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  • Monthly, Founded in 1950
    Supervisor:Chinese Academy of Sciences
    Sponsor:Institute of Zoology,Chinese Academy of Sciences
    The Entomological Society of China
    Domestic postal code: 2-153
    Foreign issuance code: Q61
    ISSN 0454-6296
    CN 11-1832/Q
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Response of miRNAs related to wing differentiation and their predicted target genes to ecdysone and the confirmation of target gene of miR-92a-1-p5 in the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphidae)
MA Tian-Tian, YANG Zong-Lin, CHANG Mei-Ling, HUO Chun-Yue, KAN Yun-Chao, LI Dan-Dan
Acta Entomologica Sinica    2021, 64 (4): 419-427.   DOI: 10.16380/j.kcxb.2021.04.001
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 Abstract: 【Aim】 Ecdysone plays important roles in wing dimorphism of the parthenogenetic aphid. In the previous study we found that five microRNAs (miRNAs) also play pivotal roles in the wing dimorphism of the pea aphid, Acyrthosiphon pisum, but whether ecdysone and miRNAs have interaction in wing differentiation of aphids is unclear. This study aims to explore the effect of ecdysone on the expression of miRNAs and their predicted target genes, and to reveal the interaction of ecdysone and miRNAs in wing differentiation of aphids. 【Methods】 The five miRNAs (Let-7, miR-92a, miR-92b, miR-92a-1-p5 and miR-277) related to the wing dimorphism of A. pisum were selected. The 2nd instar nymphs of parthenogenetic A. pisum were exposed to 0.1 mol/L ecdysone analog 20-hydroxyecdysone (20E) for 10 min and 30 min, respectively, and sampled at 48 h after treatment. The expression levels of the five miRNAs and their predicted target genes after 20E treatment were detected by qPCR. The predicted target gene of miR-92a-1-p5, flightin, was verified by dual luciferase activity assay. Finally, the expression of miR-92a-1-p5 in the 4th instar nymphs of parthenogenetic A. pisum was knocked down with nanocarrier/detergent to verify the interaction between miR-92a-1-p5 and its predicted target gene flightin. 【Results】 The expression of the five miRNAs in the 2nd instar nymphs of parthenogenetic A. pisum could be extremely significantly induced by the treatment of 0.1 mol/L 20E for 30 min. But when the 2nd instar nymphs were exposed to 0.1 mol/L 20E for 10 min, the expression levels of miR-92a-1-p5 and miR-92b extremely significantly decreased, while those of Let-7 and miR-277 increased extremely significantly compared with the control. The expression trends of Let-7 and its predicted target gene abrupt were opposite in the 2nd instar nymphs of parthenogenetic A. pisum after 20E treatment. The expression levels of wingless and Uba1, which are the predicted target genes of miR-92a and miR-277, respectively, decreased extremely significantly in the 2nd instar nymphs of parthenogenetic A. pisum exposed to 0.1 mol/L 20E for 10 min, showing the opposite trend with those of the corresponding two miRNAs. The expression level of flightin, the predicted target gene of miR92a-1-p5, decreased extremely significantly in the 2nd instar nymphs of parthenogenetic A. pisum exposed to 0.1 mol/L 20E for 30 min, exhibiting an opposite expression trend to that of miR-92a-1-p5. Dual luciferase activity assay results showed that after co-transfection of the mimics of miR-92a-1-p5 and the flightin CDS overexpression vector pmirGlO [flightin] the luciferase activity was extremely significantly decreased by 40% compared to the control transfected with NC mimics. Knocking down the expression of miR-92a-1-p5 in the 4th instar nymphs of parthenogenetic A. pisum extremely significantly decreased its expression by 83%, while extremely significantly enhanced the expression of its predicted target gene flightin by 48%, confirming that flightin is the target gene of miR-92a-1-p5. 【Conclusion】 Ecdysone can induce the expression of miRNAs in A. pisum. miR-92a1-p5 may be involved in wing differentiation of parthenogenetic aphids by regulating flightin gene. Nanocarrier/detergent can achieve effective miRNA interference in aphids. This study lays a foundation for further exploring the interaction of ecdysone and miRNAs in wing differentiation of parthenogenetic aphids.
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Research progress of olfactory binding proteins in insects 
WU Fan, ZHANG Li, QIU Yi-Lei, LI Hong-Liang
Acta Entomologica Sinica    2021, 64 (4): 523-535.   DOI: 10.16380/j.kcxb.2021.04.011
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 As the first participant in the olfactory system, olfactory binding proteins are mainly expressed in the hemolymph of peripheral olfactory system and play roles in recognizing, binding and transporting odors or pheromones to odor receptors. In recent years, with the application of novel modern biological technology, more olfactory binding proteins have been identified, and their diverse functions have been revealed. In this article, we summarized the research progress in the molecular characteristics, structure, functions and application of olfactory binding proteins in recent years. Generally, olfactory binding proteins are classified into three families, odorantbinding proteins (OBPs), chemosensory proteins (CSPs) and Niemann-Pick type C2 proteins (NPC2). Based on the α-helix and β-sheet, olfactory binding proteins form stable globular structure, which is beneficial for them to adapt to a variety of environments and tasks. And diverse functions of olfactory binding proteins are particularly important for insect physiology and behavior. Nowadays, olfactory binding proteins are used in biological control, variety selection and breeding, biosensor making, and so on. This review provides references and some new ideas for further research on olfactory binding proteins in insects.
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Cloning and functional analysis of salivary protein gene Sm13498 of the grain aphid, Sitobion miscanthi (Hemiptera: Aphididae)
FU Yu, WANG Qian, ZHANG Yong, CHEN Ju-Lian
Acta Entomologica Sinica    2021, 64 (9): 1009-1019.   DOI: 10.16380/j.kcxb.2021.09.001
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【Aim】 The grain aphid, Sitobion miscanthi, is a dominant cereal aphid species in the major growing areas of wheat (Triticum aestivum) of China. Sm13498 is a salivary protein specifically expressed in the salivary glands of S. miscanthi. This study aims to investigate the potential role of the functionally unknown salivary protein Sm13498 of S. miscanthi in modulating plant defense. 【Methods】 Based on the sequencing data of the salivary gland transcriptome of S. miscanthi, the full-length cDNA sequence of Sm13498 gene was cloned by PCR and analyzed by bioinformatics. The expression levels of Sm13498 in apterous adults of S. miscanthi feeding on T. aestivum leaves for different time were determined by RT-qPCR. The secretion function of the signal peptide of Sm13498 was verified by yeast secretory system. The function of Sm13498 and its subcellular localization in Nicotiana benthamiana was examined using Agrobacterium tumefaciens-mediated transient expression technique. 【Results】 The full-length cDNA sequence of Sm13498 of S. miscanthi was cloned (GenBank accession no.: MW346655). Its open reading frame (ORF) is 783 bp in length, encoding 260 amino acids with the predicted molecular weight of 28.01 kD and amino acids 1-22 predicted to be N-terminal signal peptide. Phylogenetic analysis showed that Sm13498 was most closely related to LOC100159087 precursor, an uncharacterized protein of Acyrthosiphon pisum, deposited in GenBank under the accession no. NP_0013135481, sharing 71.7% amino acid sequence identity. The RT-qPCR results revealed that the expression level of Sm13498 reached the peak in apterous adults of S. miscanthi feeding on wheat leaves for 12 h. Saccharomyces cerevisiae strain YTK12 containing the signal peptide fragment of Sm13498 grew normally on the YPRAA medium in the yeast secretion system, and catalyzed the conversion of colorless 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble dark-red-colored triphenylformazan (TTF), confirming the secretion activity of the predicted signal peptide. The transiently expressed Sm13498 in N. benthamiana mediated by A. tumefaciens could suppress the programmed cell death induced by Bcl-2-associated X protein (BAX) and pathogen elicitor INF1. Subcellular localization results indicated that the fusion protein  Sm13498-GFP was localized in the cytomembrane of N. benthamiana leaves. 【Conclusion】 The results suggest that the salivary protein Sm13498 of S. miscanthi may be involved in the suppression of plant defense responses. This study lays a foundation for identifying the salivary effectors of S. miscanthi and understanding the high adaptability of wheat aphids to wheat varieties.
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Knockout of ebony gene leads to melanin pigmentation in the rice stem borer, Chilo suppressalis (Lepidoptera: Crambidae) (In English)
SUN Hao, HUANG Jing-Mei, LIU Yan, GE Wen-Chao, WANG Shuai, YANG Feng-Xia, GAO Cong-Fen, WU Shun-Fan
Acta Entomologica Sinica    2021, 64 (12): 1367-1376.   DOI: 10.16380/j.kcxb.2021.12.002
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【Aim】 The rice stem borer, Chilo suppressalis, is a destructive rice pest in China and other Asian countries. However, due to lack of genetic tools, the functional genomic studies in C. suppressalis are seriously constrained. The aim of the study is to use a marker gene, ebony, to establish a gene editing system based on CRISPR/Cas9 technology in C. suppressalis. 【Methods】 With the amino acid sequences of Bombyx mori ebony protein as a query, the putative C. suppressalis ebony gene was obtained on its genomic database by the TBLASTN program. The full-length cDNA of ebony gene of C. suppressalis was cloned by PCR and subjected to bioinformatical analysis. The expression patterns of Csebony at different developmental stages (egg, larval, pre-pupal, pupal, and male and female adult stages) and in multiple tissues (head, epidermis, fat body, gut, and Malpighian tubules) of the 4th instar larvae of C. suppressalis were analyzed by qRT-PCR. Finally, we performed targeted knockout of ebony gene in C. suppressalis by microinjecting the ribonucleoprotein complexes specific guide RNA/Cas9 protein into the newly laid eggs within 2 h based on the CRISPR/Cas9 gene editing technology. 【Results】 The full-length cDNA of Csebony gene (GenBank accession no.: MZ846208) of C. suppressalis was cloned. It contains a 2 586 bp ORF encoding 861 amino acids, with the molecular mass of 9.5 kD and theoretical isoelectric point of 5.10. Csebony has no signal peptide sequence at the N-terminus. Domain analysis showed that Csebony has three conserved domains. Phylogenetic analysis indicated that Csebony is most closely related to Ostrinia furnacalis ebony. The qRT-PCR results showed that Csebony was highly expressed in the pupal stage and head. Knockout of Csebony caused melanin pigmentation in larvae, pupae, and adults of C. suppressalis. 【Conclusion】 The results showed that Csebony is involved in regulating cuticle pigmentation of C. suppressalis, and CRISPR/Cas9-based genome editing technology is effective in C. suppressalis. We can use visible marker gene to establish CRISPR/Cas9-based genome editing system in non-model organisms, so as to offer a valuable genetic tool for the study of functional genomics in C. suppressalis.

