【Aim】 Aminopeptidases N (APNs) are a class of important proteases in the digestive system in insects. This study aims to verify the expression of two apn genes (nlapn1 and nlapn4) with high transcription level in the midgut epithelium of the brown planthopper, Nilaparvata lugens, and to identify and analyze the characteristics of their proteins. 【Methods】 Phylogenetic analysis of both NLAPN1 and NLAPN4 of N. lugens was conducted by maximum likelihood method. Western blotting and LC-ESI-MS/MS were respectively conducted to localize and identify NLAPN1 and NLAPN4 in the midgut brush border membrane vesicles (BBMVs) of N. lugens. NLAPN1 and NLAPN4 were respectively expressed in Drosophila S2 cells. Lacolization of both NLAPN1 and NLAPN4 in S2 cells was analyzed by Western blot and immunofluorescence. The enzymatic activities of NLAPN1 and NLAPN4 were determined through enzyme assays using leucine-p-NA, Ala-p-NA, Met-p-NA and Lys-p-NA as the substrate, respectively. 【Results】 Phylogenetic tree analysis showed that both NLAPN1 and NLAPN4 of N. lugens were clustered together with the APN proteins highly expressed in the midgut of other hemipteran insects. NLAPN1 and NLAPN4 with the molecular weight of ~160 kD were identified in the midgut BBMV of N. lugens by Western blot and LC-ESI-MS/MS. Western blot and immunofluorescence analysis showed that NLAPN1 and NLAPN4 were expressed on the cytomembrane of transfected S2 cells, while that of NLAPN4lackG without glycosylphosphatidylinositol (GPI) anchor site at the C-terminal end was distributed in the cytoplasm. Enzyme assay results revealed that both NLAPN1 and NLAPN4 showed certain enzymatic activity using Ala-p-NA and Lys-p-NA as the substrate, while using leucine-p-NA as the substrate, NLAPN1 showed extremely high enzymatic activity (＞60 U/mg). 【Conclusion】 NLAPN1 and NLAPN4 are both highly expressed GPI-anchored membrane-bound aminopeptidases N located on the epithelial membrane of the midgut of N. lugens. Both NLAPN1 and NLAPN4 show similar structure and enzymatic characteristics to the previous identified membrane-bound APN proteins in lepidotperans, coleopterans and dipterans. Physiological and biochemical functions of membrane-bound APNs in the midgut of N. lugens and their interaction with exogenous pathogenic microorganisms need to be further studied.

【Aim】 This study aims to further understand the immune defense mechanism of Plutella xylostella and to provide new ideas for the biological control of P. xylostella by studying the function of lysozyme in P. xylostella. 【Methods】 RACE technique was used to clone lysozyme gene of P. xylostella. The prokaryotic expression vector pET-29a-Pxlys was constructed. The recombinant Pxlys was expressed by prokaryotic expression system and purified by nickel column affinity chromatography. The antibacterial activities of the recombinant Pxlys against Corynebacterium stationis, Micrococcus luteus, Staphyloccocus aureus, Escherichia coli, Shigella sp., Salmonella sp. and Bacillus thuringiensis were detected by Oxford cup method, and the lysis characteristics of the recombinant Pxlys against C. stationis and E. coli were observed under scanning electron microscope. 【Results】 The 423 bp ORF sequence of lysozyme gene Pxlys (GenBank accession number: MN702780) of P. xylostella was obtained by cloning. It encodes 140 aa with the relative molecular weight of 15.79 kD. The antibacterial activity test showed that the recombinant Pxlys had strong antibacterial activities not only against Gram-positive bacteria C. stationis, M. luteus and S. aureus, with the inhibition zone diameter of 20.0±1.1, 19.0±0.5 and 16.5±0.5 mm, respectively, but also against Gram-negative bacteria E. coli, Shigella sp. and Salmonella sp., with the inhibition zone diameter of 16.3±0.5, 15.0±0.5 and 14.0±1.1 mm, respectively. The recombinant Pxlys showed stronger antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. Besides, the recombinant Pxlys also showed the bactericidal activity against B. thuringiensis. Under scanning electron microscope, the lysis characteristics of the recombinant Pxlys against C. stationis and E. coli were different. 【Conclusion】 Pxlys has a broad-spectrum antimicrobial activity, and may have different antibacterial mechanisms against Gram-positive bacteria and Gramnegative bacteria. The results provide the basis for further understanding the immune defense system of P. xylostella. 

