Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

Estrogen-related receptor, a molecular target against lepidoptera pests.

Until recently, chemical pesticides were one of the most effective means of controlling agricultural pests; therefore, the search for insecticide targets for agricultural pests has been an ongoing problem. Estrogen-related receptors (ERRs) are transcription factors that regulate cellular metabolism and energy homeostasis in animals. Silkworms are highly sensitive to chemical pesticides, making them ideal models for pesticide screening and evaluation. In this study, we detected ERR expression in key organs involved in pesticide metabolism in silkworms (Bombyx mori), including the fat body and midgut. Using ChIP-seq technology, many estrogen- related response elements were identified in the 2000-bp promoter region upstream of metabolism-related genes, almost all of which were potential ERR target genes. The ERR inhibitor, XCT-790, and the endocrine disruptor, bisphenol A, significantly inhibited expression of the ERR target genes, BmTreh-1, BmTret-1, BmPK, BmPFK, and BmHK, in the fat bodies of silkworms, resulting in pupation difficulties in silkworm larvae that ultimately lead to death. In addition, based on the clarification that the ERR can bind to XCT-790, as observed through biofilm interferometry, its three-dimensional spatial structure was predicted, and using molecular docking techniques, small-molecule compounds with a stronger affinity for the ERR were identified. In summary, utilizing the powerful metabolic regulatory function of ERR in Lepidoptera pests, the developed small molecule inhibitors of ERR can be used for future control of Lepidoptera pests.

Fatty Acid Desaturase Bmdesat5, Suppressed in the Salivary Glands by Domestication, is Involved in Regulation of Food Intake in Silkworms.

Understanding the evolutionary genetics of food intake regulation in domesticated animals has relevance to evolutionary biology, animal improvement, and obesity treatment. Here, we observed that the fatty acid desaturase gene (Bmdesat5), which regulates food intake, is suppressed in domesticated silkworms, but expressed in the salivary glands of the wild silkworm Bombyx mandarina. The content of its catalytic product, cis-vaccenic acid, was related to the expression levels of Bmdesat5 in the salivary glands of domesticated and wild silkworm strains. These two strains also showed significant differences in food intake. Using orally administering cis-vaccenic acid and transgenic-mediated overexpression, we verified that cis-vaccenic acid functions as a satiation signal, regulating food intake and growth in silkworms. Selection analysis showed that Bmdesat5 experienced selection, especially in the potential promoter, 5'-untranslated, and intron regions. This study highlights the importance of the decrement of satiety in silkworm domestication and provides new insights into the potential involvement of salivary glands in the regulation of satiety in animals, by acting as a supplement to gut-brain nutrient signaling.

Identification of long non-coding RNAs in response to microsporidia infection in Silkworm, Bombyx mori.

Microsporidia Nosema bombycis (Nb) is a cellular parasite responsible for pébrine disease in silkworms, significantly impacting the sericulture industry. Long non-coding RNAs (lncRNAs), which are RNA fragments longer than 200 nucleotides, are pivotal in a range of cellular and physiological functions. However, the potential role of silkworm lncRNAs in response to Nb infection remains unknown. This study conducted transcriptome sequencing on both larvae and Nb-infected midguts of silkworms, identifying 1,440 lncRNAs across all examined midgut samples. Within the Nb-infected group, 42 differentially expressed lncRNAs (DElncRNAs) and 305 differentially expressed mRNAs (DEmRNAs) were detected. Functional annotation and pathway analysis showed that these DEmRNAs are mostly involved in metabolism, apoptosis, autophagy, and other key pathways. The co-expression network of DEmRNAs and DElncRNAs illustrates that 1 gene could be regulated by multiple lncRNAs and 1 lncRNA may target multiple genes, indicating that the regulation of lncRNA is intricate and networked. In addition, the DElncRNA-miRNA-mRNA network showed that some DElncRNAs may be involved in the immune response and metabolism through miRNA. Notably, the study observed an increase in lncRNA MSTRG857.1 following Nb infection, which may promote Nb proliferation. These findings offer insights into the complex interplay between insects and microsporidia.

Perilipin1 inhibits Nosema bombycis proliferation by promoting Domeless- and Hop-mediated JAK-STAT pathway activation in Bombyx mori.

