Disruption of microRNA pathway core genes inhibits molting and reproduction of the cigarette beetle, Lasioderma serricorne.

BACKGROUND: MicroRNA (miRNA) pathway genes have been widely reported to participate in several physiological events in insect lifecycles. The cigarette beetle Lasioderma serricorne is an economically important storage pest worldwide. However, the functions of miRNA pathway genes in L. serricorne remain to be clarified. Herein, we investigated the function of molting and reproduction of the miRNA pathway in L. serricorne. RESULTS: LsDicer-1, LsArgonaute-1, LsLoquacious and LsExportin-5 were universally expressed in adults, whereas LsPasha and LsDrosha were mainly expressed in the pupae. The genes presented different patterns in various tissues. Silencing of LsDicer-1, LsArgonaute-1, LsDrosha and LsExportin-5 resulted in a high proportion of wing deformities and molting defects. Silencing of LsDicer-1, LsArgonaute-1, LsPasha and LsLoquacious affected the development of the ovary and the maturation of oocytes, resulting in a significant decrease in fecundity. Further investigation revealed that the decreases in LsDicer-1 and LsArgonaute-1 expression destroyed follicular epithelia and delayed vitellogenesis and oocyte development. In addition, the expression levels of several miRNAs (let-7, let-7-5p, miR-8-3p, miR-8-5p, miR-9c-5p, miR-71, miR-252-5p, miR-277-3p, miR-263b and Novel-miR-50) were decreased significantly after knockdown of these miRNA pathway core genes, indicating that they played important roles in regulating miRNA-mediated gene expression. CONCLUSION: The results indicate that miRNA pathway genes play important roles in the molting, ovarian development and female fecundity of L. serricorne, and thus are potentially suitable target genes for developing an RNAi strategy against a major pest of stored products. © 2024 Society of Chemical Industry.

The W-Plasty Serial Excision Method for Treating Medium Congenital Melanocytic Nevi: A Retrospective Analytical Study.

BACKGROUND: Serial excision remains the most commonly used surgical procedure for treating congenital melanocytic nevus (CMN). It is critical to remove as much of the lesion as possible with each procedure to reduce the number of procedures and to shorten the treatment duration. OBJECTIVE: To investigate the clinical efficacy of W-plasty serial excision for the repair of postoperative CMN defects. METHODS: A retrospective analysis of patients with medium CMN was conducted from April 2018 to March 2022. Treatment options were divided into elliptical serial excision (10 cases) and W-plasty serial excision (10 cases). RESULTS: Follow-up occurred over 6 months. The number of elliptical excision procedures was 2 to 4 (mean 2.9). The scar-to-lesion length ratio was 1.5 to 2.0 (mean 1.7). The mean Vancouver Scar Scale (VSS) score was 5.40 ± 0.42. The number of W-plasty excision procedures was 2 to 3 (mean 2.2). The scar-to-lesion length ratio was 1.2 to 1.5 (mean 1.4). The mean VSS score was 2.70 ± 0.26. W-plasty excision was superior to elliptical excision regarding the number of procedures and the effect on postoperative scars. CONCLUSION: W-plasty serial excision can be considered a suitable option for the excision of medium CMN, leading to excellent results.

Functional analysis of nuclear receptor genes in molting and metamorphosis of the cigarette beetle, Lasioderma serricorne.

Nuclear receptors (NRs) are ligand-regulated transcription factors that are important for the normal growth and development of insects. However, systematic function analysis of NRs in the molting process of Lasioderma serricorne has not been reported. In this study, we identified and characterized 16 NR genes from L. serricorne. Spatiotemporal expression analysis revealed that six NRs were mainly expressed in 3-d-old 4th-instar larvae; five NRs were primarily expressed in 5-d-old adults and four NRs were predominately expressed in prepupae. All the NRs were highly expressed in epidermis, fat body and foregut. RNA interference (RNAi) experiments revealed that knockdown of 15 NRs disrupted the larva-pupa-adult transitions and caused 64.44-100 % mortality. Hematoxylin-eosin staining showed that depletion of 12 NRs prevented the formation of new cuticle and disrupted apolysis of old cuticle. Silencing of LsHR96, LsSVP and LsE78 led to newly formed cuticle that was thinner than the controls. The 20E titer and chitin content significantly decreased by 17.67-95.12 % after 15 NR dsRNA injection and the gene expression levels of 20E synthesis genes and chitin metabolism genes were significantly reduced. These results demonstrated that 15 NR genes are essential for normal molting and metamorphosis of L. serricorne by regulating 20E synthesis and chitin metabolism.