【Aim】 This study aims to explore the role of heat shock protein 70 (HSP70) genes in the process of resisting high temperature stress and to provide a theoretical basis for revealing the expansion mechanism of Hyphantria cunea and predicting its potential distribution area. 【Methods】 HSP70 genes of H. cunea were cloned by PCR and subjected to bioinformatical analysis. The expression characteristics of HSP70 genes in the day-2 4th instar newly molted larvae of H. cunea under 25, 30, 35 and 40℃ were detected by qPCR. The prokaryotic expression vector of HSP70 of H. cunea was constructed and induced to express in Escherichia coli BL21. The protein was purified by Ni²⁺-His column and verified by Western blot. Then, the ATPase activity of the recombinant protein obtained by prokaryotic expression was determined by in vitro experiments. 【Results】 The two HSP70 genes of H. cunea including HcHSP70 (GenBank accession no.: MT995848) and HcHSC70 (GenBank accession no.: MT261583) were cloned and sequenced. Their ORFs are 1 917 and 2 061 bp in length, encoding 637 and 687 amino acids with the predicted molecular weights of about 69.66 and 74.96 kD, and the isoelectric points of 5.90 and 5.96, respectively. The structure prediction conformed to the characteristics of the heat shock protein 70 family, which contains three highly conserved regions GIDLGTTYS, IFDLGGGTFDVSIL, and VGGSTRIPKVQ. The 3D structure of the two HSP70 proteins is composed of the N-terminal ATPase functional domain and C-terminal substrate binding domain. The phylogenetic tree showed that HcHSP70 and other members of the HSP70 family of Lepidoptera were clustered into one branch, while HcHSC70 and other members of the HSC70 family of Lepidoptera were clustered into another branch. The qPCR results showed that the expression of HcHSP70 in the day-2 4th instar newly molted larvae of H. cunea was significantly upregulated under heat stress and reached the peak under 35℃ for 2 h, while HcHSC70 had a weak expression response under heat stress. The prokaryotic expression vector of HcHSP70 was successfully constructed, and HcHSP70 was expressed in vitro. The purified recombinant protein HcHSP70 had ATPase activity, which was stable under high temperature stress. 【Conclusion】 In this study, HcHSP70 and HcHSC70 of H. cunea were cloned, and their expression characteristics under high temperature were confirmed. The prokaryotic expression and purification of HcHSP70 were successfully performed. The recombinant HcHSP70 has stable ATPase activity under high temperature, suggesting that it may play an important role in the response of H. cunea to high temperature stress.