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Research progress of the influence of microorganisms on insect behavior
CHENG Dai-Feng, LI Hui-Jing, LU Yong-Yue
Acta Entomologica Sinica    2021, 64 (6): 743-756.   DOI: 10.16380/j.kcxb.2021.06.010
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 In the long evolutionary process, a variety of forms of interaction between microorganisms and insects have been formed. The wide distribution of microorganisms provides background conditions for their contact with insects and influence on insect behavior. To further explore the phenomenon and mechanism of microorganisms influencing insect behaviors, the research progress of the influence of microorganisms on insect behaviors was reviewed in this article. Host location and selection of insects can be influenced by chemical signal substances produced by microorganisms, and the synthesis of semiochemicals in insects and host plants can also be influenced by microorganisms. Microorganisms have also been found to play important roles in intraspecific and interspecific relationships of insects. Microorganisms can even influence the reproductive behavior of insects by altering, for example, insect sex pheromones. Besides, social and aggregation behaviors of insects are also found to be influenced by semiochemicals synthesized by microorganisms. Considering the current research status of the influence of microorganisms on insect behaviors, the following aspects are suggested to be further investigated: (1) How are semiochemicals that influence insect behavior synthesized in the processes of influencing insect behavior by microorganisms? (2) Do microorganisms involve more interspecies interaction in influencing insect behavior? (3) How do host insects acquire and maintain symbiotic microoganisms that influence insect behavior at certain stages?
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Dynamic distribution of histone H3 Ser10 phosphorylation during meiosis in spermatocytes of the silkworm, Bombyx mori
ZHANG Bing, QIU Reng, KAN Yun-Chao
Acta Entomologica Sinica    2021, 64 (3): 302-308.   DOI: 10.16380/j.kcxb.2021.03.002
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【Aim】 To explore the function of histone H3 Ser10 phosphorylation  (H3Ser10ph) during meiosis in spermatocytes of the silkworm, Bombyx mori. 【Methods】 The testis tissues of B. mori from the 4th instar larval stage to pupal stage weredissected and separated. The slides of testis tissues at different stages of meiosis in spermatocytes were prepared by encapsulation of acrylamide gel and then the localization characteristics of the H3Ser10ph antibody was detected by using immunofluorescence staining. 【Results】 The phosphorylation of histone H3 Ser10 occurred in the specific position of pachytene chromosomes during meiosis in eupyrene spermatocytes of B. mori. The signal of H3Ser10ph was gradually weakened in the diplotene, and no phosphorylation signal was detected in the chromosomes at the diakinesis. With the progress of cell cycle, phosphorylation signal began to increase, and reached the highest level at the metaphase of the first meiosis. When the cells entered the prometaphase II of meiosis, the signal of H3Ser10ph on the chromosome arm disappeared, and there was holo-shaped fluorescent signal of the H3Ser10ph antibody close to the spindle microtubulin attaching side. At the end of meiosis II, a weak H3Ser10ph signal remained in the specific position of chromatin. During apyrene spermatocyte meiosis, the signal of H3Ser10ph was aggregated uniformly along the whole chromosomes from metaphase I to telophase I, and the spindle microtubules were parallel to the equatorial plane at anaphase I. 【Conclusion】 Histone H3 Ser10 phosphorylation is correlated with the dynamic changes of chromatin in eupyrene spermatocytes and apyrene spermatocytes of the silkworm. 
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Feeding preference and adaptability of Spodoptera frugiperda (Lepidoptera: Noctuidae) on different wheat cultivars in relation to leaf biochemical contents#br#
LIU Huan, ZHANG Yong, CHEN Ju-Lian
Acta Entomologica Sinica    2021, 64 ( 2): 230-239.   DOI: 10.16380/j.kcxb.2021.02.010
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【Aim】 This study aims to determine the feeding preference and adaptability of the fall armyworm (FAW), Spodoptera frugiperda, on different wheat cultivars collected from the Huang-Huai-Hai wheat growing areas in North China, to explore the relationship between insect pest damage and wheat cultivars, and to clarify the resistance levels of different wheat cultivars to FAW, so as to provide a theoretical basis for the arrangement of insect resistant cultivars and integrated pest management in wheat fields. 【Methods】 The feeding preference of the 1st and 3rd instar larvae of FAW to 15 wheat cultivars was compared. Four wheat cultivars with high feeding preference and two wheat cultivars with low feeding preference for the 1st and 3rd instar larvae of FAW were screened out, the contents of biochemical components (total protein, soluble sugar, total phenol and tannin) in leaves of these six wheat cultivars were determined, the relationship between the feeding preference of the larvae and the contents of biochemical components in leaves of these six wheat cultivars was clarified by Pearson correlation analysis, and the adaptability of FAW on the six wheat cultivars was observed. 【Results】 The results revealed that the 1st and 3rd instar larvae of FAW preferred to feeding on Huaimai 46, Huaimai 33, KOK1679 and Tongmai 6, but not on Luyuan 502 or Yannong 21. The feeding preference of the 1st and 3rd instar larvae to leaves of different wheat cultivars was positively correlated with the total protein content and the soluble sugar content in wheat leaves, but negatively correlated with the total phenol content and the tannin content. The development of FAW was significantly different on leaves of different wheat cultivars, with the highest host suitability index and the shortest larval duration on Tongmai 6, and with the longest larval duration on Yannong 21. 【Conclusion】 The FAW showed different adaptability to different wheat cultivars, and Yannong 21 showed higher resistance to this insect pest. The FAW larvae prefer wheat leaves with higher contents of total protein and soluble sugar, while the feeding can be inhibited by high contents of total phenol and tannin in wheat leaves. It is speculated that the difference of resistance levels of different wheat cultivars to FAW is resulted from the coordination of nonpreference and antibiosis in wheat by the metabolism of nutrients and secondary substances.
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Identification and expression profiling of odorant receptor genes based on the transcriptomes of two sibling species of Eucryptorrhynchus (Coleoptera: Curculionidae) at different developmental stages
LU Yi, WANG Qian, WEN Jun-Bao
Acta Entomologica Sinica    2021, 64 (6): 655-665.   DOI: 10.16380/j.kcxb.2021.06.001
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【Aim】 To complement the odorant receptor (OR) information of two sibling species of Eucryptorrhynchus, E. scrobiculatus and E. brandti, and to provide theoretical basis for subsequent functional studies by the identification and expression profile analysis of OR genes based on the transcriptome data of these two weevil species at different developmental stages. 【Methods】 The OR gene sequences were screened from the transcriptome databases of different developmental stages of E. scrobiculatus (larva, pupa and adult) and E. brandti (egg, larva, pupa and adult). Interspecific and intraspecific sequence alignments of the screened ORs were performed based on the antennal transcriptome data of the two weevil species. Phylogenetic analysis of the newly identified OR genes was performed using the maximum likelihood method. Expression abundance analysis was performed according to FPKM (fragments per kilobase per million mapped fragments) values of the newly identified OR genes in the transcriptomes of two weevil species at different developmental stages. The expression profiles of the newly identified OR genes at different developmental stages and in different adult tissues of E. scrobiculatus and E. brandti were detected by qPCR. 【Results】 Six and four OR genes with complete open reading frames were identified from the transcriptome databases of different developmental stages of E. scrobiculatus and E. brandti, respectively. According to the sequence alignment results with the antennal transcriptome data, four (EscrOR50-53) and two (EbraOR46-47) OR genes were newly identified from E. scrobiculatus and E. brandti, respectively. We also found a pair of potential homologous genes, EscrOR53 and EbraOR45, between the two weevil species. The phylogenetic tree indicated that the six newly identified ORs belong to the subfamilies 2B and 7 of coleopteran ORs. According to the FPKM data of the transcriptomes of different developmental stages of the two weevil species and the spatiotemporal expression profiles detected by qPCR, three of the newly identified OR genes of E. scrobiculatus and the two newly identified OR genes of E. brandti were all highly expressed in non-olfactory tissues of adults, and also expressed in egg, larval or pupal stages. 【Conclusion】 In this study, the OR genes and their spatiotemporal expression profiles in the transcriptomes of different developmental stages of E. scrobiculatus and E. brandti have been clarified, and it is found that non-olfactory functional ORs possibly exist in the reproductive organs, suggesting that they may play a role in the early stage of development.
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Identification of salivary gland proteins of nymphs and male and female adults of Sogatella furcifera (Hemiptera: Delphacidae)
LI Fei, YI Jin-Yu, LIU Yu-Di, HOU Mao-Lin
Acta Entomologica Sinica    2021, 64 (3): 281-301.   DOI: 10.16380/j.kcxb.2021.03.001
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 【Aim】 To identify and functionally annotate the salivary gland proteins of Sogatella furcifera and to explore their differences and correlation between different developmental stages and sexes. 【Methods】 Proteins were extracted from salivary gland tissues dissected from anesthetized nymphs, and male and female adults of S. furcifera, subjected to reductive alkylation and enzymolysis, and identified by liquid chromatography tandem mass spectrometry. Then, the salivary gland proteins were functionally annotated and classified by comparison with the Unigene protein database and KOG analysis. 【Results】There were 385, 168 and 82 salivary gland proteins specifically associated with nymphs, and male and female adults of S. furcifera, respectively. In total 319 salivary gland proteins were shared by nymphs and male adults, 60 by nymphs and female adults, and 60 by male and female adults. The KOG functional annotation showed the highest number of proteins associated with cellular processes and signaling, with 81, 22, and 70 proteins of this type specifically associated with nymphs, male adults, and female adults, respectively, and 19, 21 and 12 proteins of this type shared by nymphs and male adults, nymphs and female adults, and male and female adults, respectively, and these proteins are principally involved in post-translational modification, protein turnover, chaperones, intracellular trafficking, secretion and vesicular transport, and signal transduction.【Conclusion】 The salivary gland proteins of S. furcifera are quite active in signal transduction, which may be connected to its piercing and sucking damage to host plants.

