Insect Biochemistry and Molecular Biology最新文献

Txp40 is a ubiquitous, conserved, and novel toxin from Xenorhabdus and Photorhabdus bacteria, toxic to a wide range of insect pests. However, the three-dimensional structure and toxicity mechanism for Txp40 or any of its sequence homologs are not yet known. Here, we are reporting the crystal structure of the insecticidal protein Txp40 from Xenorhabdus nematophila at 2.08 Å resolution. The Txp40 was structurally distinct from currently known insecticidal proteins. Txp40 consists of two structurally different domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), primarily joined by a 33-residue long linker peptide. Txp40 displayed proteolytic propensity. Txp40 gets proteolyzed, removing the linker peptide, which is essential for proper crystal packing. NTD adopts a novel fold composed of nine amphipathic helices and has no shared sequence or structural homology to any known proteins. CTD has structural homology with RNases of type II toxin-antitoxin (TA) complex belonging to the RelE/ParE toxin domain superfamily. NTD and CTD were individually toxic to Galleria mellonella larvae. However, maximal toxicity was observed when both domains were present. Our results suggested that the Txp40 acts as a two-domain binary toxin, which is unique and different from any known binary toxins and insecticidal proteins. Txp40 is also unique because it belongs to the prokaryotic RelE/ParE toxin family with a toxic effect on eukaryotic organisms, in contrast to other members of the same family. Broad insect specificity and unique binary toxin complex formation make Txp40 a viable candidate to overcome the development of resistance in insect pests.

The binary toxin Cry48Aa1/Tpp49Aa1 produced by Lysinibacillus sphaericus exhibits potent toxicity against Culicidae larvae. Both Cry48Aa1 and Tpp49Aa1 toxins are crucial for binding to the toxin receptor in Culex quinquefasciatus larvae, albeit with different binding sites. Previous studies have identified Glu71, a membrane-bound α-glucosidase, as a putative binding protein for the Cry48Aa1 toxin, involved in the Cry48Aa1/Tpp49Aa1 toxicity. In this study, we employed pulldown assays to identify a group of Tpp49Aa1-binding proteins from C. quinquefasciatus solubilized midgut brush-border membrane proteins (BBMFs). RNA interference assays revealed that the silencing of an alkaline phosphatase gene (referred to as ALP1263) in C. quinquefasciatus resulted in a significant reduction in larval mortality upon exposure to Cry48Aa1/Tpp49Aa1 toxin in vivo. Furthermore, the ALP1263 protein exhibited specific and high-affinity binding to the Tpp49Aa1 toxin, with a dissociation constant (Kd) of approximately 57.3 nM. The dot blot analysis demonstrated that Tpp49Aa1 C-terminal region was essential for its interaction with the ALP1263 protein. In summary, our findings establish ALP1263 as a functional receptor for Tpp49Aa1 and emphasize its role in the toxicity of Cry48Aa1/Tpp49Aa1.

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most intriguing insect P450s in terms of structure and function. In Drosophila, CYP4G1 is highly expressed in the oenocytes and is the last enzyme in the biosynthesis of cuticular hydrocarbons, while CYP4G15 is expressed in the brain and is of unknown function. Both proteins have a CYP4G-specific and characteristic amino acid sequence insertion corresponding to a loop between the G and H helices whose function is unclear. Here we address these enigmatic structural and functional features of Drosophila CYP4Gs. First, we used reverse genetics to generate D. melanogaster strains in which all or part of the CYP4G-specific loop was removed from CYP4G1. We showed that the full loop was not needed for proper folding of the P450, but it is essential for function, and that just a short stretch of six amino acids is required for the enzyme’s ability to make hydrocarbons. Second, we confirmed by immunocytochemistry that CYP4G15 is expressed in the brain and showed that it is specifically associated with the cortex glia cell subtype. We then expressed CYP4G15 ectopically in oenocytes, revealing that it can produce of a blend of hydrocarbons, albeit to quantitatively lower levels resulting in only a partial rescue of CYP4G1 knockdown flies. The CYP4G1 structural variants studied here should facilitate the biochemical characterization of CYP4G enzymes. Our results also raise the question of the putative role of hydrocarbons and their synthesis by cortex glial cells.

