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Understanding metabolic plasticity of animal evolution is a fundamental challenge in evolutionary biology. Owing to the diversification of insect wing morphology and dynamic energy requirements, the molecular adaptation mechanisms underlying the metabolic pathways in wing evolution remain largely unknown. This study reveals the pivotal role of the duplicated Apolipoprotein D (ApoD) gene in lipid and energy homeostasis in the lepidopteran wing. ApoD underwent significant expansion in insects, with gene duplication and consistent retention observed in Lepidoptera. Notably, duplicated ApoD2 was highly expressed in lepidopteran wings and encoded a unique C-terminal tail, conferring distinct ligand-binding properties. Using Bombyx mori as a model organism, we integrated evolutionary analysis, multiomics, and in vivo functional experiments to elucidate the way duplicated ApoD2 mediates lipid trafficking and homeostasis via the AMP-activated protein kinase pathway in wings. Moreover, we revealed the specific expression and functional divergence of duplicated ApoD as a key mechanism regulating lipid homeostasis in the lepidopteran wing. These findings highlight an evolutionary scenario in which neofunctionalization conferred a novel role of ApoD in shaping adaptive lipid metabolic regulatory networks during wing phenotypic evolution. Overall, we provide in vivo evidence for the functional differentiation of duplicate genes in shaping adaptive metabolic regulatory networks during phenotypic evolution. [ABSTRACT FROM AUTHOR]

Baculoviruses are virulent pathogens that infect a wide range of insects. They initiate infections via specific interactions between the structural proteins on the envelopes of occlusion-derived virions (ODVs) and the midgut cell surface receptors in hosts. However, host factors that are hijacked by baculoviruses for efficient infection remain largely unknown. In this study, we identified a membrane-associated protein sucrose hydrolase (BmSUH) as an ODV binding factor during Bombyx mori nucleopolyhedrovirus (BmNPV) primary infection. BmSUH was specifically expressed in the midgut microvilli where the ODV-midgut fusion happened. Knockout of BmSUH by CRISPR/Cas9 resulted in a significantly higher survival rate after BmNPV orally infection. Liquid chromatography-tandem mass spectrometry analysis and co-immunoprecipitation analysis demonstrated that PIF protein complex required for ODV binding could interact with BmSUH. Furthermore, fluorescence dequenching assay showed that the amount of ODV binding and fusion to the midgut decreased in BmSUH mutants compared to wild-type silkworm, suggesting the role of BmSUH as an ODV binding factor that mediates the ODV entry process. Based on a multilevel survey, the data showed that BmSUH acted as a host factor that facilitates BmNPV oral infection. More generally, this study indicated that disrupting essential protein-protein interactions required for baculovirus efficient entry may be broadly applicable to against viral infection. Author summary: Baculoviridae is a large family of pathogens that infect insects and frequently cause fatal diseases. Bombyx mori nucleopolyhedrovirus (BmNPV) is a major threat to the sericulture industry. Although we have learned a lot about baculoviruses over the past several decades, the detailed interaction patterns between host proteins and viral proteins that lead to infection remain underexplored. Here, we determined that BmSUH, a midgut microvilli protein, was required for the efficient oral infection of BmNPV. Our research suggests that BmSUH mediates the entry of occlusion-derived virions into the midgut epithelia by interacting with per os infectivity factors. According to the findings, inhibition of viral binding to host cells is an attractive strategy to prevent infection. This study provides an approach for preventing BmNPV infection through developing genetic resistance to viruses by using CRISPR/Cas9 system to abolish the host factors that are essential for viral entry. [ABSTRACT FROM AUTHOR]

