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The silkworm (Bombyx mori) is an important model lepidopteran insect and can be used to identify pesticide resistance‐related genes of great significance for biological control of pests. Uridine diphosphate glucosyltransferases (UGTs), found in all organisms, are the main secondary enzymes involved in the metabolism of heterologous substances. However, it remains uncertain if silkworm resistance to fenpropathrin involves UGT. This study observes significant variations in BmUGT expression among B. mori strains with variable fenpropathrin resistance post‐feeding, indicating BmUGT's role in fenpropathrin detoxification. Knockdown of BmUGT with RNA interference and overexpression of BmUGT significantly decreased and increased BmN cell activity, respectively, indicating that BmUGT plays an important role in the resistance of silkworms to fenpropathrin. In addition, fenpropathrin residues were significantly reduced after incubation for 12 h with different concentrations of a recombinant BmUGT fusion protein. Finally, we verified the conservation of UGT to detoxify fenpropathrin in Spodoptera exigua: Its resistance to fenpropathrin decreased significantly after knocking down SeUGT. In a word, UGT plays an important role in silkworm resistance to fenpropathrin by directly degrading the compound, a function seen across other insects. The results of this study are of great significance for breeding silkworm varieties with high resistance and for biological control of pests. [ABSTRACT FROM AUTHOR]

BACKGROUND: Voltage‐dependent anion‐selective channels (VDACs) serve as pore proteins within the mitochondrial membrane, aiding in the regulation of cell life and cell death. Although the occurrence of cell death is crucial for defense against virus infection, the function played by VDAC in Bombyx mori, in response to the influence of Bombyx mori nucleopolyhedrovirus (BmNPV), remains unclear. RESULTS: BmVDAC was found to be relatively highly expressed both during embryonic development, and in the Malpighian tubule and midgut. Additionally, the expression levels of BmVDAC were found to be different among silkworm strains with varying levels of resistance to BmNPV, strongly suggesting a connection between BmVDAC and virus infection. To gain further insight into the function of BmVDAC in BmNPV, we employed RNA interference (RNAi) to silence and overexpress it by pIZT/V5‐His‐mCherry. The results revealed that BmVDAC is instrumental in developing the resistance of host cells to BmNPV infection in BmN cell‐line cells, which was further validated as likely to be associated with initiating programmed cell death (PCD). Furthermore, we evaluated the function of BmVDAC in another insect, Spodoptera exigua. Knockdown of the BmVDAC homolog in S. exigua, SeVDAC, made the larvae more sensitive to BmNPV. CONCLUSION: We have substantiated the pivotal role of BmVDAC in conferring resistance against BmNPV infection, primarily associated with the initiation of PCD. The findings of this study shine new light on the molecular mechanisms governing the silkworm's response to BmNPV infection, thereby supporting innovative approaches for pest biocontrol. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The silkworm Bombyx mori L. is a model organism of the order Lepidoptera. Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control. In this study, comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6, 12, and 24 h after feeding with fenpropathrin. Twenty‐six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways: glycerophospholipid metabolism, sulfur metabolism, glycolysis, amino acid metabolism, the urea cycle, and the tricarboxylic acid (TCA) cycle. After analyzing the percentage changes in the metabolic pathways at the 3 time points, sulfur metabolism, glycolysis, and the TCA cycle showed significant responses to fenpropathrin. Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways. The combination of iron(II) fumarate + folic acid (IF‐FA) enhanced fenpropathrin resistance in silkworms 6.38 fold, indicating that the TCA cycle is the core pathway associated with resistance. Furthermore, the disruption of several energy‐related metabolic pathways caused by fenpropathrin was shown to be recovered by IF‐FA in vitro. Therefore, IF‐FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways. [ABSTRACT FROM AUTHOR]

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an [F.sub.1] male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an [F.sub.1] female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of [approximately equal to]3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated. silkworm | microsatellite

Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real‐time polymerase chain reaction results confirmed that tim‐knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim‐knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms. [ABSTRACT FROM AUTHOR]

Insect wings are developed from the wing disc during metamorphosis. Bombyx mori, a model lepidopteran insect, loses flight ability after long‐term domestication from the wild silkworm, Bombyx mandarina. The mw mutant (u11 strain) shows minute wings compared to wild type (e.g., p50 strain) wings. RNA sequencing analysis previously revealed differential Hippo‐pathway‐related gene expression between the u11 and p50 strains. The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals. In this study, the function of BmSd which has been characterized as one of the Hippo‐pathway‐related genes was analyzed for silkworm wing development. We found that mats, warts, and hippo expression levels were higher in u11 compared to p50 wing discs. BmSd (scalloped) expression, which encodes a prominent transcriptional partner to Yorkie (Yki), gradually decreased during the wandering stage in u11, but exhibited the opposite expression pattern in p50. When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain, 57.9% of the individuals showed minute wings. Additionally, ex, kibra, and wingless expression levels decreased in the BmSd knockdown mutant. Further, BmSd deletion mediated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR‐associated protein 9 induced 50% of individuals with minute wings, a phenotype similar to the mw mutant. This result demonstrates that BmSd plays pivotal roles in silkworm wing development. Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development, and our findings provide a basis for further research on B. mori wing development. [ABSTRACT FROM AUTHOR]