MicroR-9c-5p and novel-mir50 co-target Akt to regulate Lasioderma serricorne reproduction.

High fecundity is a common characteristic of insect pests which increases the difficulty of population control. Serine/threonine kinase Akt is an indispensable component of the insulin signaling pathway. Silencing of LsAkt severely hinders reproduction in Lasioderma serricorne, a stored product insect pest. However, the post-transcriptional pathway of LsAkt in L. serricorne remains unknown. This study identified 2 binding sites of miR-9c-5p and novel-mir50 in the coding sequences of LsAkt. The expression profiles of 2 microRNAs (miRNAs) and LsAkt displayed an opposite pattern during the adult stages. Luciferase reporter assay showed that novel-mir50 and miR-9c-5p could downregulate the expression of LsAkt. Overexpression of miR-9c-5p and novel-mir50 by injection of mimics inhibited the expression of LsAkt and reduced oviposition, decreased egg hatchability, and blocked ovarian development. It also decreased the expression of genes involved in ovarian development (LsVg and LsVgR) and the nutritional signaling pathway (LsTOR, LsS6K, and Ls4EBP), and reduced the phosphorylation of Akt. Conversely, injection of miR-9c-5p and novel-mir50 inhibitors induced the expressions of LsAkt, LsVg, LsVgR, LsTOR, LsS6K, and Ls4EBP, enhanced Akt phosphorylation level, and accelerated ovarian development. Injection of bovine insulin downregulated the expression of miR-9c-5p and novel-mir50 and upregulated the LsAkt expression. It also rescued the reproductive development defects associated with miR-9c-5p/novel-mir50 overexpression, forming a positive regulatory loop of insulin signaling. These results indicate that miR-9c-5p/novel-mir50 regulates the female reproduction of L. serricorne by targeting Akt in response to insulin signaling. The data also demonstrate the effects of the insulin/miRNA/Akt regulatory axis in insect reproduction.

Identification and immune analysis of antimicrobial peptides from the cigarette beetle (Lasioderma serricorne).

Antimicrobial peptides (AMPs) in insects are endogenous peptides that are effector components of the innate defense system of the insect. AMPs may serve as antimicrobial agents because of their small molecular weight and broad-spectrum antimicrobial activity. In this study, we performed transcriptome analysis of cigarette beetle (Lasioderma serricorne) larvae, parasitized by the ectoparasitic wasp, Anisopteromalus calandrae. Several AMP genes were significantly upregulated following A. calandrae parasitism, postulating the hypothesis that the parasitization enhanced the host's resistance against pathogenic microorganisms through the regulation of host AMP genes. Specifically, 3 AMP genes (LsDef1, LsDef2, and LsCole) were significantly upregulated and we studied their immune function in L. serricorne. Immune challenge and functional analysis showed that LsCole was responsible for the immune response against Gram-negative and Gram-positive bacteria, while LsDef1 and LsDef2 were involved in insect defense against Gram-positive bacteria. Purified recombinant LsCole exhibited antimicrobial activities against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. LsDef2 showed an antibacterial effect against S. aureus. LsCole and LsDef2 exhibited antibiofilm activity against S. aureus. The 2 AMPs disrupted cell membranes and caused leakage of S. aureus cell contents. The results indicated that the 3 AMPs in L. serricorne are involved in the innate immunity of this pest insect. These AMPs may have potential as antimicrobial agents for bacterial infection chemotherapy. Hence, data are discussed in relation to new control strategies with greater biosafety against pest insects with use of microbial biocontrol agents in combination with RNA interference against the insect's defensive AMP genes.

Four peptidoglycan recognition proteins are indispensable for antibacterial immunity in the cigarette beetle Lasioderma serricorne (Fabricius).