Insects evolved adaptive plasticity to minimize the effects of the chemical defenses of their host plants. Nevertheless, the expressional response and adaptation of phytophagous specialists for long‐term adaption and short‐term response to host phytochemicals remains largely unexplored. The mulberry (Morus alba)–silkworm (Bombyx mori) interaction is an old and well‐known model of plant–insect interaction. In this study, we examined the long‐term adaption and short‐term response of the mulberry‐specialist silkworm to two sugar‐mimic alkaloids in mulberry: the commonly encountered 1‐deoxynojirimycin (1‐DNJ) and occasionally encountered 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol (D‐AB1), respectively. Global transcriptional patterns revealed that the physiological responses induced by the selective expression of genes involved in manifold cellular processes, including detoxification networks, canonical digestion processes, target enzymes, and other fundamental physiological processes, were crucial for regulating metabolic homeostasis. Comparative network analysis of the effects of exposure to D‐AB1 and 1‐DNJ supported the contention that B. mori produced similar and specific trajectories of changed gene expression in response to different sugar‐mimic alkaloids. D‐AB1 elicited a substantial proportion of downregulated genes relating to carbohydrate metabolism, catabolic process, lipid metabolism, and glycan biosynthesis and metabolism. This study dramatically expands our knowledge of the physiological adaptations to dietary sugar‐mimic alkaloid intake and uncovered both metabolic evolutionarily responses and unique adaptive mechanisms previously unknown in insects. [ABSTRACT FROM AUTHOR]

Dinotefuran, a third-generation neonicotinoid insecticide, is widely utilized in agriculture for pest control; however, its environmental consequences and risks to non-target organisms remain largely unknown. Bombyx mori is an economically important insect and a good toxic detector for environmental assessments. In this study, ultrastructure analysis showed that dinotefuran exposure caused an increase in autophagic vesicles in the silk gland. Dinotefuran exposure triggered elevated levels of oxidative stress in silk glands. Reactive oxygen species, oxidized glutathione disulfide, glutathione peroxidase, the activities of UDP glucuronosyl-transferase and carboxylesterase were induced in the middle silk gland, while malondialdehyde, reactive oxygen species, superoxide dismutase ， oxidized glutathione disulfide were increased in the posterior silk gland. Global transcription patterns revealed the physiological responses were induced by dinotefuran. Dinotefuran exposure substantially induced the expression levels of many genes involved in the mTOR and PI3K - Akt signaling pathways in the middle silk gland, whereas many differentially expressed genes involved in fatty acid and pyrimidine metabolism were found in the posterior silk gland. Additionally, functional, ultrastructural, and transcriptomic analysis indicate that dinotefuran exposure induced an increase of autophagy in the silk gland. This study illuminates the toxicity effects of dinotefuran exposure on silkworms and provides new insights into the underlying molecular toxicity mechanisms of dinotefuran to nontarget organisms. [Display omitted] • Dinotefuran exposure had toxic effects on silk glands of Bombyx mori. • Dinotefuran increased detoxification activity and caused oxidative stress damage in the silkworm. • Dinotefuran exposure induced autophagic vesicles in the silk gland. • Dinotefuran caused metabolic disorders and autophagy induction in the silk gland. [ABSTRACT FROM AUTHOR]

Although membrane-associated sucrase activity has been detected in the midgut of various lepidopteran species, it has not yet been identified and characterized at the molecular level. In the present study, we identified a novel sucrose hydrolase (SUH) gene from the following three bombycoid silkworms: Bombyx mori , Trilocha varians , and Samia cynthia ricini and named them BmSuh , TvSuh , and ScSuh , respectively. The EST dataset showed that BmSuh is one of the major glycoside hydrolase genes in the larval midgut of B. mori . These genes were almost exclusively expressed in the larval midgut in all three species, mainly at the feeding stage. SUHs are classified into the glycoside hydrolase family 13 and show significant homology to insect maltases. Enzymatic assays revealed that recombinant SUHs were distinct from conventional maltases and exhibited substrate specificity for sucrose. The recombinant BmSUH was less sensitive to sugar-mimic alkaloids than TvSUH and ScSUH, which may explain the reason why the sucrase activity in the B. mori midgut was less affected by the sugar-mimic alkaloids derived from mulberry. [ABSTRACT FROM AUTHOR]