Lipid droplets (LDs) are dynamic organelles that participate in the regulation of lipid metabolism and cellular homeostasis inside of cells. LD-associated proteins, also known as perilipins (PLINs), are a family of proteins found on the surface of LDs that regulate lipid metabolism, immunity, and other functions. In silkworms, pébrine disease caused by infection by the microsporidian Nosema bombycis (Nb) is a severe threat to the sericultural industry. Although we found that Nb relies on lipids from silkworms to facilitate its proliferation, the relationship between PLINs and Nb proliferation remains unknown. Here, we found Nb infection caused the accumulation of LDs in the fat bodies of silkworm larvae. The characterized perilipin1 gene (plin1) promotes the accumulation of intracellular LDs and is involved in Nb proliferation. plin1 is similar to perilipin1 in humans and is conserved in all insects. The expression of plin1 was mostly enriched in the fat body rather than in other tissues. Knockdown of plin1 enhanced Nb proliferation, whereas overexpression of plin1 inhibited its proliferation. Furthermore, we confirmed that plin1 increased the expression of the Domeless and Hop in the JAK-STAT immune pathway and inhibited Nb proliferation. Taken together, our current findings demonstrate that plin1 inhibits Nb proliferation by promoting the JAK-STAT pathway through increased expression of Domeless and Hop. This study provides new insights into the complicated connections among microsporidia pathogens, LD surface proteins, and insect immunity.IMPORTANCELipid droplets (LDs) are lipid storage sites in cells and are present in almost all animals. Many studies have found that LDs may play a role in host resistance to pathogens and are closely related to innate immunity. The present study found that a surface protein of insect lipid droplets could not only regulate the morphological changes of lipid droplets but also inhibit the proliferation of a microsporidian pathogen Nosema bombycis (Nb) by activating the JAK-STAT signaling pathway. This is the first discovery of the relationship between microsporidian pathogen and insect lipid surface protein perilipin and insect immunity.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression.

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Ectopic expression of BmeryCA in Bombyx mori increases silk yield and mechanical properties by altering the pH of posterior silk gland.

The silk glands are the specialized tissue where silk protein synthesis, secretion, and conformational transitions take place, with pH playing a critical role in both silk protein synthesis and fiber formation. In the present study, we have identified erythrocyte carbonic anhydrase (BmeryCA) belonging to the α-CA class in the silk gland, which is a Zn2+ dependent metalloenzyme capable of efficiently and reversibly catalyzing the hydrated reaction of CO2 to HCO3-, thus participating in the regulation of acid-base balance. Multiple sequence alignments revealed that the active site of BmeryCA was highly conserved. Tissue expression profiling showed that BmeryCA had relatively high expression levels in hemolymph and epidermis but is barely expressed in the posterior silk gland (PSG). By specifically overexpressing BmeryCA in the PSG, we generated transgenic silkworms. Ion-selective microelectrode (ISM) measurements demonstrated that specifically overexpression of BmeryCA in the PSG led to a shift in pH from weakly alkaline to slightly neutral conditions. Moreover, the resultant PSG-specific BmeryCA overexpression mutant strain displayed a significant increase in both silk yield and silk fiber mechanical properties. Our research provided new insights into enhancing silk yield and improving the mechanical properties of silk fibers.

Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

Contrasting gut bacteriomes unveiled between wild Antheraea assamensis Helfer (Lepidoptera: Saturniidae) and domesticated Bombyx mori L. (Lepidoptera: Bombycidae) silkworms.

