suffers significant losses due to various virus infections. Bombyx mori bidensovirus (BmBDV) is one of the pathogens that cause flacherie disease in silkworms. Most silkworm strains die after BmBDV infection. However, certain silkworm strains show resistance to the virus, which is determined by a single recessive gene, nsd-2. The +nsd-2 gene (allele of nsd-2; the susceptibility gene) encodes a putative amino acid transporter expressed only in the insect's midgut, where BmBDV can infect, suggesting that this membrane protein may function as a receptor for BmBDV. Interestingly, the expression analysis revealed no changes in the +nsd-2 gene expression levels in virus-uninfected silkworms, whereas the gene expression drastically decreased in the virus-infected silkworm. This condition indicates that the host factor's expression, the putative virus receptor, is affected by BmBDV infection. It has recently been reported that the expression levels of some host genes encoding cuticle, antioxidant, and immune response-related proteins were significantly regulated by BmBDV infection. In this review, we discuss the host response against virus infection based on our knowledge and long-term research experience in this field.

Epicardial adipose tissue is tightly associated with exercise intolerance in patients with type 2 diabetes mellitus with asymptomatic left ventricular structural and functional abnormalities.

AIMS: This study aimed to elucidate whether the volume of epicardial adipose tissue (EAT) is associated with left ventricular (LV) structural and functional abnormalities and exercise capacity in patients with type 2 diabetes mellitus (T2DM). METHODS: EAT thickness and LV structural and functional abnormality components (e.g., global longitudinal strain, E/e', LV mass index, relative wall thickness) were measured using echocardiography in 176 patients with asymptomatic stage A and B heart failure (SAHF and SBHF, respectively) and 62 healthy controls (HC). Peak oxygen uptake (peakVO2) was measured by using cardiopulmonary exercise testing. RESULTS: Even when matching study participants for age, sex, and body mass index, the EAT was thicker (HCs 5.5 ±â€¯1.2 versus SAHF 6.4 ±â€¯1.0 and SBHF 9.3 ±â€¯1.7 mm) and peakVO2 was lower (HC 24.1 ±â€¯3.3 versus SAHF 19.1 ±â€¯2.0 and SBHF 16.9 ±â€¯3.1 ml/kg/min) in the heart failure (HF) group than in the HC group (p < 0.001). EAT thickness (ß = -0.189, p < 0.001) and peakVO2 were significantly associated, even after adjusting for multivariates (R2 = 0.457). CONCLUSIONS: In T2DM patients with asymptomatic HF, EAT may be associated with LV structural and functional abnormalities and exercise intolerance.

Pheromonal activities of the bombykol isomer, (10E,12E)-10,12-hexadecadien-1-ol, in the pheromone gland of the silkmoth Bombyx mori.

Bombykol (EZ) is the single component of the female sex pheromone in the silkmoth Bombyx mori. EZ alone evokes full courtship behaviors from conspecific males; however, its geometric isomer (EE) was consistently detected in the pheromone glands (PG) of 16 B. mori strains and a field population of the wild silkmoth Bombyx mandarina, which also uses EZ as the single pheromone component. We investigated the pheromonal activities of EE using a commercial hybrid strain of B. mori, Kinshu × Showa. The behavioral assay demonstrated that a 104-105-fold higher dose of EE than EZ was able to elicit behavioral responses from males. To elucidate whether the trace contaminant of EZ in the EE standard is responsible for these responses, we examined the responses of male antennae to EE using a gas chromatograph-electroantennographic detector system (GC-EAD). The EE, at high doses elicited marginal responses from the male antennae. We next examined antennal and behavioral responses of B. mori whose BmOR1 gene, which is responsible for the reception of bombykol, was knocked out. The knockout of BmOR1 resulted in the complete loss of antennal and behavioral responses to EE and EZ, demonstrating that if EE itself is active, it induces these responses via the incidental stimulation of BmOR1, not via the stimulation of EE-specific receptors. The existence of EE in the PG of B. mori and B. mandarina is discussed from the viewpoints of pheromone biosynthesis and the evolution of pheromone communication systems.

Decrease in the expression level of the gene encoding the putative Bombyx mori bidensovirus receptor during virus infection.