【Aim】 To identify the expression patterns of caspase in Spodoptera exigua induced by apoptosis inducer and under pathogenic microorganism stress, so as to lay a foundation for further study on the mechanism of apoptosis in Lepidoptera insects. 【Methods】 RT-PCR was used to amplify the full-length coding regions of two caspase genes (SeCasp-3 and SeCasp-4) from the 3rd instar larvae of S. exigua. qPCR was used to detect the expression patterns of the two SeCasp genes in the fat body cells of S. exigua after induction by apoptosis inducers hydrogen peroxide (H₂O₂) (100 μmol/L), actinomycin D (ActD) (10 μg/mL) and dexamethasone (DEX) (50 μg/mL), and in the 3rd instar larvae of S. exigua infected by pathogens Bacillus thuringiensis kurstaki (10⁸ colonies/mL), Escherichia coli TG1 (10⁸ colonies/mL), Heliothis virescens ascovirus 3h (HvAV-3h) (1.16×10¹¹genome copies/mL) and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (5 000 OBs/μL). 【Results】 The coding regions of SeCasp-3 (GenBank accession no.: MW183334) and SeCasp-4 (GenBank accession no.: MW183335) are 942 and 843 bp in length, encoding 313 and 280 amino acids, respectively. The putative protein sequences of SeCasp-3 and SeCasp-4 show 45.54% and 58.46% amino acid sequence identities with Dronc of Bombyx mori, respectively, and SeCasp-3 and SeCasp-4 show high homology. SeCasp-3 and SeCasp-4 exhibited different expression patterns in the fat body cells of S. exigua induced by different chemicals. The relative expression levels of both SeCasp-3 and SeCasp-4 in the fat body cells treated by 100 μmol/L H₂O₂ and 10 μg/mL ActD for 24 and 48 h were significantly up-regulated. In the fat body cells of S. exigua treated by 50 μg/mL DEX for 24 and 48 h, the relative expression level of SeCasp-3 showed no significant change, but that of SeCasp-4 was significantly enhanced by several thousand folds as compared to the control. The expression patterns of SeCasp-3 and SeCasp-4 in the 3rd instar larvae of S. exigua infected by different pathogens were basically similar. The general linear model analysis showed that the infection of B. thuringiensis kurstaki and E. coli TG1 caused no significant change in the expression levels of SeCasp-3 and SeCasp-4 in the 3rd instar larvae of S. exigua, while the infection of HvAV-3h and AcMNPV significantly inhibited the expression of the two genes. 【Conclusion】 Two S. exigua caspase genes were identified and their expression responses to apoptosis inducers and pathogenic microorganism infection were assayed in this study, laying a foundation for further exploring the function of caspase and process of insect apoptosis.

【Aim】 This study aims to analyze the effect of imidacloprid treatment on the olfactory learning behavior and the gene transcription in the brain of Apis mellifera ligustica so as to provide evidence for the negative effects of neonicotinoid insecticides on honeybees. 【Methods】 Under laboratory conditions, A. m. ligustica adult workers were fed with 50% sucrose solution containing 4 ng imidacloprid at one time, with those fed with 50% sucrose solution without imidacloprid as the control, and its effect on the olfactory learning behavior of A. m. ligustica adult workers was measured via proboscis extension response (PER) behavior test. Total RNA was extracted from the brain of A. m. ligustica workers tested above for RNA-Seq sequencing and bioinformatics analysis. To verify the RNA-Seq sequencing results, the expression levels of six selected differentially expressed genes (DEGs) in the brain of A. m. ligustica adult workers were detected by real-time fluorescent quantitative PCR. 【Results】 The olfactory learning ability of A. m. ligustica adult workers fed with 50% sucrose solution containing 4 ng imidacloprid was significantly decreased as compared to the control group (fed with 50% sucrose solution). RNA-Seq sequencing results showed that there were 123 DEGs [adjusted P-value (padj)<0.05］ between the treatment group and the control group, including 82 down-regulated DEGs and 41 up-regulated DEGs. GO enrichment analysis revealed that the down-regulated DEGs were mainly enriched in S-adenosylmethionine-dependent methyltransferase activity, acid phosphatase activity, oxidoreductase activity, and protein heterodimerization activity. The up-regulated DEGs were mainly enriched in functional items such as transmembrane receptor activity, molecular transducer activity, and neurological system processes. KEGG enrichment analysis showed that the down-regulated DEGs were mainly enriched in such organelles as ribosome and lysosome, metabolism pathways like carbon metabolism and tryptophan metabolism, and Toll and IMD signaling pathways, while the up-regulated DEGs were not enriched in KEGG pathways. Real-time fluorescent quantitative PCR results showed that the relative expression levels of the six DEGs tested showed the same trend with the RNA-Seq sequencing results of FPKM (fragments per kilobase million) value, verifying the reliability of the sequencing results. 【Conclusion】 Exposure of sublethal dose of imidacloprid significantly reduces the olfactory learning ability of A. m. ligustica adult workers, and also affects the expression of immune and detoxification related genes, enzyme activity, redox and other biological metabolic processes in the brain of A. m. ligustica. Short-term stress of sublethal dose of imidacloprid can stimulate the olfactory sensory process and nerve signal transduction process of A. m. ligustica.