BACKGROUND: Insect gut microbiomes play a fundamental role in various aspects of insect physiology, including digestion, nutrient metabolism, detoxification, immunity, growth and development. The wild Muga silkworm, Antheraea assamensis Helfer holds significant economic importance, as it produces golden silk. METHODS AND RESULTS: In the current investigation, we deciphered its intricate gut bacteriome through high-throughput 16S rRNA amplicon sequencing. Further, to understand bacterial community dynamics among silkworms raised under outdoor environmental conditions, we compared its gut bacteriomes with those of the domesticated mulberry silkworm, Bombyx mori L. Most abundant bacterial phyla identified in the gut of A. assamensis were Proteobacteria (78.1%), Bacteroidetes (8.0%) and Firmicutes (6.6%), whereas the most-abundant phyla in B. mori were Firmicutes (49-86%) and Actinobacteria (10-36%). Further, Gammaproteobacteria (57.1%), Alphaproteobacteria (10.47%) and Betaproteobacteria (8.28%) were the dominant bacterial classes found in the gut of A. assamensis. The predominant bacterial families in A. assamensis gut were Enterobacteriaceae (27.7%), Comamonadaceae (9.13%), Pseudomonadaceae (9.08%) Flavobacteriaceae (7.59%) Moraxellaceae (7.38%) Alteromonadaceae (6.8%) and Enterococcaceae (4.46%). In B. mori, the most-abundant bacterial families were Peptostreptococcaceae, Enterococcaceae, Lactobacillaceae and Bifidobacteriaceae, though all showed great variability among the samples. The core gut bacteriome of A. assamensis consisted of Pseudomonas, Acinetobacter, Variovorax, Myroides, Alteromonas, Enterobacter, Enterococcus, Sphingomonas, Brevundimonas, Oleispira, Comamonas, Oleibacter Vagococcus, Aminobacter, Marinobacter, Cupriavidus, Aeromonas, and Bacillus. Comparative gut bacteriome analysis revealed a more complex gut bacterial diversity in wild A. assamensis silkworms than in domesticated B. mori silkworms, which contained a relatively simple gut bacteriome as estimated by OTU richness. Predictive functional profiling of the gut bacteriome suggested that gut bacteria in A. assamensis were associated with a wide range of physiological, nutritional, and metabolic functions, including biodegradation of xenobiotics, lipid, amino acid, carbohydrate metabolism, and biosynthesis of secondary metabolites and amino acids. CONCLUSIONS: These results showed great differences in the composition and diversity of gut bacteria between the two silkworm species. Both insect species harbored core bacterial taxa commonly found in insects, but the relative abundance and composition of these taxa varied markedly.

The mechanoreceptor Piezo is required for spermatogenesis in Bombyx mori.

BACKGROUND: The animal sperm shows high diversity in morphology, components, and motility. In the lepidopteran model insect, the silkworm Bombyx mori, two types of sperm, including nucleate fertile eupyrene sperm and anucleate unfertile apyrene sperm, are generated. Apyrene sperm assists fertilization by facilitating the migration of eupyrene spermatozoa from the bursa copulatrix to the spermatheca. During spermatogenesis, eupyrene sperm bundles extrude the cytoplasm by peristaltic squeezing, while the nuclei of the apyrene sperm bundles are discarded with the same process, forming matured sperm. RESULTS: In this study, we describe that a mechanoreceptor BmPiezo, the sole Piezo ortholog in B. mori, plays key roles in larval feeding behavior and, more importantly, is essential for eupyrene spermatogenesis and male fertility. CRISPR/Cas9-mediated loss of BmPiezo function decreases larval appetite and subsequent body size and weight. Immunofluorescence analyses reveal that BmPiezo is intensely localized in the inflatable point of eupyrene sperm bundle induced by peristaltic squeezing. BmPiezo is also enriched in the middle region of apyrene sperm bundle before peristaltic squeezing. Cytological analyses of dimorphic sperm reveal developmental arrest of eupyrene sperm bundles in BmPiezo mutants, while the apyrene spermatogenesis is not affected. RNA-seq analysis and q-RT-PCR analyses demonstrate that eupyrene spermatogenic arrest is associated with the dysregulation of the actin cytoskeleton. Moreover, we show that the deformed eupyrene sperm bundles fail to migrate from the testes, resulting in male infertility due to the absence of eupyrene sperm in the bursa copulatrix and spermatheca. CONCLUSIONS: In conclusion, our studies thus uncover a new role for Piezo in regulating spermatogenesis and male fertility in insects.

An engineered Pichia pastoris platform for the biosynthesis of silk-based nanomaterials with therapeutic potential.

Silk fibroin (SF) from the cocoon of silkworm has exceptional mechanical properties and biocompatibility and is used as a biomaterial in a variety of fields. Sustainable, affordable, and scalable manufacturing of SF would enable its large-scale use. We report for the first time the high-level secretory production of recombinant SF peptides in engineered Pichia pastoris cell factories and the processing thereof to nanomaterials. Two SF peptides (BmSPR3 and BmSPR4) were synthesized and secreted by P. pastoris using signal peptides and appropriate spacing between hydrophilic sequences. By strain engineering to reduce protein degradation, increase glycyl-tRNA supply, and improve protein secretion, we created the optimized P. pastoris chassis PPGSP-8 to produce BmSPR3 and BmSPR4. The SF fed-batch fermentation titers of the resulting two P. pastoris cell factories were 11.39 and 9.48 g/L, respectively. Protein self-assembly was inhibited by adding Tween 80 to the medium. Recombinant SF peptides were processed to nanoparticles (NPs) and nanofibrils. The physicochemical properties of nanoparticles R3NPs and R4NPs from the recombinant SFs synthesized in P. pastoris cell factories were similar or superior to those of RSFNPs (Regenerated Silk Fibroin NanoParticles) originating from commercially available SF. Our work will facilitate the production by microbial fermentation of functional SF for use as a biomaterial.

Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Physiological and transcriptomic responses of silkworms to graphene oxide exposure.

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis-Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO.

Bombyx moriSuppressor of Hairless is involved in the regulation of the silkworm cell cycle and endoreplication of the silk glands.

The Notch signaling pathway is important in cell cycle regulation and cell proliferation. The transcriptional repressor Suppressor of Hairless [Su(H)] is a molecular switch for downstream target genes of the Notch signaling pathway but the regulatory mechanism of the Su(H) gene in the cell cycle is unclear. We determined the function of the Notch signaling pathway and Bombyx mori Su(H) [BmSu(H)] in the regulation of the silkworm cell cycle. Inhibition of Notch signaling promoted the replication of DNA in silkworm gland cells and expression of the BmSu(H) gene was significantly reduced. Overexpression of the BmSu(H) gene inhibited DNA replication and cell proliferation of silkworm cells, whereas knockout of the BmSu(H) gene promoted DNA replication and cell proliferation. Knockout of the BmSu(H) in silkworms improved the efficiency of silk gland cell endoreplication and increased important economic traits. We demonstrated that BmSu(H) protein can directly bind to the promoters of BmCyclinA, BmCyclinE and BmCDK1 genes, inhibiting or promoting their transcription at the cell and individual level. This study identified molecular targets for genetic improvement of the silkworm and also provided insights into the regulatory mechanism of the cell cycle.

The dual role of Spn-E in supporting heterotypic ping-pong piRNA amplification in silkworms.

The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.

The BTB-ZF gene Bm-mamo regulates pigmentation in silkworm caterpillars.

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor of dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermal features in some insects.

BmNPV p35 regulates apoptosis in Bombyx mori via a novel target of interaction with the BmVDAC2-BmRACK1 complex.

Voltage-dependent anion channel 2 (VDAC2) is an important channel protein that plays a crucial role in the host response to viral infection. The receptor for activated C kinase 1 (RACK1) is also a key host factor involved in viral replication. Our previous research revealed that Bombyx mori VDAC2 (BmVDAC2) and B. mori RACK1 (BmRACK1) may interact with Bombyx mori nucleopolyhedrovirus (BmNPV), though the specific molecular mechanism remains unclear. In this study, the interaction between BmVDAC2 and BmRACK1 in the mitochondria was determined by various methods. We found that BmNPV p35 interacts directly with BmVDAC2 rather than BmRACK1. BmNPV infection significantly reduced the expression of BmVDAC2, and activated the mitochondrial apoptosis pathway. Overexpression of BmVDAC2 in BmN cells inhibited BmNPV-induced cytochrome c (cyto c) release, decrease in mitochondrial membrane potential as well as apoptosis. Additionally, the inhibition of cyto c release by BmVDAC2 requires the involvement of BmRACK1 and protein kinase C. Interestingly, overexpression of p35 inhibited cyto c release during mitochondrial apoptosis in a RACK1 and VDAC2-dependent manner. Even the mutant p35, which loses Caspase inhibitory activity, could still bind to VDAC2 and inhibit cyto c release. In summary, our results indicated that BmNPV p35 interacts with the VDAC2-RACK1 complex to regulate apoptosis by inhibiting cyto c release. These findings confirm the interaction between BmVDAC2 and BmRACK1, the interaction between p35 and the VDAC2-RACK1 complex, and a novel target that BmNPV p35 regulates apoptosis in Bombyx mori via interaction with the BmVDAC2-BmRACK1 complex. The result provide an initial exploration of the function of this interaction in the BmNPV-induced mitochondrial apoptosis pathway.

[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Study on anti-BmNPV mechanism of branched-chain amino acid aminotransferases in silkworm.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.