The peptidoglycan recognition protein (PGRP), an important pattern recognition receptor of insects, is significant for reducing innate immunity and effective pest control. We cloned four PGRP genes (LsPGRP-LB, LsPGRP-LB1, LsPGRP-LE, and LsPGRP-SC2) from the cigarette beetle, Lasioderma serricorne (Fabricius), which encoded proteins of 216, 197, 317, and 190 amino acids, respectively. Three LsPGRPs were predominantly expressed in the larval and pupal stages, whereas LsPGRP-LE displayed high expression in adults. All the four LsPGRPs genes were highly expressed in the midgut and integument. Pathogen inoculation revealed that the four LsPGRPs actively responded to Escherichia coli and its peptidoglycan. The transcription levels of LsPGRP-LE and LsPGRP-SC2 increased significantly after Staphylococcus aureus stimulation. RNA interference-mediated knockdown of the four LsPGRPs led to increased larval mortality when challenged by E. coli, and the expression of four antimicrobial peptide genes (LsCole, LsAtt2, LsDef1 and LsDef2) had a significant decrease. Higher mortality and lower AMP expression were also observed in L. serricorne under S. aureus infection after silencing LsPGRP-LE and LsPGRP-SC2. Our results suggest that the four LsPGRP genes play important and distinct regulatory roles in the antibacterial defense response of L. serricorne.

Disruption of the Serine/Threonine Kinase Akt Gene Affects Ovarian Development and Fecundity in the Cigarette Beetle, Lasioderma serricorne.

Serine/threonine kinase Akt, an important component of the insulin signaling pathway, plays an essential role in many physiological processes. In this study, we identified and characterized an Akt gene (designated LsAkt) from the cigarette beetle, Lasioderma serricorne. LsAkt contains a 1614 bp open reading frame encoding a 537 amino acid protein that possesses a conserved pleckstrin homology domain and a serine/threonine kinase domain. The expression of LsAkt was high in pupal stages and peaked in day-4 female pupae. In adult tissues, LsAkt was highly expressed in the thorax, ovary, and midgut. The expression of LsAkt was induced by methoprene or bovine insulin in vivo, but significantly decreased by 20-hydroxyecdysone. RNA interference-mediated knockdown of LsAkt resulted in severely blocked ovarian development and reduced fecundity and hatchability. The vitellogenin (Vg) content and juvenile hormone (JH) titers of LsAkt-depletion beetles were decreased, and expressions of Vg and four JH signaling and biosynthetic genes were significantly decreased. Silencing of LsAkt reduced the amounts of glucose, glycogen, and trehalose in female adults and affected the expressions of seven key carbohydrate metabolic genes. Taken together, it is inferred that Akt implicates in L. serricorne reproduction by modification of Vg synthesis, juvenile hormone production and carbohydrate metabolism.

Role of Chitin Deacetylase 1 in the Molting and Metamorphosis of the Cigarette Beetle Lasioderma serricorne.

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval-pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval-pupal and pupal-adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.

Knockdown of ß-N-acetylglucosaminidase 2 Impairs Molting and Wing Development in Lasioderma serricorne (Fabricius).

ß-N-acetylglucosaminidases (NAGs) are carbohydrate enzymes that degrade chitin oligosaccharides into N-acetylglucosamine monomers. This process is important for chitin degradation during insect development and metamorphosis. We identified and evaluated a ß-N-acetylglucosaminidase 2 gene (LsNAG2) from the cigarette beetle, Lasioderma serricorne (Fabricius). The full-length open reading frame of LsNAG2 was 1776 bp and encoded a 591 amino acid protein. The glycoside hydrolase family 20 (GH20) catalytic domain and an additional GH20b domain of the LsNAG2 protein were highly conserved. Phylogenetic analysis revealed that LsNAG2 clustered with the group II NAGs. Quantitative real-time PCR analyses showed that LsNAG2 was expressed in all developmental stages and was most highly expressed in the late larval and late pupal stages. In the larval stage, LsNAG2 was predominantly expressed in the integument. Knockdown of LsNAG2 in fifth instar larvae disrupted larval-pupal molting and reduced the expression of four chitin synthesis genes (trehalase 1 (LsTRE1), UDP-N-acetylglucosamine pyrophosphorylase 1 and 2 (LsUAP1 and LsUAP2), and chitin synthase 1 (LsCHS1)). In late pupae, LsNAG2 depletion resulted in abnormal adult eclosion and wing deformities. The expression of five wing development-related genes (teashirt (LsTSH), vestigial (LsVG), wingless (LsWG), ventral veins lacking (LsVVL), and distal-less (LsDLL)) significantly declined in the LsNAG2-depleted beetles. These findings suggest that LsNAG2 is important for successful molting and wing development of L. serricorne.