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Host adaptability of Bemisia tabaci on tomato plants with ToCV single infection and TYLCV&ToCV co-infection and the changes in the nutrient contents and defense responses of host plants
DING Tian-Bo, ZHOU Xue, YANG Nan, YANG Yang, TANG Yao, CHU Dong
Acta Entomologica Sinica    2021, 64 (3): 384-391.   DOI: 10.16380/j.kcxb.2021.03.010
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【Aim】 This study focuses on two important tomato viruses, tomato chlorosis virus (ToCV) and tomato yellow leaf curl virus (TYLCV). It aims to explore the effects of ToCV single infection and TYLCV&ToCV co-infection on the host adaptability of Bemisia tabaci MED, and to reveal the physiological mechanism from the perspective of host plant nutrient contents and defense responses. 【Methods】 After ToCV single infection andTYLCV&ToCV co-infection, the survival rates and number of eggs laid of B. tabaci MED adults on the infected tomato plants, and the contents of amino acids and total sugar in the infected tomato plants were detected. Additionally, the expression profiles of key genes related to jasmonic acid (JA) (FAD7 and PI II) and salicylic acid (SA) (NPR1 and PR1) signal pathways in tomato plants in response to ToCV single infection and TYLCV&ToCV co-infection were analyzed by RT-qPCR. 【Results】 The survival rate and number of eggs laid of B. tabaci MED adults feeding on the tomato plants infected by tomato viruses decreased significantly compared to those feeding on the healthy tomato plants. Feeding onthe TYLCV&ToCV coinfected tomato plants could reduce the survival rate and number of eggs laid of B. tabaci MED adults to the lowest level. The contents of total amino acids, fourteen types of hydrolyzed amino acids and total sugar in the TYLCV&ToCV co-infected tomato plants were significantly lower than those in the ToCV-singly infected tomato plants. The expression levels of FAD7 and PI II were significantly downregulated in tomato plants infected by ToCV and TYLCV&ToCV, and the expression levels of these two genes were the lowest in the TYLCV&ToCV co-infected tomato plants. However, the expression levels of NPR1 and PR1 were upregulated in tomato plants infected by ToCV and TYLCV&ToCV. The expression levels of NPR1 in the TYLCV&ToCV co-infected tomato plants and PR1 in the ToCV-singly infected tomato plants were all significantly higher than those in the healthy tomato plants. 【Conclusion】 Both ToCV single infection and TYLCV&ToCV co-infection could decrease the host adaptability of B. tabaci MED on tomato plants, and TYLCV&ToCV co-infection brought a more adverse effect on the survival of B. tabaci MED. Compared with the healthy tomato plants, the nutritional condition and defense system in ToCV-singly infected and TYLCV&ToCV co-infected tomato plants have obviously altered and are different between both. The results provide a basis for revealing the interaction between B. tabaci and plant viruses.

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Regulation of histone methylation modification in 20E signaling transduction detected by CRISPR/Cas9 system in Drosophila cells
ZHANG Wen-Hao, LONG Shi-Hui, NI Yi-Lu, ZHANG Jia-Hui, LI Sheng, LI Kang
Acta Entomologica Sinica    2021, 64 (5): 549-557.   DOI: 10.16380/j.kcxb.2021.05.001
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【Aim】 To screen and detect the regulation of histone methylation modification in 20-hydroxyecdysone (20E) signaling transduction by CRISPR/Cas9 knockout system in Drosophila melanogaster cells. 【Methods】 Histone methyltransferases of D. melanogaster and their modification sites were analyzed and summarized from Flybase website. After transfection of the constructed knockout vector pAc-sgRNA-Cas9 inserted with the sgRNA of five histone methyltransferase genes [trr, Mes-4, ash1, Su(var)3-9 and egg] into Kc cells of D. melanogaster, the gene mutation was detected by TA cloning and sequencing. Taking Trr inducing 20E primary response gene Br-C as a positive control, qRT-PCR and Western blotting were used to test the validity of pAc-sgRNA-Cas9 knockout system. The dual luciferase assay of 20E response element (EcRE) was used to determine whether trr, Mes-4, ash1, Su(var)3-9 and egg participate in 20E signaling transduction. 【Results】 Drosophila histone methylation modification occurs mainly on histone H3 lysine residues and less on H3 arginine residues. Besides, there was less methylation modification on histone H4. After the transfection of its knockout vector pAc-trr-sgRNA-Cas9 in Kc cells, trr was mutated successfully, the tri-methylation level of H3K4 was reduced, and the 20E-induced transcriptional level of its primary response gene Br-C was inhibited, indicating the validity of this knockout system. Besides trr, Mes-4 and egg knockouts also reduced the dual luciferase activity of EcRE. 【Conclusion】 The pAc-sgRNA-Cas9 knockout system inserted with sgRNA of target gene is effective and fast for gene mutation in Drosophila Kc cells. Using this knockout system, in addition to Trr, Mes-4 and egg were found to participate in the regulation of 20E signaling transduction via manipulating the activity of EcRE. This study lays the theoretical basis and work foundation for CRISPR/Cas9-mediated gene knockout in insect cells and further studying histone methylation modification involved in regulating 20E signaling transduction.
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Morphology and distribution of antennal sensilla of Thrips hawaiiensis (Thysanoptera: Thripidae)
LIU Yu-Yan, WANG Liang, ZHANG Xiang-Qin, CHEN Yi-Xin, TIAN Hou-Jun, LIN Shuo, LI Heng, YU Yun, LIN Ling-Hong, ZHANG Jie, CHEN Yong, WEI Hui
Acta Entomologica Sinica    2021, 64 (4): 498-509.   DOI: 10.16380/j.kcxb.2021.04.009
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【Aim】 The aim of this study is to determine the type, morphology and distribution of antennal sensilla of Thrips hawaiiensis (Thysanoptera: Thripidae) at various developmental stages. 【Methods】 The antennal morphology and structure and the type, morphology and distribution of antennal sensilla of female and male adults, nymphs, pre-pupae and pupae of T. hawaiiensis were observed by using the scanning electron microscopy (SEM). 【Results】 The antenna of adult T. hawaiiensis is composed of scape, pedicel and a long flagellum with five flagellomeres (I-V). The mean length of antennae of female and male adults is 263.70±5.78 and 225.79±8.92 μm, respectively. The length of antennae increases with the growth of T. hawaiiensis. There are seven types of sensilla, i.e., Bhm bristles (BB), sensilla campaniformia (SCa), sensilla chaetica (SChI, SChII), sensilla trichodea (ST), sensilla basiconica (SBI, SBII, SBIII), sensilla coeloconica (SCo) and sensilla cavity (SCav), and two cuticular structures, i.e., microtrichia (mt) and cuticular denticles (cd), on the antenna of adults. The antenna of prepupae is conical, has no distinct segmentation and can move freely, with the mean length of 138.81±6.29 μm. The pupal antenna is cylindricalshaped pressing against the cephalo-thoracic back and immobile, and has no obvious segmentation, with a mean length of 213.07±6.30 μm. The antenna of the 1st and 2nd instar nymphs is composed of scape, pedicel and a flagellum with four flagellomeres (I-IV), with the mean length of 122.48±1.72 and 134.58±3.75 μm, respectively. There are eight types of sensilla [BB, SCa, SCh (SChI, SChII), SB (SBI, SBII), SCo, SCav, ST, and unusual sensillum (US)] and two cuticular structures [cd and ligulate structure (LS)] on the antenna of the 1st instar nymphs. There are seven types of sensilla [BB, SCa, SCh (SChI, SChII), ST, SB (SBI, SBII), SCo, and SCav] and one cuticular structure (cd) on the antenna of the 2nd instar nymphs. 【Conclusion】 In this study, the antenna and the morphology and distribution of antennal sensilla of T. hawaiiensis at various developmental stages were observed and comprehensively described, and the functions of antennal sensilla were inferred. The results lay a theoretical foundation for further research on the physiological functions of antennal sensilla of thrips.

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Diversity and sequence characteristics of the OCT family genes in Anopheles sinensis (Diptera: Culicidae)
LEI Dan, YAN Zhen-Tian, ZHANG Xiao-Xiao, CHEN Bin
Acta Entomologica Sinica    2021, 64 (3): 337-347.   DOI: 10.16380/j.kcxb.2021.03.006
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 【Aim】 To identify, classify and name genes of the organic cation transporter (OCT) family of Anopheles sinensis at the whole-genome level, so as to provide an information frame for the OCT genes of insects. 【Methods】 The amino acid sequences encoded by OCT genes in An. gambiae, Drosophila melanogaster and Caenorhabditis elegans were downloaded from NCBI, VectorBase and EMBL databases, and used as queries to search for the OCT genes in An. sinensis genome using the local Blast program based on the genome and transcriptome sequencing data of An. sinensis. The characteristics of these OCT genes in An. sinensis, including the gene structure, Scaffold location and conservative motifs, were predicted using bioinformatics methods. The phylogenetic tree of OCT genes of An. sinensis was constructed by the maximum likelihood method. 【Results】 The An. sinensis genome contains 54 OCT genes, which are divided into three subfamilies, OCTA, OCTB and OCTC, with 33, 15 and 6 genes, respectively. The encoded OCT amino acid sequences, except for AsOCTA20 and AsOCTB2, have MFS_1 and Sugar_tr transmembrane domains (TMDs), and most of the OCT amino acid sequences have 12 TMDs. All the OCT amino acid sequences have three conserved motifs, GRK-(PT)-VL, PES-(APVS) and EQFPT-(VI)-RN, and each subfamily has its own conservative motifs. Four pairs of genes (AsOCTB4a and AsOCTB4b, AsOCTA10a and AsOCTA10b, AsOCTA19a and AsOCTA19b, and AsOCTA23a and AsOCTA23b) are derived from gene duplication events. AsOCTC genes are relatively primitive, while AsOCTB genes are relatively evolved, both forming an obvious monophyly. AsOCTA genes are between AsOCTC genes and AsOCTB genes in evolution, with more complicated evolutionary relationship. 【Conclusion】 This study enriches the genome data of An. sinensis, and lays the foundation for the subsequent research of the functions of OCT genes in An. sinensis, especially their functions in insecticide resistance mechanism.