The midgut is an important barrier against microorganism invasion and proliferation, yet is the first tissue encountered when a baculovirus naturally invades the host. However, only limited knowledge is available how different midgut cell types contribute to the immune response and the clearance or promotion of viral infection. Here, single-nucleus RNA sequencing (snRNA seq) was employed to analyze the responses of various cell subpopulations in the silkworm larval midgut to B. mori nucleopolyhedrovirus (BmNPV) infection. We identified 22 distinct clusters representing enteroendocrine cells (EEs), enterocytes (ECs), intestinal stem cells (ISCs), Goblet cell-like and muscle cell types in the BmNPV-infected and uninfected silkworm larvae midgut at 72 h post infection. Further, our results revealed that the strategies for immune escape of BmNPV in the midgut at the late stage of infection include (1) inhibiting the response of antiviral pathways; (2) inhibiting the expression of antiviral host factors; (3) stimulating expression levels of genes promoting BmNPV replication. These findings suggest that the midgut, as the first line of defense against the invasion of the baculovirus, has dual characteristics of "resistance" and "tolerance". Our single-cell dataset reveals the diversity of silkworm larval midgut cells, and the transcriptome analysis provides insights into the interaction between host and virus infection at the single-cell level.

β-Glucosidases play an important role in the chemical defense of many insects by hydrolyzing and thereby activating glucosylated pro-toxins that are either synthesized de novo or sequestered from the insect's diet. The horseradish flea beetle, Phyllotreta armoraciae, sequesters pro-toxic glucosinolates from its brassicaceous host plants and possesses endogenous β-thioglucosidase enzymes, known as myrosinases, for glucosinolate activation. Here, we identify three myrosinase genes in P. armoraciae (PaMyr) with distinct expression patterns during beetle ontogeny. By using RNA interference, we demonstrate that PaMyr1 is responsible for myrosinase activity in adults, whereas PaMyr2 is responsible for myrosinase activity in larvae. Compared to PaMyr1 and PaMyr2, PaMyr3 was only weakly expressed in our laboratory population, but may contribute to myrosinase activity in larvae. Silencing of PaMyr2 resulted in lower larval survival in a predation experiment and also reduced the breakdown of sequestered glucosinolates in uninjured larvae. This suggests that PaMyr2 is involved in both activated defense and the endogenous turnover of sequestered glucosinolates in P. armoraciae larvae. In activity assays with recombinant enzymes, PaMyr1 and PaMyr2 preferred different glucosinolates as substrates, which was consistent with the enzyme activities in crude protein extracts from adults and larvae, respectively. These differences were unexpected because larvae and adults sequester the same glucosinolates. Possible reasons for different myrosinase activities in Phyllotreta larvae and adults are discussed.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

Functional annotation is a critical step in the analysis of genomic data, as it provides insight into the function of individual genes and the pathways in which they participate. Currently, there is no consensus on the best computational approach for assigning functional annotation. This study compares three functional annotation methods (BLAST, eggNOG-Mapper, and InterProScan) in their ability to assign Gene Ontology terms in two species of Insecta with differing levels of annotation, Bombyx mori and Manduca sexta. The methods were compared for their annotation coverage, number of term assignments, term agreement and non-overlapping terms. Here we show that there are large discrepancies in gene ontology term assignment among the three computational methods, which could lead to confounding interpretations of data and non-comparable results. This study provide insight into the strengths and weaknesses of each computational method and highlight the need for more standardized methods of functional annotation.