Dinotefuran is a third-generation neonicotinoid pesticide and is increasingly used in agricultural production, which has adverse effects on nontarget organisms. However, the research on the impact of dinotefuran on nontarget organisms is still limited. Here the toxic effects of dinotefuran on an important economic species and a model lepidopteran insect, Bombyx mori , were investigated. Exposure to different doses of dinotefuran caused physiological disorders or death. Cytochrome P450, glutathione S-transferase, carboxylesterase, and UDP glycosyl-transferase activities were induced in the fat body at early stages after dinotefuran exposure. By contrast, only glutathione S-transferase activity was increased in the midgut. To overcome the lack of sensitivity of the biological assays at the individual organism level, RNA sequencing was performed to measure differential expressions of mRNA from silkworm larvae after dinotefuran exposure. Differential gene expression profiling revealed that various detoxification enzyme genes were significantly increased after dinotefuran exposure, which was consistent with the upregulation of the detoxifying enzyme. The global transcriptional pattern showed that the physiological responses induced by dinotefuran toxicity involved multiple cellular processes, including energy metabolism, oxidative stress, detoxification, and other fundamental physiological processes. Many metabolism processes, such as carbon metabolism, fatty acid biosynthesis, pyruvate metabolism, and the citrate cycle, were partially repressed in the midgut or fat body. Furthermore, dinotefuran significantly activated the MAPK/CREB, CncC/Keap1, PI3K/Akt, and Toll/IMD pathways. The links between physiological, biochemical toxicity and comparative transcriptomic analysis facilitated the systematic understanding of the integrated biological toxicity of dinotefuran. This study provides a holistic view of the toxicity and detoxification metabolism of dinotefuran in silkworm and other organisms. [Display omitted] • Dinotefuran significantly inhibited the growth and development of Bombyx mori. • P450, GST, CarE and UGT are involved in dinotefuran detoxification. • Dinotefuran inhibited fundamental metabolism and induced oxidative stress damage. • Dinotefuran affected MAPK/CREB, CncC/Keap1 and PI3K/Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Apolipoprotein D (ApoD) plays important roles in response to injury, cell differentiation, lifespan extension, and increasing stress resistance. However, the evolutionary mechanism of ApoD in insects remains largely unelucidated. We conducted a comprehensive study of the molecular evolution and functional divergence of ApoD in insects. A type I functional divergence analysis revealed significant differences among insect ApoD homologs, suggesting that they underwent functional divergence. We demonstrated that lepidopteran insects have three genes that are close homologs to ApoD and show divergences in sequence, expression pattern, and protein-protein interaction. Furthermore, positive selection was detected in lepidopteran ApoD2, and positively selected sites were located around the pocket and loop domains, which might result in conformational changes and affect binding properties. Moreover, we showed that the three ApoD s in Bombyx mori were significantly regulated by environmental stress. Thus, this work illustrates the dialectical relationship between genetic diversity and functional conservation of ApoD and highlights its unique functions in the stress response of Lepidoptera. • Lepidopteran insect ApoD genes have universally expanded via gene duplication. • Duplicated ApoD genes show divergence in sequence and exon/intron structure. • Three homologous ApoD genes show marked divergence in their expression. • Positive selection was detected in lepidopteran ApoD2 after gene duplication. [ABSTRACT FROM AUTHOR]

The widespread use of pesticides hampers the immune system of non-target organisms, however, there is a lack of common biomarkers to detect such effects. Myeloid differentiation primary response factor 88 (MyD88) is a crucial junction protein in the Toll-like receptor signaling pathway, which plays an important role in the inflammatory response. In this study, we investigated MyD88 as a potential biomarker for pesticide-induced stress. Phylogenetic analysis revealed that MyD88 was a conserved protein in the evolution of vertebrates and invertebrates. MyD88s usually have death domain (DD) and Toll/interleukin-1 receptor (TIR) domain. Bombyx mori (B. mori) is an important economic insect that is sensitive to toxic substances. We found microbial pesticides enhanced the expression level of MyD88 in B. mori. Transcriptome analysis demonstrated that MyD88 expression level was increased in the fatbody after dinotefuran exposure, a third-generation neonicotinoid pesticide. Moreover, the expression of MyD88 was upregulated in fatbody and midgut by imidacloprid, a first-generation neonicotinoid pesticide. Additionally, insect growth regulator (IGR) pesticides, such as methoprene and fenoxycarb, could induce MyD88 expression in the fatbody of B. mori. These results indicated that MyD88 is a potential biomarker for pesticide-induced stress in B. mori. This study provides novel insights into screening common biomarkers for multiple pesticide stresses and important implications for the development of more sustainable pest management strategies. [Display omitted] • The protein structure of MyD88 is conserved from vertebrates to invertebrates. • Many genes in the TLR pathway could be induced by dinotefuran in Bombyx mori. • MyD88 was induced by multiple chemical pesticides in the silkworm. • MyD88 is a potential biomarker for pesticides exposure. [ABSTRACT FROM AUTHOR]