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Research advances of salivary effectors and elicitors in herbivorous insects
DONG Yu-Mei, ZHANG Mei-Qian, SHEN Hui, HUANG Xing-Ge, YANG Yu-Xia, LI Ji-Fen, ZHANG Wen-Dan, SHEN Dan-Yu, JING Mao-Feng, DOU Dao-Long, XIA Ai
Acta Entomologica Sinica    2021, 64 (8): 982-997.   DOI: 10.16380/j.kcxb.2021.08.010
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 Herbivorous insects and host plants have developed complicated defense and counter defense mechanisms through co-evolution. In this article, we systematically reviewed the roles of insect saliva effectors and elicitors in the interactions between insects and plants and their mechanisms. The salivary elicitors secreted by insects during feeding can be recognized by plants to trigger early plant immunity, while insect effectors released from oral secretion can inhibit plant immune defenses. Resistant plants further evolved R proteins to recognize insect avirulence effectors and initiate effector-triggered immunity. Phytophagous insects can avoid the recognition by plant R proteins through different strategies. Therefore, in this arms race, insect saliva determines whether insects can succeed to feed on plants. During feeding process, chewing insects secrete a large number of enzymes into plants, and piercing-sucking insects secrete sheath saliva and water saliva into plants, but both of them utilize effectors and elicitors to manipulate plant immune responses. By analyzing the reported insect effectors, it was found that the molecular mechanisms of insect effectors are different. They affect plant early defense signals, regulate plant hormone pathways or others, or target small RNA pathways. Recent advances in insect elicitors were also reviewed in this article, revealing that elicitors can induce the release of plant secondary metabolites, and regulate hormone levels, Ca2+ influx and reactive oxygen species (ROS) burst to enhance plant resistance. Finally, we analyzed the secretory characteristics, host specificity and multifunctionality of insect effectors, and discussed research prospects on avirulence effectors and their plant R genes as well as pattern recognition receptors of elicitors. 
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Distribution and structure of wax glands in various body forms of the horned gall aphid, Schlechtendalia chinensis (Hemiptera: Aphididae)
WEI Hong-Yuan, FENG Guo-Rui, XU Xin, SHAO Shu-Xia, CHEN Xiao-Ming, YANG Zi-Xiang
Acta Entomologica Sinica    2021, 64 (4): 490-497.   DOI: 10.16380/j.kcxb.2021.04.008
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【Aim】 The horned gall aphid, Schlechtendalia chinensis, is the major productive species of Chinese gallnuts. S. chinensis exhibits complex life cycles, including alteration of sexual and asexual reproduction via host switch between Chinese sumac (Rhus chinensis) and mosses. There are six body forms, i.e., fundatrix, fundatrigeniae, fundatrispuriae, apterae, sexuaparae, and sexual forms, alternating inside and outside a gall to finish its whole life cycle. 【Methods】 Specimens of various body forms of S. chinensis were obtained from S. chinensis reared in the laboratory based on the original population of S. chinensis collected from the fields in Yanjin, Yunnan, southwestern China. The distribution, morphology, and structure of wax glands in S. chinensis were investigated using light microscope, electron microscope, and technique of ultrathin section. 【Results】 All body forms of S. chinensis except the 1st instar fundatrix possess wax glands on the dorsum, including 2 columns and 2 rows on the head, 4 columns and 1 row on the thorax, 6 columns and 8 rows on the abdomen, with a total of 56 wax glands. No wax glands appear on the dorsum of the 1st instar fundatrix (living outside a gall), but there are 2 columns and 8 rows of wax glands (in total 16 wax glands) on the dorsum of the 2nd instar fundatrix (living inside a gall). Each wax gland is composed of 2-22 polygonal depressions, and the number and morphology of depressions are obviously different among various body forms. The wax glands of fundatrigeniae, sexuaparae, and fundatrispuriae are the most complicated, and followed by apterae. Furthermore, the structure of wax glands in different parts of the sexual forms is significantly different: the wax gland structure of the dorsal plate and dorsolateral plate near the midline is simple, while that near the lateral line is complex. The wax gland structure of the 2nd instar fundatrix is simple as well. Corresponding to the distribution of wax glands, waxes appear on the body surface of all body forms except the 1st instar fundatrix. Fundatrigeniae, sexuaparae, and fundatrispuriae secrete a large amount of wax, followed by apterae. There is a small amount of wax on the body surface of the sexual forms and the 2nd instar fundatrix, whereas no wax exists on the body surface of the 1st instar fundatrix. 【Conclusion】 The number, arrangement, and development of wax glands on various body forms of S. chinensis are different. These differences are closely related to the microenvironments in which they live and the biological characteristics of each stage, being probably the long-term adaptation of the horned gall aphid to environmental conditions.
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Effects of silencing insulin like peptide (Ilp) genes on the wing and ovarian development and trehalose metabolism in Nilaparvata lugens (Hemiptera: Delphacidae)
YU Wei-Dong, LIU Yong-Kang, LUO Yu-Jia, HUANG Zhen, ZHOU Tai, YE Lin, TANG Bin, WANG Shi-Gui
Acta Entomologica Sinica    2021, 64 (4): 428-438.   DOI: 10.16380/j.kcxb.2021.04.002
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【Aim】 Insulin-like peptide (Ilp) is located in the upstream of insulin signaling pathway and plays a key role in regulating sugar metabolism. This study aims to explore the function of Ilp in regulating the trehalose metabolism balance in the brown planthopper, Nilaparvata lugens. 【Methods】 The expression of Ilp genes in the 5th instar nymphs of N. lugens was inhibited by RNAi technology, and the phenotype and ovarian development of the female adults of N. lugens were observed after RANi.The changes in the contents of trehalose, glycogen and glucose and the trehalase activity were determined by biochemical methods, and the changes in the expression levels of trehalase genes (TRE1-1, TRE1-2 and TRE2) and trehalose synthase genes (TPS1 and TPS2) in the 5th instar nymphs of N. lugens at 48 h and 72 h after RNAi were tested by qPCR. 【Results】 The dsRNA effectively inhibited the expression of Ilp genes and resulted in abnormal wing in N. lugens, and the injection of dsIlp3 and dsIlp1+dsIlp2+dsIlp3+dsIlp4 caused underdeveloped ovaries in the 2-day-old female adults of N. lugens. The glucose levels were significantly increased at 48 h after injection of dsIlp1-4 and dsIlp1+dsIlp2+dsIlp3+dsIlp4, respectively, and the glycogen levels were also significantly increased at 48 h after injection of dsIlp2, dsIlp3 and dsIlp4, respectively. The trehalose content was significantly increased at 48 h after injection of dsIlp3 and dsIlp4, respectively, while the trehalose content decreased significantly at 72 h after inhibition of Ilp2 and Ilp4, respectively, and increased significantly both at 48 h and 72 h after injection of dsIlp1+dsIlp2+dsIlp3+dsIlp4. In addition, the soluble trehalase activity increased significantly at 72 h after injection of dsIlp1+dsIlp2+dsIlp3+dsIlp4, while the membrane-bound trehalase activity decreased significantly at 72 h after injection of dsIlp3, dsIlp4 and dsIlp1+dsIlp2+dsIlp3+dsIlp4, respectively. The expression levels of TRE1-1, TRE1-2, TRE2, TPS1 and TPS2 were significantly decreased after injection of dsIlp1, dsIlp2 and dsIlp4, respectively. 【Conclusion】 Silencing of Ilp genes has an inhibitory effect on the development and reproduction of N. lugens, and can increase the contents of glucose and glycogen, downregulate the expression of genes of trehalase and trehalose synthase, and increase the soluble trehalase activity, thus regulating the trehalose metabolism in N. lugens.
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Research progress of insect sodium channels#br#
WU Shao-Ying, DUAN Wen-Bo, LI Fen, YANG Lei, WANG Hao, WANG Li-Kui
Acta Entomologica Sinica    2021, 64 (7): 862-874.   DOI: 10.16380/j.kcxb.2021.07.010
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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.
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Resistance selection and biological characteristics of Neoseiulus barkeri (Acari: Phytoseiidae) to chlorpyrifos
WANG Li, CHENG Ming-Ming, FU Yun-Mei, FANG Yun-Hong, WEI Zhi-Tang, CHENG Lu-Yan, LEI Shuang, DING Li-Li, YU Shi-Jiang, CONG Lin, RAN Chun
Acta Entomologica Sinica    2021, 64 (3): 374-383.   DOI: 10.16380/j.kcxb.2021.03.009
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 【Aim】 To clarify the effects of acquired chlorpyrifos resistance in the predatory mite Neoseiulus barkeri on its biological characteristics, so as to provide a theoretical basis for its field application. 【Methods】 The residual film method was adopted to test the toxicity of chlorpyrifos to N. barkeri in the laboratory, and the lethal medium concentration (LC50) was chosen as the selection pressure. Life table was used to assess the effects of acquired chlorpyrifos resistance on the relative fitness (Rf)of N. barkeri. In addition, Holling type II model was used to analyze the differences in the predation functional response of the chlorpyrifos-susceptible and resistant strains of N. barkeri to eggs and female adults of Panonychus citri at different temperatures.【Results】 A chlorpyrifos-resistant strain of N. barkeri with the resistance ratio of 34.77-fold was obtained after selection of 21 generations. The acquired resistance had no significant effects on the developmental duration, proportion of female offspring and predation functional response of N. barkeri, but shortened the female adult longevity and oviposition period. The susceptible and resistant strains of N. barkeri could not complete the whole generation at 15℃. The fecundity (number of eggs laid per female) of the susceptible and resistant strains of N. barkeri was the highest at 25℃, and the fecundity was significantly higher for the susceptible strain (41.64±1.04 eggs/female) than for the resistant strain (38.80±0.93 eggs/female). Likewise the oviposition periods of the susceptible and resistant strains of N. barkeri were the longest at 25℃, and the oviposition period was significantly longer for the susceptible strain (24.82±1.50 d) than for the resistant strain (21.34±1.26 d). Further, the shortest developmental duration of the susceptible and resistant strains of N. barkeri was recorded at 30℃, being 6.62±0.23 d for the susceptible strain and 6.53±0.13 d for the resistant strain. Meanwhile, the strongest predation ability of the susceptible and resistant strains of N. barkeri to eggs or female adults of P. citri was recorded at 30℃, with the daily maximum predation amounts of the susceptible and resistant strains of N. barkeri to P. citri eggs being 156.25 and 140.85, respectively, and to female adults of P. citri being 23.10 and 22.32, respectively. The relative fitness (Rf) of the resistant strain of N. barkeri was lower than that of the susceptible strain at 25 and 30℃, but slightly higher than that of the susceptible strain at 20 and 35℃. 【Conclusion】 Generally, chlorpyrifos resistance in N. barkeri has little effect on its biological characteristics under different temperatures. The study results provide a theoretical basis for screening resistant strains of N. barkeri and their field application.