Identification and Expression Analysis of Four Small Heat Shock Protein Genes in Cigarette Beetle, Lasioderma serricorne (Fabricius).

Small heat shock proteins (sHsps) are molecular chaperones that play crucial roles in the stress adaption of insects. In this study, we identified and characterized four sHsp genes (LsHsp19.4, 20.2, 20.3, and 22.2) from the cigarette beetle, Lasioderma serricorne (Fabricius). The four cDNAs encoded proteins of 169, 180, 181, and 194 amino acids with molecular weights of 19.4, 20.2, 20.3, and 22.2 kDa, respectively. The four LsHsp sequences possessed a typical sHsp domain structure. Quantitative real-time PCR analyses revealed that LsHsp19.4 and 20.3 transcripts were most abundant in pupae, whereas the transcript levels of LsHsp20.2 and 22.2 were highest in adults. Transcripts of three LsHsp genes were highly expressed in the larval fat body, whereas LsHsp20.2 displayed an extremely high expression level in the gut. Expression of the four LsHsp genes was dramatically upregulated in larvae exposed to 20-hydroxyecdysone. The majority of the LsHsp genes were significantly upregulated in response to heat and cold treatments, while LsHsp19.4 was insensitive to cold stress. The four genes were upregulated when challenged by immune triggers (peptidoglycan isolated from Staphylococcus aureus and from Escherichia coli 0111:B4). Exposure to CO2 increased LsHsp20.2 and 20.3 transcript levels, but the LsHsp19.4 transcript level declined. The results suggest that different LsHsp genes play important and distinct regulatory roles in L. serricorne development and in response to diverse stresses.

Clip-Domain Serine Protease Gene (LsCLIP3) Is Essential for Larval-Pupal Molting and Immunity in Lasioderma serricorne.

Clip-domain serine proteases (CLIPs) play crucial roles in insect development and innate immunity. In this study, we identified a CLIP gene (designated LsCLIP3) from the cigarette beetle Lasioderma serricorne. LsCLIP3 contains a 1,773-bp open reading frame (ORF) encoding a 390-amino-acid protein and shows a conserved clip domain and a trypsin-like serine protease domain. Phylogenetic analysis indicated that LsCLIP3 was orthologous to the CLIP-B subfamily. LsCLIP3 was prominently expressed in larva, pupa, and early adult stages. In larval tissues, it was highly expressed in the integument and fat body. The expression of LsCLIP3 was induced by 20-hydroxyecdysone. A similar induction was also found by peptidoglycans from Escherichia coli and Staphylococcus aureus. RNA interference (RNAi)-mediated silencing of LsCLIP3 disrupted larval-pupal molting and specifically reduced the expression of genes in 20-hydroxyecdysone synthesis and signaling pathway. The chitin amounts of LsCLIP3 RNAi larvae were greatly decreased, and expressions of six chitin metabolic-related genes were significantly reduced. Knockdown of LsCLIP3 increased larval sensitivity to Gram-negative and Gram-positive bacteria. There was significantly decreased expression of four antimicrobial peptide (AMP) genes. The results suggest that LsCLIP3 is an important component of the larva to pupa molt and for the immunity of L. serricorne.

The complete mitochondrial genome of the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae).