【Aim】 The study aims to establish a suitable olfactory behavior assay system for small insects such as Drosophila melanogaster based on trajectory tracking in four-quadrant olfactometer by analyzing the olfactory behavior of D. melanogaster under different experimental conditions. 【Methods】 The movement trajectory of D. melanogaster in the four-quadrant olfactometer was tracked, and the residence time and moving speed within each field arena was analyzed using Ethovision XT software. Meanwhile, the effects of airflow velocity and ordor placement on the attraction of apple vinegar and the repellence of Eucalyptus globulus leaf oil to D. melanogaster were investigated. 【Results】 The airflow velocity suitable for olfactory behavior analysis of D. melanogaster was from 300 to 1 200 mL/min, and 300 mL/min airflow was recommended as the optimal airflow velocity at which the moving speed of D. melanogaster individuals was more uniform. Ordor placement had no effect on the olfactory behavior of D. melanogaster, but had some impact on the attraction index of ordor. Single ordor showed better efficiency. Meanwhile, the efficiency of single individual test was better than that of multiple individual test. 【Conclusion】 The olfactory behavior assay system based on trajectory tracking developed in this study can be used for screening and identifying effective components in semiochemical compounds of insects. Better efficiency can be obtained by testing each insect individually with a suitable airflow velocity and single ordor. Multiple individual test could be used in the primary screening of compounds.

 【Aim】 The darkling beetle plays an important role in maintaining biodiversity and ecosystem function in desert semi-desert habitats. This study aims to reveal the niche and interspecific associations of darkling beetles so as to lay a basis for studying the mechanism of community construction in desert grasslands of alluvial fans. 【Methods】 A field survey was carried out to investigate darkling beetle communities using pitfall in a desert grassland of alluvial fans in Helan Mountain, northwestern China from May to October 2019. The sampling transect of about 200 m×200 m was divided equally into 100 square plots. Based on 2×2 contingency table analysis, niche breadth, niche overlap, variance ratio test, Chi-square test, association coefficient (AC), percentage of cooccurrence (PC), Ochiai index (OI), Dice index (DI) and Spearman rank correlation coefficient, we analyzed the niche and interspecific association of darkling beetle species at a small scale. 【Results】 A total of 1 086 individuals of adult darkling beetles belonging to 10 species from 7 genera were collected. Microdera kraatzi kraatzi, Anatolica pandaroides and Anatolica planata were the dominant species in the whole community. The spatial niche breadth of A. planata was the largest, the temporal niche breadth of Scytosoma pygmaeum was the largest and the temporal-spatial niche breadth of M. kraatzi kraatzi was the largest. The temporal, spatial and temporal-spatial niche breadth of Opatrum subaratum was the smallest, which was at a weak competitive position. The results of niche breadth clustering showed that M. kraatzi kraatzi, A. pandaroides, S. pygmaeum and S. dissilimarginis are wide niche species. The temporal niche overlaps of species pairs of darkling beetles were more significant than their spatial niche overlaps. The variance ratio and W test results showed that there existed a significantly positive interspecific association. The results of the Chi-square test, association coefficient (AC), and the Spearman rank correlation coefficient showed that the ratio of species pairs of the positive and negative associations was greater than 1 and the overall correlation tended to be positive. The percentage of co-occurrence (PC), Ochiai index and Dice index indicated that there was a significant positive correlation among the dominant species M. kraatzi kraatzi, A. pandaroides and A. planata. According to constellation diagram of the Spearman rank correlation coefficient network, the 10 species were divided into three ecological species groups. 【Conclusion】 The dominant species have a wide niche, the temporal niche and spatial niche of species vary inconsistently, and the niche overlaps of species pairs with significant positive connection are also relatively higher. Clustering of ecological species groups reflects the differences in the ecological adaptability of species pairs. The results provide references for studying the succession of darkling beetle community in desert grasslands of alluvial fans.

