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Chitin deacetylases (CDAs) are chitin degradation enzymes that strictly regulate growth and development in insects. In this study, we identified and characterized a full-length cDNA of the CDA gene (SpCDA1) in the drugstore beetle, Stegobium paniceum. The open reading frame of SpCDA1 (1614 bp) encoded a 537 amino acid protein, which possessed typical domain structures of CDAs. Phylogenetic comparison to other insect CDAs revealed that SpCDA1 belongs to Group Ib CDAs. Quantitative real-time PCR analyses showed that SpCDA1 was highly expressed in late larval stages. Significant increase of SpCDA1 transcript level in the larvae was observed upon the exposure of 20-hydroxyecdysone. Injection of double-stranded RNA (dsRNA) of SpCDA1 into the late larvae significantly reduced SpCDA1 transcript levels, resulted in larval-pupal molting difficulty and produced high larval mortality. After 15 days, the survival rate of S. paniceum in dsSpCDA1 group was significantly reduced by 72% compared to the control. The results demonstrated that SpCDA1 is essential for successful larval-pupal transition in S. paniceum and this gene may be a potential target for pest control., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator in the insulin signaling pathway. It belongs to a class of non-receptor phosphatases of protein tyrosine phosphatase and can catalyze the dephosphorylation of tyrosine to regulate cell differentiation, growth, and metabolism. However, few studies have focused on the role of PTP1B in regulating energy metabolism of insects. In this study, we investigated the expression profiles and the functions of a PTP1B gene (designated TcPTP61F) in the red flour beetle Tribolium castaneum. Quantitative real-time PCR analyzed showed that TcPTP61F was highly expressed in the pupal and adult stages. In adult tissues, TcPTP61F was prominently expressed in the tarsus and head. RNA interference-mediated silencing of TcPTP61F reduced the expression of eight genes in trehalose metabolic and glycolytic pathways. TcPTP61F depletion also caused a significant change in the distribution of trehalose, glucose, and glycogen. Additionally, knockdown of TcPTP61F inhibited the pyruvate kinase (PK) activity and significantly decreased the adenosine triphosphate (ATP) level. The results suggest that TcPTP61F is indispensible for trehalose and energy metabolism of T. castaneum. [ABSTRACT FROM AUTHOR]

Clip-domain serine proteases (CLIPs) play crucial roles in insect development and innate immunity. In this study, we identified a CLIP gene (designated Ls CLIP3) from the cigarette beetle Lasioderma serricorne. Ls CLIP3 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 Ls CLIP3 was orthologous to the CLIP-B subfamily. Ls CLIP3 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 Ls CLIP3 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 Ls CLIP3 disrupted larval–pupal molting and specifically reduced the expression of genes in 20-hydroxyecdysone synthesis and signaling pathway. The chitin amounts of Ls CLIP3 RNAi larvae were greatly decreased, and expressions of six chitin metabolic-related genes were significantly reduced. Knockdown of Ls CLIP3 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 Ls CLIP3 is an important component of the larva to pupa molt and for the immunity of L. serricorne. [ABSTRACT FROM AUTHOR]

Abstract 3-Hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is an important rate-limiting enzyme in the mevalonate pathway, which is critical for the synthesis of juvenile hormone and insect development. In this study, a full-length cDNA encoding HMGR ( BdHMGR ) was cloned from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frame of BdHMGR encoded 928 amino acids and shared a high degree of identity with other known HMGRs. The expression levels of BdHMGR were high during the early and lately pupal stages, and were low at the larval and mid pupal stages. In the adult stages, the highest level of BdHMGR was detected on day-10 of the adult lifecycle, and the expression levels in females were greater than those in males. The highest tissue-specific expression was in the ovary, followed by fat body and other tissues. Injection of double-stranded RNA (dsRNA) of BdHMGR into adult females significantly reduced BdHMGR transcript levels, and inhibited ovarian development and affected the expression of two yolk protein genes. After dissection, we found that one or two of the spheroids of ovaries from the BdHMGR dsRNA-injected flies were amorphous and reduced in size as compared with the controls. The results suggest that BdHMGR is required for ovarian development in B. dorsalis and may be a potential target for pest control. Graphical abstract Unlabelled Image Highlights • A HMGR gene was identified in Bactrocera dorsalis. • BdHMGR was highly expressed in the pupal and adult stages. • BdHMGR was mainly distributed in the ovary. • BdHMGR is potentially involved in ovarian development. [ABSTRACT FROM AUTHOR]