ATP binding cassette (ABC) transporters are a diverse family of transmembrane proteins. Specific subfamily members expressed in the lepidopteran midgut can act as susceptibility determinants for several insecticidal Bt Cry proteins. However, the susceptibility determinants to many Cry toxins still remain unclear. Therefore, we knocked out a series of ABC transporters that are highly expressed in the midgut of Bombyx mori larvae by transcription activator-like effector nuclease (TALEN)-mediated gene editing, and the lineages that became resistant to Cry toxins were searched by toxin overlay bioassay. As a result, the B. mori ABC transporter subfamily B1 (BmABCB1) knockout lineage showed 19.17-fold resistance to Cry1Ba, 876.2-fold resistance to Cry1Ia, and 29.1-fold resistance to Cry9Da, suggesting that BmABCB1 is the determinant of susceptibility to these toxins. BmABCC2 and BmABCC3 have been shown to be susceptibility determinants based on their function as receptors. Therefore, we next heterologously expressed these ABC transporters in HEK293T cells and performed a cell swelling assay to examine whether these molecules could exert receptor functions. As a result, BmABCB1-expressing cells showed swelling response to Cry1Ia and Cry9Da, and cells expressing PxABCB1, which is the Plutella xylostella ortholog of BmABCB1, showed swelling for Cry1Ba, suggesting that ABCB1 is a susceptibility determinant by functioning as a receptor to these toxins. Furthermore, in order to clarify how high binding affinity is based on receptor function, we performed surface plasmon resonance analysis and found that each KD of Cry1Ba, Cry1Ia, and Cry9Da to BmABCB1 were 7.69 × 10⁻⁸ M, 2.19 × 10⁻⁹ M, and 4.17 × 10⁻⁶ M respectively.

We previously demonstrated that Aedes aegypti pyruvate kinase (AaPK) plays a key role in the regulation of both carbon and nitrogen metabolism in mosquitoes. To further elucidate whether AaPK can be post-translationally regulated by Ae. aegypti sirtuin 2 (AaSirt2), an NAD⁺-dependent deacetylase that catalyzes the removal of acetyl groups from acetylated lysine residues, we conducted a series of analysis in non-starved and starved female mosquitoes. Transcriptional and protein profiles of AaSirt2, analyzed by qPCR and western blots, indicated that the AaSirt2 is differentially modulated in response to sugar or blood feeding in mosquito tissues dissected at different times during the first gonotrophic cycle. We also found that AaSirt2 is localized in both cytosolic and mitochondrial cellular compartments of fat body and thorax. Multiple lysine-acetylated proteins were detected by western blotting in both cellular compartments. Furthermore, western blotting of immunoprecipitated proteins provided evidence that AaPK is lysine-acetylated and bound with AaSirt2 in the cytosolic fractions of fat body and thorax from non-starved and starved females. In correlation with these results, we also discovered that RNAi-mediated knockdown of AaSirt2 in the fat body of starved females significantly decreased AaPK protein abundance. Notably, survivorship of AaSirt2-deficient females maintained under four different nutritional regimens was not significantly affected. Taken together, our data reveal that AaPK is post-translationally regulated by AaSirt2.

The midgut of Zabrotes subfasciatus (Coleoptera) and other insects may have regions lacking a peritrophic membrane (matrix, PM) and covered with a jelly-like material known as peritrophic gel. This work was undertaken to test the hypothesis that the peritrophic gel is a vertebrate-like mucus. By histochemistry we identified mucins along the whole midgut, which contrasts with the known occurrence of PM only at the posterior midgut. We also analyzed the expression of the genes coding for mucus-forming mucins (Mf-mucins), peritrophins, chitin synthases and chitin deacetylases along the midgut and carcass (insect without midgut) by RNA-seq. Mf-mucins were identified as proteins with high O-glycosylation and multiple tandem repeats of Pro/Thr/Ser residues. Peritrophins were separated into PM proteins, cuticular proteins analogous to peritrophins (CPAPs) and ubiquitous-chitin-binding domain-(CBD)-containing proteins (UCBPs). PM proteins have at least 3, CPAP one or 3, and UCBPs have a varied number of CBDs. PM proteins are more expressed at midgut, CPAP at the carcass, and UCBP at both. The results showed that most PM proteins are mainly expressed at the posterior midgut, together with midgut chitin synthase and chitin deacetylase, and in agreement with the presence of PM only at the posterior midgut by visual inspection. The excretion of most midgut chitinase is avoided, suggesting that the shortened PM is functional. Mf-mucins are expressed along the whole midgut, probably forming the extracellular mucus layer observed by histochemistry. Thus, the lack of PM at anterior and middle midgut causes the exposure of a mucus, which may correspond to the previously described peritrophic gel. The putative functional interplay of mucus and PM is discussed. The major role of mucus is proposed to be tissue protection and of PM to enhancing digestive efficiency by allowing enzyme recycling.