The cigarette beetle, Lasioderma serricorne (Fabricius), is an important pest of stored commodities and distributed widely in the world. Here, we report the complete mitochondrial genome of L. serricorne which was 15,958 bp and composed of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. The gene order and orientation of L. serricorne were identical to those of other Coleopteran mitogenomes. ATG, ATA, ATT, ATC, TTG were initiation codons and TAA, TAG, T were termination codons. All 22 tRNA genes were predicted with a typical cloverleaf structure except for trnS1 (AGN). Phylogenetic analysis performed using 13 PCGs with 14 other beetles showed that L. serricorne is closely related to Stegobium paniceum, which agree with the conventional taxonomy.

Two Chitin Biosynthesis Pathway Genes in Bactrocera dorsalis (Diptera: Tephritidae): Molecular Characteristics, Expression Patterns, and Roles in Larval-Pupal Transition.

Glucose-6-phosphate isomerase (G6PI) and UDP-N-acetylglucosamine pyrophosphorylase (UAP), two key components in the chitin biosynthesis pathway, are critical for insect growth and metamorphosis. In this study, we identified the genes BdG6PI and BdUAP from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frames (ORFs) of BdG6PI (1,491 bp) and BdUAP (1,677 bp) encoded 496 and 558 amino acid residues, respectively. Multiple sequence alignments showed that BdG6PI and BdUAP had high amino acid sequence identity with other insect homologues. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated that BdG6PI was mainly expressed in the early stages of third-instar larvae and adults, while significantly higher expression of BdUAP was observed in adults. Both transcripts were expressed highly in the Malpighian tubules, but only slightly in the tracheae. The expression of both BdG6PI and BdUAP was significantly up-regulated by 20-hydroxyecdysone exposure and down-regulated by starvation. Moreover, injection of double-stranded RNAs of BdG6PI and BdUAP into third-instar larvae significantly reduced the corresponding gene expressions. Additionally, silencing of BdUAP resulted in 65% death and abnormal phenotypes of larvae, while silencing of BdG6PI had a slight effect on insect molting. These findings provide some data on the roles of BdG6PI and BdUAP in B. dorsalis and demonstrate the potential role for BdUAP in larval-pupal transition.

Electroacupuncture attenuates collagen-induced arthritis in rats through vasoactive intestinal peptide signalling-dependent re-establishment of the regulatory T cell/T-helper 17 cell balance.

OBJECTIVE: Imbalance between T-helper 17 (Th17) cells and regulatory T (Treg) cells is causally linked to the development of rheumatoid arthritis (RA). In this study, we tested the hypothesis that electroacupuncture (EA) confers therapeutic benefits in RA through activation of vasoactive intestinal peptide (VIP)-dependent signalling and restoration of the Th17/Treg cell balance. MATERIALS AND METHODS: A collagen-induced arthritis (CIA) model was induced in Sprague-Dawley rats by injection of bovine type II collagen in incomplete Freund's adjuvant on day 0 and day 7. Three days after the second injection, EA was given at acupuncture points GB39 and ST36 three times per week for 4 weeks. To block VIP signalling, [D-P-Cl-Phe(6)-Leu(17)]-VIP, a VIP receptor antagonist, was administered intraperitoneally 30 min before EA. Inflammatory and pathological responses in the joint were assessed. Synovial VIP receptor mRNA levels and Treg and Th17 cell frequencies in the spleen were determined. RESULTS: EA significantly reduced the severity of CIA, as evidenced by reduced paw volumes, arthritis scores and inflammation scores. EA significantly increased mRNA expression of the VIP receptor VPAC1 and led to an elevation in CD4(+)FOXP3(+) Treg cell frequency and a reduction in CD4(+)IL17(+) Th17 cell frequency. Pre-injection of a VIP receptor antagonist significantly reversed EA-induced expansion of Treg cells, but did not alter the frequencies of Th17 cells. CONCLUSIONS: EA exerts anti-inflammatory effects in a collagen-induced rat model of arthritis. These effects appear to be mediated through activation of VIP signalling and re-establishment of the Th17/Treg cell balance.

[Mechanism of regulating deacetylase SIRT1 expression and activity.].

SIRT1 is an NAD+-dependent deacetylase in mammalian cells and plays important roles in senescence, cell death and tumorigenesis. To clarify the regulation of SIRT1 expression and activity is crucial for scientists to understand the biological function of SIRT1. This review mainly focuses on the mechanisms by which SIRT1 expression and activity are regulated.