Insulin signaling pathways have integral roles in regulating organ growth and body size of insects. Here, we identified and characterized six insulin signaling pathway components— InR , IRS , PI3K92E , PI3K21B , Akt , and PDK —from Bactrocera dorsalis . Quantitative real-time polymerase chain reaction was used to establish gene expression profiles for the insulin signaling pathway components for different developmental stages and tissues, and in response to 20-hydroxyecdysone (20E) and starvation. IRS , PI3K92E , and PI3K21B were highly expressed in the head, while InR , Akt , and PDK were most abundant in Malpighian tubules. Both IRS and PI3K92E were highly expressed during the larval–pupal and pupal–adult transition, while the remaining four genes were highly expressed only during the pupal–adult transition. Following initial exposure to 20E, the expression levels of most genes were significantly decreased. However, the expression levels of IRS , PI3K92E , and PI3K21B were significantly increased at 8 and 12 h post-treatment compared with the control. Moreover, we found that most insulin signaling pathway genes in B. dorsalis were up-regulated in response to starvation, but decreased when re-fed. On the contrary, transcript levels of PI3K21B decreased significantly during starvation. Furthermore, injection of IRS dsRNA into adult females significantly reduced IRS transcript levels. Suppression of IRS expression inhibited ovarian development, and the average ovary size was reduced by 33% compared with the control. This study provides new insight into the roles of insulin signaling pathway components in B. dorsalis , and demonstrates an important role for IRS in ovarian development. [ABSTRACT FROM AUTHOR]

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 deacetylase1 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. [ABSTRACT FROM AUTHOR]

β-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 β-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. [ABSTRACT FROM AUTHOR]

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 LsPGRP s 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. • RNAi suppression of four LsPGRPs increased the mortality of L. serricorne after Escherichia coli infection. • Silencing of LsPGRP-LE and LsPGRP-SC2 increased the mortality of larvae exposed to Staphylococcus aureus. • Four LsPGRPs regulated the expression of AMPs in the antibacterial defense response. [ABSTRACT FROM AUTHOR]

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. • Sixteen NR genes from L. serricorne were identified and characterized. • RNAi-aided knockdown of 15 NR genes disrupted the larva–pupa–adult transition and caused high mortality. • NRs are critical for L. serricorne molting by regulating 20E synthesis and chitin metabolism. [ABSTRACT FROM AUTHOR]

The forkhead box O transcription factor ( FoxO ) is an important downstream transcription factor in the well-conserved insulin signaling pathway, which regulates the body size and development of insects. In this study, the FoxO gene ( BdFoxO ) was identified from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frame of BdFoxO (2732 bp) encoded a 910 amino acid protein, and the sequence was well conserved with other insect species. The BdFoxO was highly expressed in larvae and pupae among different development stages, and the highest tissue-specific expression level was found in the fat bodies compared to the testis, ovary, head, thorax, midgut, and Malpighian tubules of adults. Interestingly, we found BdFoxO expression was also up-regulated by starvation, but down-regulated when re-fed. Moreover, the injection of BdFoxO double-stranded RNAs into third-instar larvae significantly reduced BdFoxO transcript levels, which in turn down-regulated the expression of other four genes in the insulin signaling pathway. The silencing of BdFoxO resulted in delayed pupation, and the insect body weight increased significantly compared with that of the control. These results suggested that BdFoxO plays an important role in body size and development in B. dorsalis . [ABSTRACT FROM AUTHOR]