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.

[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.

[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.

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.

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.

Gene expression and localization analysis of Bombyx mori bidensovirus and its putative receptor in B. mori midgut.

Bombyx mori bidensovirus (BmBDV), which causes fatal flacherie disease in the silkworm, replicates only in midgut columnar cells. The viral resistance expressed by some silkworm strains, which is characterized as non-susceptibility irrespective of the viral dose, is determined by a single gene, nsd-2. We previously identified nsd-2 by positional cloning and found that this gene encodes a putative amino acid transporter that might function as a receptor for BmBDV. In this study, we investigated the relationship between the part of the midgut expressing nsd-2 (resistance gene), +(nsd-2) (susceptibility gene) and BmBDV propagation. Quantitative RT-PCR (qRT-PCR) analysis using total RNA isolated from the anterior, middle, and posterior parts of the midgut showed that nsd-2 and +(nsd-2) were strongly expressed in the posterior part of the midgut. The expression levels of both genes were very low in the anterior and middle parts. The qRT-PCR analysis showed that the expression levels of BmBDV-derived transcripts were correlated with the levels of +(nsd-2) expression. However, BmBDV-derived transcripts were clearly detected in all parts of the midgut. These results suggest that the infectivity of BmBDV depends mainly on the expression level of +(nsd-2) in the midgut and that viral infection is supported even by very faint expression of +(nsd-2). By contrast, the expression levels of +(nsd-2) were exceedingly low or undetectable in the middle part of the midgut, indicating that BmBDV infection might occur via another mechanism, independent of +(nsd-2), in the middle part of the midgut.

Sex pheromone desaturase functioning in a primitive Ostrinia moth is cryptically conserved in congeners' genomes.

(E)-11- and (Z)-11-tetradecenyl acetate are the most common female sex pheromone components in Ostrinia moths. The Δ11-desaturase expressed in the pheromone gland (PG) of female moths is a key enzyme that introduces a double bond into pheromone molecules. A single Δ11-desaturase of Ostrinia nubilalis, OnubZ/E11, has been shown to produce an ∼7:3 mixture of (E)-11- and (Z)-11-tetradecenoate from the substrate tetradecanoate. In contrast, the sex pheromone of Ostrinia latipennis, a primitive species of Ostrinia, is (E)-11-tetradecenol. This pheromone is unique in that it is not acetylated, and includes no Z isomer. In the present study, through the cloning and functional analysis of a PG-specific Δ11-desaturase in O. latipennis, we showed that the absence of the Z isomer in the pheromone is attributable to the strict product specificity of the Δ11-desaturase in this species, LATPG1. Phylogenetic analysis revealed that LATPG1 was not closely related to OnubZ/E11. Rather, it was closely related to retroposon-linked cryptic Δ11-desaturases (ezi-Δ11) found in the genomes of O. nubilalis and Ostrinia furnacalis. Taken together, the results showed that an unusual Δ11-desaturase is functionally expressed in O. latipennis, although the genes encoding this enzyme appear to be cryptic in congeners.

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.

Discovery of a disused desaturase gene from the pheromone gland of the moth Ascotis selenaria, which secretes an epoxyalkenyl sex pheromone.

Female Ascotis selenaria (Geometridae) moths use 3,4-epoxy-(Z,Z)-6,9-nonadecadiene, which is synthesized from linolenic acid, as the main component of their sex pheromone. While the use of dietary linolenic or linoleic fatty acid derivatives as sex pheromone components has been observed in moth species belonging to a few families including Geometridae, the majority of moths use derivatives of a common saturated fatty acid, palmitic acid, as their sex pheromone components. We attempted to gain insight into the differentiation of pheromone biosynthetic pathways in geometrids by analyzing the desaturase genes expressed in the pheromone gland of A. selenaria. We demonstrated that a Δ11-desaturase-like gene (Asdesat1) was specifically expressed in the pheromone gland of A. selenaria in spite of the absence of a desaturation step in the pheromone biosynthetic pathway in this species. Further analysis revealed that the presumed transmembrane domains were degenerated in Asdesat1. Phylogenetic analysis demonstrated that Asdesat1 anciently diverged from the lineage of Δ11-desaturases, which are currently widely used in the biosynthesis of sex pheromones by moths. These results suggest that an ancestral Δ11-desaturase became dysfunctional in A. selenaria after a shift in pheromone biosynthetic pathways.

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the 'Black Box' of the ecdysteroid biosynthesis pathway.

In insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5beta-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5beta-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

Detailed investigation of the sequential pathological changes in silkworm larvae infected with Bombyx densovirus type 1.

Bombyx mori densovirus type 1 (BmDNV-1) is a pathogen causing flacherie disease in silkworms. BmDNV-1 multiplies only in the nuclei of the columnar cells of larval midgut epithelium. Although several immunohistochemical studies using anti-BmDNV-1 antibody have been reported to date, sequential pathological changes in BmDNV-1-infected larvae have not been completely elucidated. In this paper, sequential investigations were performed on the pathological features of BmDNV-1-infected larvae and BmDNV-1 propagation. Oral infection experiments using newly ecdysed 4th instar larvae revealed that the larvae began to die 9 days post infection (dpi), and the remaining died 10 dpi. Histological observations revealed phenotypic alterations in the midgut cells from 4 dpi, and complete disruption of the midgut structure at 9 dpi. Quantitative RT-PCR of two BmDNV-1 genes indicated that BmDNV-1 began to propagate from 4 dpi, and gradually increased until the larvae died. These expression patterns revealed marked correlation with the histological changes observed in the virus-infected midgut cells. Moreover, bioassays using larvae at various developmental stages clearly indicated that the pathogenicity of this virus is not dependent on the larval stage or the molting process.

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.

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).

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.

Positional cloning of a gene responsible for the cts mutation of the silkworm, Bombyx mori.

The larval head cuticle and anal plates of the silkworm mutant cheek and tail spot (cts) have chocolate-colored spots, unlike the entirely white appearance of the wild-type (WT) strain. We report the identification and characterization of the gene responsible for the cts mutation. Positional cloning revealed a cts candidate on chromosome 16, designated BmMFS, based on the high similarity of the deduced amino acid sequence between the candidate gene from the WT strain and the major facilitator superfamily (MFS) protein. BmMFS likely encodes a membrane protein with 11 putative transmembrane domains, while the putative structure deduced from the cts-type allele possesses only 10-pass transmembrane domains owing to a deletion in its coding region. Quantitative RT-PCR analysis showed that BmMFS mRNA was strongly expressed in the integument of the head and tail, where the cts phenotype is observed; expression markedly increased at the molting and newly ecdysed stages. These results indicate that the novel BmMFS gene is cts and the membrane structure of its protein accounts for the cts phenotype. These expression profiles and the cts phenotype are quite similar to those of melanin-related genes, such as Bmyellow-e and Bm-iAANAT, suggesting that BmMFS is involved in the melanin synthesis pathway.

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.

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.

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.