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Effects of RNAi-mediated suppression of Br-Z2/3 gene on the expression of downstream apoptotic genes and pupation in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)
HU Xiao-Han, TIAN Su-Fen, LI Ya-Qing, LIN Shuo, CHEN Yi-Xin, WEI Hui, GU Xiao-Jun, HUANG Jing-Fei, WANG Xi-Ying, LI Zhi-Hua
Acta Entomologica Sinica    2021, 64 ( 2): 141-148.   DOI: 10.16380/j.kcxb.2021.02.001
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【Aim】 This study aims to establish an in vitro prokaryotic expression system for synthesis of the dsRNA of pupa-specific expression gene Br-Z2/3 from the diamondback moth, Plutella xylostella, and to find out the effects of RNAi-mediated suppression of Br-Z2/3 on the expression of Br-Z2/3 and apoptotic genes and the pupation of the moth. 【Methods】 The L4440-Br-Z2/3 recombinant vector was constructed and transformed into competent cells of Escherichia coli HT115. The dsRNA of Br-Z2/3 was obtained after induction with IPTG, and RNAi was carried out by microinjecting Br-Z2/3 dsRNA into the 4th instar larvae of P. xylostella. At 12 and 24 h after RNAi of Br-Z2/3, the expression levels of Br-Z2/3 and its downstream apoptotic genes, reaper, caspase-9 and Gadd45g, were detected through qPCR. Simultaneously, the pupation rate, average pupation time, rate of deformed pupae and larval mortality of the 4th instar larvae of P. xylostella after RNAi of Br-Z2/3 were observed and recorded. 【Results】 The dsRNA of Br-Z2/3 was successfully expressed in the prokaryotic system. The qPCR results showed that the expression levels of Br-Z2/3 and the related apoptotic gene reaper decreased significantly, but those of caspase-9 and Gadd45g increased significantly after RNAi of Br-Z2/3 in the 4th instar larvae of P. xylostella. Compared with the control group, the dsBr-Z2/3-injected group showed significantly lower pupation rate with delayed pupation peak, and had significantly higher mortality and higher rate of deformed pupae. 【Conclusion】 An in vitro prokaryotic expression system for synthesis of the dsRNA of Br-Z2/3 from P. xylostella was successfully established. RNAi of Br-Z2/3 by microinjection demonstrates that Br-Z2/3 plays a crucial role in the pupation of P. xylostella.
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Expression patterns and pheromone-binding properties of the pheromone binding protein CpunPBP3 in Conogethes punctiferalis (Lepidoptera: Crambidae) (In English)
CHEN Qiu-Ying, YANG Xi, YOU Dong-Rui, YANG Mu, XU Zhi-Feng, XIAO Wei
Acta Entomologica Sinica    2021, 64 (3): 318-326.   DOI: 10.16380/j.kcxb.2021.03.004
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【Aim】 This study aims to better understand the sex pheromone perception mechanisms by identifying and characterizing a sex pheromone binding protein (PBP) in the yellow peach moth, Conogethes punctiferalis (CpunPBP3). 【Methods】 The cDNA sequence of CpunPBP3 of C. punctiferalis was amplified and analyzed, and the amino acid sequence was compared to those of the homologous proteins in other Crambidae species. The day-age-dependent changes and circadian fluctuations in the expression levels of CpunPBP3 in the male adult antenna of C. punctiferalis, and the changes in the expression level of CpunPBP3 in the antenna over 24 h-period following exposure of adult males to the sex pheromones E10-16∶Ald (150 ng) and Z10-16∶Ald (6 ng) were examined by qRT-PCR. The recombinant expression vector pET-30a(+)/CpunPBP3 was constructed, and the recombinant CpunPBP3 was expressed in Escherichia coli. The binding capacity of the purified recombinant protein CpunPBP3 with the above two sex pheromones was evaluated by fluorescence competitive binding assay. 【Results】 The phylogenetic analysis result revealed that CpunPBP3 and the previously identified C. punctiferalis PBP genes CpunPBP2 and CpunPBP5 clustered in different branches, but CpunPBP3 is similar to PBP genes in other insect species. The qRT-PCR results showed that the expression level of CpunPBP3 in the male adult antenna increased first and then decreased from day 0 to 8 after adult eclosion, with significantly higher expression level at 17∶00 than at 1∶00, but with no significant difference at other time points within 24-h photoperiod. However, the expression level of CpunPBP3 in the male adult antenna significantly decreased after induction by 150 ng E10-16∶Ald for 3 and 6 h, and significantly increased after induction by 6 ng Z10-16∶Ald for 6 and 24 h. Fluorescence competitive binding assay result showed that the recombinant CpunPBP3 had strong binding capacity with E10-16∶Ald and Z10-16∶Ald, with the Ki values of 9.267 and 8.656 μmol/L, respectively. 【Conclusion】 The study determined the nucleotide and amino acid sequences and the expression pattern of CpunPBP3, and CpunPBP3 was expressed in response to the sex pheromone induction. The recombinant CpunPBP3 has strong binding capacity with sex pheromone, indicating that CpunPBP3 is a sex pheromone binding protein in C. punctiferalis.