Bombyx mori bidensovirus (BmBDV) is a pathogen that replicates only in the midgut columnar cells of silkworms, causing fatal disease. Resistance to BmBDV, which does not depend on the viral dose, is determined by a single gene, nsd-2 (resistance gene). Previously, we identified nsd-2 by positional cloning using B. mori genome information and found that this gene encodes a putative amino acid transporter that may function as a receptor for BmBDV. In this study, to understand the relationship between BmBDV and the putative virus receptor, we performed expression analysis of +nsd-2 (allele of nsd-2; susceptibility gene) after virus infection. Quantitative RT-PCR analysis using total RNA isolated from the midgut of an uninfected and a virus-infected silkworm revealed no change in the expression levels of +nsd-2 in the uninfected silkworm, whereas the expression levels of +nsd-2 drastically decreased in the virus-infected silkworm. Moreover, comparison of the expression pattern between the BmBDV-derived transcript and +nsd-2 revealed that the expression level of +nsd-2 decreased with an increase in the virus-derived transcript. In addition, expression analysis of 26 genes encoding other transporters in the midgut demonstrated that the expression levels of three other genes also decreased similarly to the decrease of the expression levels of +nsd-2 after virus infection. Thus, our results suggest that some transporters, including +nsd-2, are affected by BmBDV infection.

A single amino acid substitution in the Bombyx-specific mucin-like membrane protein causes resistance to Bombyx mori densovirus.

Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date. Here, we isolated the nsd-1 gene by positional cloning and characterized the properties of its product, NSD-1. Sequence and biochemical analyses revealed that this gene encodes a Bombyx-specific mucin-like glycoprotein with a single transmembrane domain. The NSD-1 protein was specifically expressed in the larval midgut epithelium, the known infection site of BmDV. Sequence analysis of the nsd-1 gene from 13 resistant and 12 susceptible strains suggested that a specific arginine residue in the extracellular tail of the NSD-1 protein was common among susceptible strains. Germline transformation of the susceptible-type nsd-1 (with a single nucleotide substitution) conferred partial susceptibility to resistant larvae, indicating that the + nsd-1 gene is required for the susceptibility of B. mori larvae to BmDV and the susceptibility is solely a result of the substitution of a single amino acid with arginine. Taken together, our results provide striking evidence that a novel membrane-bound mucin-like protein functions as a cell-surface receptor for a densovirus.

Characterization and genome comparison of an Indian isolate of bidensovirus infecting the silkworm Bombyx mori.

The bipartite genome of an Indian isolate of Bombyx mori bidensovirus (BmBDV), one of the causative agents of the fatal silkworm disease 'Flacherie', was cloned and completely sequenced. Nucleotide sequence analysis of this Indian isolate of BmBDV revealed two viral DNA segments, VD1 and VD2 as well as a DNA polymerase motif which supports its taxonomical status as the type species of a new family of Bidnaviridae. The Indian isolate of BmBDV was found to have a total of six putative ORFs four of which were located on the VD1 with the other two being on the VD2 DNA segment. The VD1 DNA segment was found to code for three non-structural proteins including a viral DNA polymerase as well as one structural protein, while the VD2 DNA segment was found to code for one structural and one non-structural protein, similar to that of the Japanese and Zhenjiang isolates of BmBDV. A BmBDV ORF expression study was done through real time qPCR wherein the VD2 ORF 1 and 2 showed the maximum transcript levels. This is the first report of the genome characterization of an Indian isolate of BmBDV, infecting silkworm B. mori.

Splice Variants of pH-Sensitive Chloride Channel Identify a Key Determinant of Ivermectin Sensitivity in the Larvae of the Silkworm Bombyx mori.

The pH-sensitive chloride channels (pHCls) are broadly expressed in insects, but little is known about their physiologic role, diversity, and sensitivity to insecticides acting on relevant chloride channels. Here we have sequenced 50 transcripts of the pHCl-1 gene from the brain, third thoracic ganglion (T3G), and midgut of larvae of silkworm Bombyx mori It was found that >50 variants were expressed with distinct splicing in the T3G compared with the brain and midgut. Of the variants detected, variant 9, which was expressed most abundantly in the larvae, was reconstituted in Xenopus laevis oocytes to characterize its pH and ivermectin sensitivity. Variant 9 formed a functional pHCl with half-maximal activation at a pH of 7.87, and was activated by ivermectin irrespective of the extracellular pH. This was in contrast to variant 1, which was activated more profoundly at acidic rather than basic pH. To identify a key determinant for such differential ivermectin sensitivity, different amino acids in variants 1 and 9 were swapped, and the effects of the mutations on ivermectin sensitivity were investigated. The V275S mutation of variant 1 enhanced ivermectin sensitivity, whereas the S275V mutation of variant 9 caused a reduction in sensitivity. In homology models of the Bombyx pHCls, Val275 of variant 1 interacted more strongly with Ala273 than Ser275 of variant 9 at the channel gate. This interaction is likely to prevent ivermectin-induced opening of the channel, accounting, at least partially, for the differential macrolide action on the two variants.

Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.

BACKGROUND: Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. RESULTS: Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. CONCLUSIONS: We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.