Recent studies highlighted that apolipoprotein D (ApoD) and its homologs exert neuroprotective and antioxidant functions in mammals and Drosophila . Unlike mammals and Drosophila , lepidopteran insects possess three distinct ApoD homologs. However, few information on their functions in lepidopteran insects are available. In this study, we investigated the protective potential of a novel ApoD homolog, BmApoD1, in Bombyx mori . Quantitative PCR analyses demonstrated that BmApoD1 is extensively expressed at low levels during the larval stage but abundantly expressed in the testis during the pupal and adult stages. Tryptophan fluorescence titration demonstrated that recombinant BmApoD1 protein can bind retinoic acid and ergosterol. In addition, we provided evidence that N-linked glycans of BmApoD1 are essential to BmApoD1 secretion, and three residues, namely, Asp69, Asp104, and Asp196, are the glycosylation sites of BmApoD1. Furthermore, we showed that BmApoD1 is significantly up-regulated in the larvae after oxidant or starvation treatment. The recombinant BmApoD1 protein can protect cells from oxidative stress induced by H 2 O 2 and reduce actinomycin D-induced cell apoptosis. These observations, together with the transcriptional up-regulation of BmApoD1 in several tissues upon oxidative insult, identify BmApoD1 as a potent antioxidant. Our results demonstrate that BmApoD1 is critical for metabolic adaptation of B. mori to environmental challenges. [ABSTRACT FROM AUTHOR]

As a third-generation neonicotinoid insecticide, dinotefuran is extensively used in agriculture, and its residue in the environment has potential effects on nontarget organisms. However, the toxic effects of dinotefuran exposure on nontarget organism remain largely unknown. This study explored the toxic effects of sublethal dose of dinotefuran on Bombyx mori. Dinotefuran upregulated reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the midgut and fat body of B. mori. Transcriptional analysis revealed that the expression levels of many autophagy and apoptosis-associated genes were significantly altered after dinotefuran exposure, consistent with ultrastructural changes. Moreover, the expression levels of autophagy-related proteins (ATG8-PE and ATG6) and apoptosis-related proteins (BmDredd and BmICE) were increased, whereas the expression level of an autophagic key protein (sequestosome 1) was decreased in the dinotefuran-exposed group. These results indicate that dinotefuran exposure leads to oxidative stress, autophagy, and apoptosis in B. mori. In addition, its effect on the fat body was apparently greater than that on the midgut. In contrast, pretreatment with an autophagy inhibitor effectively downregulated the expression levels of ATG6 and BmDredd, but induced the expression of sequestosome 1, suggesting that dinotefuran-induced autophagy may promote apoptosis. This study reveals that ROS generation regulates the impact of dinotefuran on the crosstalk between autophagy and apoptosis, laying the foundation for studying cell death processes such as autophagy and apoptosis induced by pesticides. Furthermore, this study provides a comprehensive insight into the toxicity of dinotefuran on silkworm and contributes to the ecological risk assessment of dinotefuran in nontarget organisms. [Display omitted] • Dinotefuran exposure affected the ultrastructure and oxidative stress of Bombyx mori. • Dinotefuran affected many genes related to autophagy and apoptosis. • Autophagy induced by dinotefuran may facilitate apoptosis. • The fat body responded greater to dinotefuran than the midgut. [ABSTRACT FROM AUTHOR]