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Advances and perspectives of epigenetic regulation of insect diapause
AN Hao-Min, LIU Wen, WANG Xiao-Ping
Acta Entomologica Sinica    2021, 64 (4): 510-522.   DOI: 10.16380/j.kcxb.2021.04.010
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 Diapause is a strategy for insect to avoid harsh environment, and has a great significance for continuation of insect population. In particular, facultative diapause can be affected by periodic seasonal changes, in which epigenetics may play critical roles. Epigenetics refers to heritable variations independent of DNA sequence, including various modifications at DNA, RNA, protein and chromatin levels, and may be involved in development plasticity. The research of epigenetic regulation of insect diapause mainly focuses on two aspects: one is how epigenetic regulation respond to environmental signals, and the other is how environmental signal induces epigenetic regulation in insect diapause. Although it has been reported that DNA methylation can respond to photoperiodic signal and histone acetylation can be coupled with endocrine systems, the detail mechanisms of epigenetic regulations of insect diapause, however, are not completely revealed. Regulations of diapause induced by epigenetics have been reported in multiple types of insect diapause. For the same diapause process, there may be co-regulation between different epigenetic mechanisms. However, how this synergy responds to environmental signals and how it precisely regulates insect diapause are still unknown. In conclusion, the previous research only indicated the possibility of epigenetic regulation in insect diapause, but the molecular mechanisms are scarcely known and need further study. Especially the following aspects might be critical in the future research: the molecular mechanism of epigenetics responding to diapause-induced environmental signals, the molecular mechanism of epigenetics coupled with endocrine regulation, the cell signal transduction of epigenetic regulation, and the synergetic regulation of epigenetics in insect diapause.
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Influences of gall-inducing insects on the physiology and metabolism of host plants
YANG Meng-Ke, LIU Sai, QIAO Hai-Li, GUO Kun, XU Rong, XU Chang-Qing, CHEN Jun
Acta Entomologica Sinica    2021, 64 (4): 536-548.   DOI: 10.16380/j.kcxb.2021.04.012
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Plant galls, the abnormal outgrowths of plant tissues induced by gallinducing insects, are ideal materials to investigate the co-evolution between plants and insects. Gall-inducing insects are also important pests in agriculture and forestry. Investigations into the effects of gall-inducing insects on their host plants are useful to reveal the relationships between gall-inducing insects and plants, and can also help reveal the growth process of host plants. Besides, investigations into the responses of galled plants to gall-inducing insects are helpful to screen out the resistance indexes, resistance genes and sensitive genes of plants, providing a theoretical basis for resistance breeding. This review focuses mainly on the effects of gall-inducing insects on the photosynthesis, physiology and metabolism of their host plants. Gall-inducing insects generally reduce the photosynthetic pigments and photosynthetic rate of host plants, raise the contents of primary metabolites such as sugars and amino acids in inner tissues of galls, raise the contents of secondary metabolites such as non-volatile phenols and flavonoids and volatile terpenoids in outer tissues of galls, raise the activities of protective enzymes such as POD and SOD, and raise the contents of phytohormones such as IAA, SA and JA in host plants. The current research data indicate that investigations into the influences of gallinducing insects on the physiology and metabolism of host plants are even in their infancy, and the influencing mechanisms still need to be further explore.
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Research progress of insect miRNAs
YANG Jie, XIE Miao, XU Xue-Jiao, BAI Jian-Lin, YOU Min-Sheng
Acta Entomologica Sinica    2021, 64 ( 2): 259-280.   DOI: 10.16380/j.kcxb.2021.02.013
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 MicroRNAs (miRNAs) are a class of 19-24 nt endogenous non-coding small RNAs universally existing in various organisms. MiRNAs can regulate gene expression mainly by the combination between its seed region and the ORF or 3′UTR of target genes at the posttranscriptional level, and play critical roles in multiple biological processes including cell differentiation, proliferation and apoptosis. As miRNAs have eventually become a research hotspot in life sciences, great advances have been made in the study of insect miRNAs. The development of highthroughput sequencing and bioinformatics have accelerated the identification of miRNAs in different species, providing the theoretical basis for subsequent research. MiRNAs can be identified with direct cloning, bioinformatics prediction and highthroughput sequencing while their expression levels can be detected through miRNA gene chip analysis, Northern blot and realtime quantitative PCR (RT-qPCR). Inhibition of expression or overexpression of miRNAs can be applied to further reveal their biological functions. MiRNAs exert significant influence on insect growth and reproduction process by participating in ecdysone pathway and regulating the expression of some related genes such as genes related to ecdysone receptor, sex differentiation, wing development, lipid metabolism and ovarian development. MiRNAs are also involved in the circadian rhythm, memory formation and leaning capacity of some insects. In the progress of insect-virus interaction, a number of virus-encoded miRNAs disturb the immune response of host insects by regulating target gene expression while the insectencoded miRNAs influence virus replication. MiRNAs have also been found to participate in insect immune response against exogenous pathogens via regulating the expression of immunerelated genes, which affect innate immune response of insects. Additionally, it has been reported that miRNAs develop or enhance insecticide resistance by negatively regulating the expression of detoxification-related genes, altering insect susceptibility to insecticides and contributing to insecticide resistance in insects. This review provides a theoretical basis for further understanding of insect miRNAs and their potential applications in integrated pest management.
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Insect metamorphosis: Nature, history, evolution and regulation-A review of the book Insect Metamorphosis: From Natural History to Regulation of Development and Evolution
LI Kang, SONG Jia-Sheng, ZHOU Shu-Tang, LI Sheng
Acta Entomologica Sinica    2021, 64 (6): 769-770.   DOI: 10.16380/j.kcxb.2021.06.012
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Selection of reference genes for real-time fluorescence quantitative PCR of miRNAs in Sitobion avenae (Hemiptera: Aphididae)
YANG Chao-Xia, YAN Yi, ZHANG Fang-Mei, ZHU Xun, ZHANG Yun-Hui, LI Xiang-Rui
Acta Entomologica Sinica    2021, 64 (4): 439-448.   DOI: 10.16380/j.kcxb.2021.04.003
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【Aim】 This study aims to select the stably expressed reference genes for expression analysis of microRNAs (miRNAs) in the cereal aphid, Sitobion avenae, under specific conditions. 【Methods】 According to the Illumina sequencing results of S. avenae miRNAs, 10 candidate reference genes including miR-10-3p, miR-993, miR-276, miR-275, miR-252a, miR-1, miR-375, pc-15, pc-73 and one normal reference gene U6 were screened, and their relative expression levels in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids and in wingless aphids of S. avenae exposed to four insecticides (95.1% imidacloprid technical material, 97.8% thiacloprid technical material, 95% avermectins technical material and 40% omethoate emulsifiable concentrate) were detected by using qRT-PCR. The expression stability of the 10 candidate reference genes was evaluated by GeNorm, NormFinder, ΔCt method, BestKeeper, and RefFinder. 【Results】 The qRT-PCR results showed that the highly expressed reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to four insecticides were miR-276, miR-276, miR-10-3p, and miR-10-3p, respectively. Integrating the analysis results of five approaches, more stably expressed reference genes under the above four conditions were miR-252a, miR-993, miR-275, and miR-993, respectively. The optimal number of reference genes was two under different conditions by GeNorm. Based on the comprehensive ranking of RefFinder, the optimal combinations of stably expressed reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to four insecticides were miR-252a and miR-276, miR-993 and miR-276, miR-275 and miR-10-3p, miR-993 and miR-10-3p, respectively. 【Conclusion】 The optimal combinations of reference genes in winged and wingless aphids of S. avenae at different developmental stages, in various tissues of winged and wingless aphids, and in wingless aphids of S. avenae exposed to different insecticides are miR-252a and miR-276, miR-993 and miR-276, miR-275 and miR-10-3p, miR-993 and miR-10-3p, respectively. The results provide a basis of reference gene selection for quantitative expression analysis of miRNA genes in S. avenae.
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Oviposition preference of Serangium japonicum (Coleoptera: Coccinellidae) to eggplant varieties with different leaf trichome densities
MEI Wen-Juan, YAO Feng-Luan, LIN Shuo, DING Xue-Ling, ZHENG Yu, LU Xue-Song, HE Yu-Xian, WENG Qi-Yong
Acta Entomologica Sinica    2021, 64 (9): 1092-1103.   DOI: 10.16380/j.kcxb.2021.09.009
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 【Aim】 Oviposition is vital for the survival and development of insect populations. Our previous study demonstrated that Serangium japonicum prefer to lay eggs on eggplant (Solanum melongena) leaves. This study aims to explore the oviposition preference of S. japonicum to eggplant varieties with different leaf trichome densities, so as to further clarify the mechanisms regarding to leaf trichome-related oviposition preference in S. japonicum. 【Methods】 Leaf discs and plants of four eggplant varieties (Heidaodi, Heiniu, Riyouchangzhizhuang and Luoxing) were provided for S. japonicum to determine the oviposition preference. The offspring performance including the growth and development, and adult fecundity, predation capability and attachment force of S. japonicum on leaves of each of the four eggplant varieties was tested. Microstructure of the abaxial leaf surface of the four eggplant varieties, the preference of S. japonicum female adults to leaf odours, the length and width of Bemisia tabaci eggs on eggplant leaves, and the cannibalism risk of S. japonicum eggs by its female adults were examined. 【Results】 S. japonicum preferred to lay eggs on Heidaodi, where the shortest offspring developmental duration (15.41±0.21 d) and the highest fecundity (877.44±15.27 eggs laid per female) were found. However, the proportion of S. japonicum eggs had no significant correlation with offspring performance (developmental duration, survival rate and adult body weight) and adult performance (fecundity and predation capability), but was significantly positively correlated with the density and length of leaf trichomes on the abaxial leaf surface (ALS). Furthermore, the proportion of S. japonicum eggs laid on leaf discs was significantly positively correlated with the attachment force of its female adults. The attachment force of S. japonicum female adults was positively correlated with leaf trichome density but had no significant correlation with adult performance. The leaf odour, prey quality, and cannibalism risk on the four eggplant varieties had no significant effects on the oviposition preference of S. japonicum. 【Conclusion】 The density of ALS leaf trichome and the attachment force of S. japonicum mediated by leaf trichomes play an important role in the oviposition preference of S. japonicum.
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Differences in the evolution of mitochondrial genome between pollinating and non-pollinating fig wasps (In English)
WANG Jian-Xia, ZHOU Yi, XIN Zhao-Zhe, ZHAO Dan, XIAO Jin-Hua, HUANG Da-Wei
Acta Entomologica Sinica    2021, 64 (4): 479-489.   DOI: 10.16380/j.kcxb.2021.04.007
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【Aim】 At present, there are few reports on the mitochondrial genomes of fig wasps. The purpose of this study is to explore whether there are some differences in the evolution of mitochondrial genome (mitogenome) between pollinating fig wasps (PFWs) and non-pollinating fig wasps (NPFWs). 【Methods】 Based on the mitogenomes from 15 fig wasp species, of which the mitogenomes of 11 species were newly determined, we used the comparative mitochondrial genomic method to analyze the sequence and evolutionary characteristics of the mitogenomes of fig wasps. 【Results】 The length of the mitogenomes of 11 fig wasps newly determined ranges from 12 768 to 17 060 bp, and the AT content in the 11 mitogenomes is more than 80%. The AT-skew is negative and the GC-skew is positive in most species except for the non-pollinating fig wasp Philotrypesis tridentata. Frequent mitochondrial gene rearrangement occurs in fig wasps, which may be valuable for phylogenetic analysis of the species. Further analysis of selection pressure indicates that the ω ratios of protein-coding genes (PCGs) in mitogenomes of fig wasps are far less than 1, suggesting that these genes have experienced purifying selection. However, most of the genes in PFWs may have accumulated more nonsynonymous mutations than those in NPFWs. Furthermore, compared with the NPFWs, the mitogenomes of PFWs have more gene rearrangements, and higher nucleotide diversity and amino acid substitution rate. 【Conclusion】 The mitogenome evolution of PFWs is faster than that of NPFWs, which may be related to the significantly different lifestyles or evolutionary histories of the two groups.

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Identification and characterization of two aminopeptidases N from the midgut of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)
LIN Li, YU Xiao-Qiang, GUAN Xiong, SHAO En-Si
Acta Entomologica Sinica    2021, 64 (7): 771-780.   DOI: 10.16380/j.kcxb.2021.07.001
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【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.
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Function of the cytoplasmic peptidoglycan recognition protein RfPGRP-L2 in maintaining the homeostasis of gut microbiota in Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) 
XIAO Rong, WANG Xing-Hong, LI Xiong-Wei, LIU Hui-Hui, LU Sheng-Ping, HOU You-Ming, SHI Zhang-Hong
Acta Entomologica Sinica    2021, 64 (3): 348-362.   DOI: 10.16380/j.kcxb.2021.03.007
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Abstract: 【Aim】 To determine the function of a cytoplasmic peptidoglycan recognition protein, RfPGRP-L2, in the maintenance and regulation of homeostasis of gut microbiota in the invasive insect pest, Rhynchophorus ferrugineus, so as to provide scientific basis and action targets for the development of new insect pest control strategies targeting to destroy the homeostasis of gut microbiota. 【Methods】 The sequence characteristics of RfPGRP-L2 were analyzed by bioinformatics methods. RT-qPCR was used to analyze the expression levels of RfPGRP-L2 in the different tissues (head, fat body, epidermis, foregut, mid-/hindgut and hemolymph) of the healthy 4th instar larvae of R. ferrugineus and in the gut and fat body of the 4th instar larvae of R. ferrugineus challenged with Escherichia coli DH5α and Staphylococcus aureus by injection with 1 μL bacterial suspension with the OD600 value of 1.6 and oral feeding with sugarcane slices smeared with 1 mL bacterial suspension with the OD600 value of 1.6, respectively. Prokaryotic expression of RfPGRP-L2 was carried out, and in vitro assays were applied to determine the agglutination and antibacterial activity of the recombinant RfPGRP-L2 to E. coli DH5α and S. aureus. After RNAi of RfPGRP-L2, the number of gut bacterial colonies of E. coli in the hemolymph and gut of the 4th instar larvae of R. ferrugineus was determined. The expression levels of antimicrobial peptide genes in the fat body and gut of the 4th instar larvae of R. ferrugineus after RNAi of RfPGRP-L2 were detected by RT-qPCR. By bacterial 16S rRNA-based high-throughput sequencing, the effect of RNAi of RfPGRP-L2 on the gut microbiota composition of the healthy 4th instar larvae of R. ferrugineus was analyzed.【Results】 The SMART analysis revealed that RfPGRP-L2 has no transmembrane domain and signal peptide, suggesting that RfPGRP-L2 is a cyoplasmic peptidoglycan recognition protein. The RT-qPCR results showed that RfPGRP-L2 was highly expressed in the immunity-related tissues, such as the hemolymph, gut and fat body of the healthy 4th instar larvae of R. ferrugineus. The expression level of RfPGRP-L2 in the fat body of the 4th instar larvae of R. ferrugineus was significantly up-regulated upon the challenge with injected E. coli and S. aureus for 6 h and 12 h, respectively. After oral feeding of E. coli for 6 h, the expression level of RfPGRP-L2 in the gut of the 4th instar larvae of R. ferrugineus was also increased significantly. Furthermore, in vitro assays revealed that the recombinant RfPGRP-L2 could result in the obvious agglutination of E. coli and S. aureus, suggesting that RfPGRP-L2 could recognize the two pathogenic bacteria. When RfPGRP-L2 was silenced in the 4th instar larvae of R. ferrugineus, the ability to clear the invaded EGFP-tagged E. coli in the gut and hemolymph was significantly impaired as compared to the control group, the expression level of antimicrobial peptide gene RfCecropin in the gut was significantly reduced, the number of gut bacterial colonies in the healthy 4th instar larvae was significantly increased compared to the control group, and the gut bacterial composition was also altered significantly. 【Conclusion】 The cyoplasmic peptidoglycan recognition protein RfPGRP-L2, acting as a pattern recognition receptor to discriminate pathogens and to activate the immune signaling pathways in intestinal epithelial cells, can promote the expression of antimicrobial peptide gene to modulate the homeostasis of gut microbiota in R. ferrugineus.