【Aim】 Raspy crickets (Orthoptera: Gryllacrididae) are a unique group in the Orthoptera, and they produce silk and use it to build shelters. The purpose of this study is to investigate the structural characteristics of their silk glands. 【Methods】 The fine structure and ultrastucture of silk glands of Capnogryllacris nigromarginata were observed by anatomy observation, immunofluorescence, hematoxylin-eosin staining, PAS-hematoxylin staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). 【Results】 The silk glands of C. nigromarginata are composed of acini and silk ducts. Each acinus is composed of a fibrous sheath enclosing four main types of cells: type Ⅰ secretory cells, type Ⅱ secretory cells, peripheral cells and canal cells. Type Ⅰ and Ⅱ secretory cells are large glandular cells in irregular shape. The secretory cells have large nuclei. The cytoplasm of secretory cells is characterized by containing abundant endoplasmic reticulum and secretory particles. Type Ⅰ secretory cells are near the center of acinus. PAS-hematoxylin staining showed that type Ⅰ secretory cells contain glycoprotein. Type Ⅱ secretory cells are at the peripheral region of acinus located between type Ⅰ secretory cells and peripheral cells or sheath cells. The canal cells are scattered between the secretory cells and form the extracellular transport canal of secretion. In contact with the sheath cells, peripheral cells have microvilli cavity formed by cell membrane invagination, and there are a large number of mitochondria in the cytoplasm. The microvilli cavity is connected to an extracellular canal surrounded by canal cells. Secretory particles are accumulated at the junction of the secretory cells and the extracellular transport canal. Then they discharge secretions to the extracellular transport canals. The extracellular canals of multiple acini converge to the silk duct composed of a single layer of cells. The cell periphery of the silk duct is involved in the organization of a series of deep invaginations of the plasma membrane. A large number of elongated mitochondria can be observed around the cell plasma membrane invaginations. The apical border of the silk duct cell near the inner lumen has continuous membrane processes that are closely aligned under the cuticle of the duct wall. 【Conclusion】 The secretory cells of silk glands of C. nigromarginata can be divided into type Ⅰ and type Ⅱ secretory cells. The production and secretion process of secretory materials in turn pass through secretory cells, extracellular canals of canal cells, branch ducts, common duct of silk glands, and salivarium. When the secretions are transported outward from the extracellular canal surrounded by the canal cells, the microfilaments in the microvilli cavity of peripheral cells may provide impetus for the excretion of secretions.

There are only one or two voltage-gated sodium channel α subunit genes in insects, but the two post-transcriptional modifications, alternative splicing and RNA editing, confer the functional diversity of insect sodium channels. The insect β accessory subunits, TipE and TEH1-4, also play important roles in the expression and regulation of sodium ion channels. Voltagegated sodium channel plays an important role in the generation and transmission of action potential and is the target site of many natural and synthetic neurotoxins and insecticides, including the pyrethroids, indoxacarb and metaflumizone. Pyrethroids can prolong the transmembrane sodium ion flow by controlling the inactivation and deactivation of sodium channels in insects, causing neuroexcitatory conduction disorders. Indoxacarb and metaflumizone block the neuronal action potential in the central and peripheral nervous system of insects. These neural agents can disturb the normal function of sodium channels in insects. Two pyrethroid binding sites have been commonly identified in sodium channels of insects, but sodium channels of different species have differences in binding sites for pyrethroids. Therefore, in this article we reviewed insect sodium channels and their interaction with insecticides, hoping to promote the research of insect nerve receptors and to provide important references for identification of mutations associated with resistance and development of effective insecticides.

Eusociality is a major characteristic of ants (Formicidae), with apparent division of labor among individuals. They use complex cooperative strategies to protect their nests from predators, pathogenic microorganisms, and ant competitors, and to capture preys. Myrmicinae is the largest group of Formicidae, with 147 living genera. They mainly spray alkaloid-rich venom secretions for defense and hunting. In this article, the composition of defensive alkaloids of Myrmicinae ants and their distribution characteristics in different genera and species were reviewed, whose chemical structure mainly includes piperidine, pyridine, pyrrole, indolizidine, pyrrolizidine and fatty amine, and their function and application were summarized and prospected. Piperidine alkaloids are the typical characteristics of the venom of Solenopsis species, while pyrrolidine, pyrroline, and pyrrolizidine alkaloids predominate in the venom of Monomorium species. Indolizidine alkaloids are the major components of the venom secretion of Myrmicaria ants and Solenopsis thief ants. In addition, fatty amines are also components of the venom of Monomorium species. These venom alkaloids possess a variety of biological activities and have great application value in development of pesticides and biomedicines.