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified. However, molecular mechanisms of most of these mutants remain to be explored. Here we report the identification of a gene Bm‐app is responsible for the silkworm minute wing (mw) mutation which exhibits exceedingly small wings during pupal and adult stages. Compared with the wild type silkworm, relative messenger RNA expression of Bm‐app is significantly decreased in the u11 mutant strain which shows mw phenotype. A 10 bp insertion in the putative promoter region of the Bm‐app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm‐app promoter activity. Furthermore, clustered regularly interspaced short palindromic repeats/RNA‐guided Cas9 nucleases‐mediated depletion of the Bm‐app induced similar wing defects which appeared in the mw mutant, demonstrating that Bm‐app controls wing development in B. mori. Bm‐app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins, indicating that it is required for cell mitosis and growth during wing development. We also discuss the possibility that Bm‐app regulates wing development through the Hippo signaling pathway in B. mori. [ABSTRACT FROM AUTHOR]

The ellipsoid egg, the second recessive gene of chocolate larvae, and melanism are controlled by three recessive genes, elp, ch-2, and mln in silkworm, respectively. Their order and genetic distance have been scheduled in established linkage group. Owing to lack of crossing over in females, the reciprocal backcrossed F1(BC1) progenies were bred for linkage analysis using the wild type silkworm strain P50(+elp+ch-2+mln /+elp+ch-2+mln) and W18 with ellipsoid egg, the second recessive gene of chocolate larvae, and melanism (elp ch-2 mln / elp ch-2 mln). In this research, we mapped three mutant genes, elp, ch-2, and mln on the chromosome 18 based on the SSR linkage map and STS markers designed based on silkworm genome sequence. The established linkage group, molecular linkage group, and the physic map of chromosome 18 had been corresponded. The genetic distance for this chromosome in this research was 94.2 cM, and the order of the mutants and molecular markers were consistent with the established silkworm linkage maps and the fine genome sequences. This research will lay important bases for map-based cloning for other mutants on chromosome 18.

The non-lepis wing of silkworm (Bombyx mori) is controlled by the recessive gene, nlw. Owning to lack of crossing over in females, the reciprocal backcrossed F(1) (BC(1)) progenies were used for linkage analysis and mapping of nlw based on the SSR linkage map and STS markers using the wild type (+(nlw)/+(nlw)) silkworm strain P50 and U06 with scaleless wing (nlw/nlw). The nlw gene was linked to eight SSR markers and one STS marker. All the individuals with the wild type in the BC1F (Using F(1) as female to backcross to the recessive parent, that is (U06xP50)xU06) showed heterozygous profile of (U06xP50) F(1), and the ones with non-lepis wing in BC1F exhibited the homozygous profile of the strain U06. Using a reciprocal BC1M (Using F1 as male to backcross to the recessive parent, that is U06x(U06xP50))cross, we constructed a linkage map of 125.6 cM, and the distance between nlw and the nearest marker cash2p was 11.4 cM.

The yellow color of silkworm (Bombyx mori) cocoon is mainly controlled by three genes, Y (yellow blood), I (yellow inhibitor) and C (out-layer yellow cocoon) genes. I gene locates on the 9th chromosome of silkworm and prevents the transport of carotenoid from epithelia of midgut into hemolymph. Owning to a lack of crossing over in females, reciprocal backcrossed F1(BC1) progenies were used for linkage analysis and mapping of the I gene based on the SSR linkage map using silkworm strains Baghdad (Ba), which express white hemolymph (II+Y+Y), and KY, which express yellow hemolymph (+I+IYY). The gene of interest was linked to three (S0904, S0905, and S0906) SSR markers. All the individuals with white hemolymph in the BC1F (BC1 was generated using F1 as female) showed heterozygous profile of (BaxKY) F1, and the yellow ones in BC1F showed the homozygous profile of the strain KY. Using a reciprocal BC1M cross, we con-structed a linkage map of 38.4 cM, and the distance between I gene and the nearest marker S0904 is 7.4 cM.

Cleaved amplified polymorphic sequence (CAPs) markers are based on PCR amplification of known genes, cDNA sequences or RAPD sequences. The PCR products are digested by restriction enzymes, generating the simple type of data as heterozygotes and homozygotes. Here we designed primers based on silkworm attacin and alpha-amylase genes, then digested the PCR products in silkworm strains P50, C108 and their progeny F1 using 4 different restriction enzymes respectively. Furthermore, the genetic diversity and phylogenetic relationship of 12 silkworm strains were investigated using the obtained two CAPs markers.