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Genetic structure and Wolbachia infection in geographical populations of Monolepta hieroglyphica (Coleoptera: Chrysomelidae) in South China
LI Jing, ZHANG Xiao-Fei, XU Ling-Ling, SHEN Yuan-Yuan, LI Xiao-Xiao, WANG Zhen-Ying
Acta Entomologica Sinica    2021, 64 (6): 730-742.   DOI: 10.16380/j.kcxb.2021.06.009
Abstract210)      PDF(pc) (1879KB)(57)    PDF(mobile) (1879KB)(7)    Save
 【Aim】 Monolepta hieroglyphica is a polyphagous pest feeding on a large number of cultivated plant species. The aim of this study is to investigate the genetic diversity, genetic structure, and levels of genetic differentiation and gene flow among geographical populations of M. hieroglyphica distributed in South China, and to clarify the diversity and prevalence of the bacterial endosymbiont Wolbachia in M. hieroglyphica geographical populations in South China. 【Methods】 The mitochondrial COII gene was used as genetic marker. The partial COII gene sequences in a total of 403 individuals from 14 geographical populations of M. hieroglyphica were amplified by PCR and sequenced. The haplotype diversity (Hd), genetic differentiation coefficient (Fst) and gene flow (Nm) between populations were analyzed, and the analysis of molecular variance (AMOVA) and Tajima’s D and Fu’s Fs neutrality tests were performed. Median-joining network and phylogenetic tree were constructed based on haplotype sequences. Wolbachia wsp gene was amplified by PCR to detect population infection rates, and the obtained wsp sequences were used for strain typing and phylogenetic analysis of Wolbachia. 【Results】 For all the 403 test individuals of M. hieroglyphica in this study, 23 COII haplotypes were observed and divided into two clusters in phylogenetic tree. The Hd of total population was 0.748, ranging from 0.394 to 0.782 within each population. The neutrality test results suggested that M. hieroglyphica populations followed the neutral evolution model and there was no evidence of population expansion in recent history. The values of Fst and Nm of total population were 0.2481 and 0.76, respectively. The AMOVA results showed that a high proportion (73.75%) of the total genetic variance attributed to variation within population. There was no significant correlation between genetic distance and geographical distance among populations (R=0.2898, P=0.0640). The Wolbachia infection rates in the 14 geographical populations of M. hieroglyphica ranged from 92.59% to 100%, with an average infection rate of 97.60%. Six Wolbachia strains (named as wMhie1-wMhie6) were identified based on wsp sequences, and these strains all belong to the supergroup A, which is clearly distinguished from other representative stains and forms a unique cluster in the phylogenetic tree. 【Conclusion】 The genetic diversity of M. hieroglyphica populations distributed in South China is comparatively high. There is significant genetic differentiation among most populations and the gene flow is low among populations. No significant correlation exists between genetic differentiation and geographical isolation. High infection rates and diversity of Wolbachia exist in M. hieroglyphica populations in South China.
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cDNA cloning, prokaryotic expression, and ligand binding characterization of the odorant binding proteins CpunOBP3 and CpunOBP4 of the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae)
GUO Hong-Gang, WEI Chun-Hua, ZHANG Min-Zhao, QIN Xiao-Chun, DU Yan-Li
Acta Entomologica Sinica    2021, 64 (3): 327-336.   DOI: 10.16380/j.kcxb.2021.03.005
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【Aim】 This study aims to determine the physiological function of odorant binding proteins (OBPs) in the chemoreception of the yellow peach moth, Conogethes punctiferalis, so as to provide a theoretical basis for selecting OBPs that may be used as targets in C. punctiferalis biological control. 【Methods】 Two OBP genes (CpunOBP3 and CpunOBP4) were cloned from antennae of C. punctiferalis by PCR based on our previous antennal transcriptome data. The nucleotide and amino acid sequences of the two OBPs were analyzed with bioinformatics software. The recombinant  xpression vectors pET-30a/CpunOBP3 and pET-30a/CpunOBP4 were constructed. and the recombinant proteins CpunOBP3 and CpunOBP4 were obtained by prokaryotic expression and purification. The binding activity of the recombinant CpunOBP3 and CpunOBP4 to 24 ligands was analyzed by fluorescence competitive binding assay.【Results】 The open reading frame of CpunOBP3 gene (GenBank accession no.: GEDO010000010.1) is 387 bp in length, encoding a protein of 128 amino acids with the predicted molecular weight of 14.72 kD. The open reading frame of CpunOBP4 gene (GenBank accession no.: GEDO010000011.1) is 438 bp in length, encoding a protein of 145 amino acids. The predicted molecular weight of  CpunOBP4 without signal peptide is 12.82 kD. CpunOBP3 and CpunOBP4 share the typical structural features of OBPs, including six conservative cysteine residues. Both the recombinant CpunOBP3 and CpunOBP4 were mainly expressed in the inclusion. Fluorescence competitive binding assay indicated that the recombinant CpunOBP3 showed the binding ability to seven plant volatiles tested, with the strongest binding ability to 3-carene(Ki=10.33 μmol/L), but not to two sex pheromones tested. The recombinant CpunOBP4 exhibited the binding ability not only to the two sex pheromones tested (cis-10-hexadecenal with the Ki value of 14.65 μmol/L and hexadecanoyl with the Ki value of 7.83 μmol/L), but also to eight plant volatiles tested, with the strongest binding ability to ethyl butyrate (Ki=4.32 μmol/L). 【Conclusion】 Based on these results, we inferred that CpunOBP3 plays an important role in the host location and shift of C. punctiferalis, while CpunOBP4 possesses dual functions in recognizing sex pheromones and plant volatiles. The results provide a theoretic basis for controlling the occurrence and damage of C. punctiferalis via disturbing its olfactory reception.
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Identification of GABA receptor genes and the role of FoRDL in spinosad resistance in Frankliniella occidentalis (Thysanoptera: Thripidae)
WANG Jing, HE Bing-Qing, HUA Deng-Ke, ZHANG Kun, YUAN Jiang-Jiang, ZHENG Xiao-Bin, XU Bao-Yun, ZHANG You-Jun, WU Qing-Jun
Acta Entomologica Sinica    2021, 64 (8): 943-955.   DOI: 10.16380/j.kcxb.2021.08.006
Abstract207)      PDF(pc) (4607KB)(38)    PDF(mobile) (4607KB)(10)    Save

【Aim】 Gammaaminobutyric acid (GABA) is an important neurotransmitter in animal nervous system. This study aims to identify the GABA receptor (GABAR) familygenesinthewestern flower thrips, Frankliniella occidentalis, and to clarify the role of ionotropic receptor (GABAAR) in the resistance evolution to spinosad in F. occidentalis. 【Methods】 Based on the genomeand transcriptome data of F. occidentalis, the GABAR genes were identified, cloned and analyzed with bioinformatics tools. The expression patterns of the GABAAR subunit genes, FoRDL, FoLCCH3, and FoGRD in the spinosad susceptible strain of F. occidentalis at different developmental stages (1st-2nd instar nymphal, pupal, and adult stages), and their expression differences between the spinosad susceptible and resistant strains of F. occidentalis at the adult stage were detected by qPCR. After treatment with 0.250 and 0.400 mg/L spinosad at 24 h post RNAi of FoRDL in the 3-day-old adults of the spinosad susceptible strain of F. occidentalis, the mortality rates of F. occidentalis adults were determined by bioassay. 【Results】 Eight GABAR genes including FoRDL, FoLCCH3, FoGRD, FoGRD-like1, FoGRD-like2, FoB1, FoB2, and FoB-like (GenBank accession numbers: MH148151-MH148158) were annotated and cloned, and their ORF lengths vary from 1 080 to 3 720 bp. Phylogenetic analysis showed that GABAR genes of F. occidentalis were clustered with the corresponding genes of other insect species, indicating high conservativeness. GABAAR subunits FoRDL, FoLCCH3 and FoGRD all have a typical nitrogen-terminal extracellular region loop structure (loop A-F) and four transmembrane regions (TM 1-4), and exon 3 of FoRDL has mutually exclusive splicing. The expression levels of FoRDL, FoLCCH3 and FoGRD in the spinosad susceptible strain of F. occidentalis increased with the developmental stage of F. occidentalis, and the expression peak occurred at the adult stage. The expression level of FoRDL in the spinosad resistant strain of F. occidentalis at the adult stage was significantly lower than that in the susceptible strain at the adult stage. After treatment with 0.250 and 0.400 mg/L spinosad following RNAi of FoRDL in the susceptible strain of F. occidentalis, the mortality rates of F. occidentalis adults decreased significantly by 55.80% and 43.00%, respectively, as compared to those of the control. 【Conclusion】 Five ionic and three metabotropic GABAR genes have been identified in F. occidentalis. FoRDL may play a role in the resistance evolution to spinosad in F. occidentalis.

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Diversity and differences of gut bacterial communities in different instar larvae and diapause prepupae of Colletes gigas (Hymenoptera: Colletidae)
KOU Ruo-Mei, LI Yue, DOU Fei-Yue, ZHOU Ze-Yang, HUANG Dun-Yuan
Acta Entomologica Sinica    2021, 64 (6): 682-693.   DOI: 10.16380/j.kcxb.2021.06.004
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【Aim】 Based on the important role of insect gut microorganisms in host health, growth and development, this study aims to preliminarily explore the diversity and differences of gut bacterial communities in different instar larvae and diapause prepupae of Colleles gigas. 【Methods】 The V3-V4 gene fragment of 16S rRNA was amplified by PCR using the bacterial DNA extracted from the gut contents of the 1st-5th instar larvae and diapause prepupae of C. gigas collected from the field, and was sequenced by IlluminaMiseq second-generation high-throughput sequencing technology. Based on the obtained sequence data, the composition, abundance and diversity of gut bacteria of larvae and diapause prepupae of C. gigas were analyzed by bioinformatics methods. 【Results】 Bacteria of 15 phyla, 23 classes, 43 orders, 80 families and 128 genera were detected in the gut bacterial communities of C. gigas larvae, of which the main phylum, order, family and genera were Proteobacteria (accounting for 93.74%), Rickettsiales (accounting for 68.68%), Anaplasmataceae (accounting for 68.64%), and Wolbachia (accounting for 68.64%), respectively. Beta diversity analysis showed that the gut bacterial community changed with the development of larvae, and could be divided into three groups: early instar group (1st-3rd instar larva), late instar group (4th-5th instar larva) and diapause prepupa group. Alpha diversity analysis showed that there was a significant difference in the gut bacterial diversity between the diapause prepupa group and the late instar group and early instar group, while the gut bacterial diversity between the late instar group and the early instar group showed no significant difference. The linear discriminant analysis results showed that there was significantly dominant class in both diapause prepupa group and early instar group, being Alphaproteobacteria and Gammaproteobacteria, respectively, but no dominant class existed in the late instar group. At the order level, Enterobacteriales and Pseudomonadales were dominant in the early instar group, and Rickettsiales was dominant in the diapause prepupa group. At the family level, Enterobacteriaceae and Moraxellaceae were dominant in the early instar group, and Anaplasmataceae was dominant in the diapause prepupa group. At the genus level, Enterobacter and Acinetobacter were dominant in the early instar group, and Wolbachia was dominant in the diapause prepupa group. The results of functional gene annotation also showed the characteristics of the three groups. 【Conclusion】 There are significant differences in the community structure of gut bacteria in different instar larvae and diapause prepupae of C. gigas. The bacterial diversity decreases gradually from the early instar group to the late instar group and then to the diapause prepupa group, and this may be related to the feeding characteristics and adaptation of gut microorganisms to the gut environment. This study lays a foundation for the study of gut microorganisms of soil-nesting wild bees and also provides a new angle and direction for the protection of this kind of wild bees.
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Inter-specific competition between invasive ant Anoplolepis gracilipes and native ant Oecophylla smaragdina (Hymenoptera: Formicidae) in Xishuangbanna, southwestern China
LÜ Xiao-Yan, LIU Xia, ZHANG Yuan
Acta Entomologica Sinica    2021, 64 (10): 1196-1204.   DOI: 10.16380/j.kcxb.2021.10.009
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【Aim】 Invasive species can affect the biodiversity of an invasive site by influencing native species populations through competition. Anoplolepis gracilipes is one of the most destructive invasive ants in the world. This study aims to identify the competitive relationship between A. gracilipes and a dominant indigenous ant species Oecophylla smaragdina in Xishuangbanna, southwestern China. 【Methods】 By combining field investigation and the controlled experiment, the body size, the patterns of foraging activity outside the nest in cold fog season and rainy season, the foraging ability (foraging time and the maximum number of recruited workers within foraging time), the fighting behavior (attack intensity and mortality in different fighting combinations), and the starvation and thirst tolerance (the mean survival time and survival rate along time when no food and water were supplied) between A. gracilipes and O. smaragdina were observed and comparatively analyzed. 【Results】 The body length of A. gracilipes workers (3.66±0.06 mm) was significantly smaller than that of O. smaragdina workers (8.27±0.16 mm). The foraging time of A. gracilipes was longer than that of O. smaragdina in the fog cold season, while the numbers of foraging individuals of both species decreased in the high temperature period of the afternoon in the rainy season. When three different foods (apple, bee honey and sausage) were used as the bait, A. gracilipes only needed 4-8 min to find food, while O. smaragdina needed 8-21 min to find food. After finding food, A. gracilipes workers had the ability to gather their companions faster than O. smaragdina. In the controlled experiments, no attack or low intensity attack occurred predominantly in the combination of one individual of A. gracilipes with one individual of O. smaragdina, and when the number of individuals of either of the two species increased to five, the fighting intensity increased significantly, and both species exhibited intraspecific cooperation. There was no significant difference in the average survival time of workers between the two species under starvation and thirst, but A. gracilipes could survive for 120 h, while O. smaragdina could only survive for 96 h. 【Conclusion】 A. gracilipes exhibits stronger ability to forage and longer activity duration in the fog cold season than the indigenous ant species O. smaragdina in Xishuangbanna, suggesting that A. gracilipes may have strong temperature adaptability. It is necessary to intensify the research on this invasive species, and its population development in this area should be paid close attention to.

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Research progress of Notch signaling pathway in insects
YANG Xi, CHEN Peng, JIANG Xia, PAN Min-Hui, LU Cheng
Acta Entomologica Sinica    2021, 64 ( 2): 250-258.   DOI: 10.16380/j.kcxb.2021.02.012
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Composed of the Notch receptor, the Notch ligand (DSL protein), the CSL [C promoter binding factor-1 (CBF1), Suppressor of hairless (Su(H)), Lag-1] transcription factors, other effectors, and the regulatory molecules of Notch, Notch signaling pathway plays a fundamental role in the development of tissues in animals and the decision of cell fate in organs. Since its discovery in Drosophilia in 1917, the research on Notch signaling pathway based on insects has been very active, and it has been proved that it mainly plays fundamental roles in embryo and organ development regulation, cell proliferation and cell cycle regulation in insects. Notch gene locus mutation can lead to embryonic death and wing loss in Drosophila. The expression of intracellular domain of Notch (NICD) can affect the development of follicular cells in the ovary of Drosophila, cockroaches and other insects. Delta mediates the formation of body segments and the normal development of nervous system in insects. Su(H) mainly affects the cell cycle process of insect cells in the form of transcription factors. Fringe plays a key role in the development of wings of such insects as Drosophila and Bombyx mori. In addition, Notch signaling pathway interacts with Hippo signaling pathway, Wnt signaling pathway and EGFR signaling pathway, indicating that Notch signaling pathway is not a single line form but a complex network structure involved in insect life process. In recent years, the research on Notch signaling pathway has been extended from insects to major human diseases, oncology medicine and molecular therapy. In view of the highly conservative nature of Notch signaling pathway, the research results of Notch signaling pathway in insects not only play a key role in exploring the developmental mechanism of insects, but also provide important references and new ideas for studying other animals and even human diseases.
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Identification and functional analysis of heat shock protein 70 gene of Hyphantria cunea (Lepidoptera: Arctiidae)
QIAO Heng, LI Hui, GENG Yi-Shu, ZHAO Xu-Dong, YU Xiao-Hang, HAO De-Jun
Acta Entomologica Sinica    2021, 64 (7): 790-799.   DOI: 10.16380/j.kcxb.2021.07.003
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【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.
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Evaluation of the effects of Beauveria bassiana on the predation of Tetranychus urticae (Acari: Tetranychidae) by Orius sauteri (Hemiptera: Anthocoridae) using functional response model
CHEN Ya-Feng, WANG Su, DI Ning, JIN Dao-Chao
Acta Entomologica Sinica    2021, 64 (8): 967-975.   DOI: 10.16380/j.kcxb.2021.08.008
Abstract203)      PDF(pc) (1459KB)(65)    PDF(mobile) (1459KB)(15)    Save
 【Aim】 The application of natural enemies and biocontrol bacteria are effective means of pest biological control, and the combined application of the two kinds of agents have strong application prospects and potential of synergistic effect on biological control in the production. However, it is essential to evaluate the safety of biocontrol bacteria on natural enemies. This study aims to assess the biocontrol potential of combined release

of Beauveria bassiana and Orius sauteri for biological control of pests. 【Methods】 We measured the emergence rate of O. sauteri adults after the 5th instar nymphs were treated with two different concentrations of B. bassiana spore suspensions (routine concentration: 1×107 conidia/mL; low concentration: 5×103 conidia/mL) and spore powder (1×107 conidia/individual) and the predation ability of the 5th instar nymphs of O. sauteri

subjected to the above treatments to female adults of Tetranychus urticae under different prey densities. 【Results】 The emergence rate of O. sauteri adults was significantly reduced after the 5th instar nymphs were treated with B. bassiana spore powder (1×107 conidia/individual), and there was no significant difference in adult emergence rate between the other treatments and control. The predation of the 5th instar nymphs of O.

sauteri in the three treatment groups on female adults of T. urticae fitted with Holling Ⅱ functional response models, and the searching efficiency reduced with the increasing of prey densities. Specifically, the 5th instar nymphs of O. sauteri treated with B. bassiana spore suspension at a low concentration (5×103 conidia/mL) showed the highest daily maximal predation amount (Na-max) and the shortest handling time (Th), and the  gression of searching efficiency showed the lowest decreasing trend with the increasing of prey density. However, the 5th instar nymphs of O. sauteri treated with B. bassiana spore powder had the lowest Na-max and the longest Th. 【Conclusion】 Low concentration of B. bassiana spore suspension does not affect the predation ability of the 5th instar nymphs of O. sauteri on female adults of T. urticae, but spore powder treatment causes adverse effect to the predation ability of the 5th instar nymphs of this natural enemy on female adults of T. urticae. This study preliminarily demonstrated the feasibility of the combined application of low concentration of B. bassiana spore suspension and O. sauteri on the control of T. urticae.

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Expression and ligand binding characterization of the odorant binding protein AcerOBP14 of Apis cerana cerana
PENG Zhu, HUANG Li, ZHAO Shu-Guo, LÜJian-Hua, ZHAO Hui-Ting
Acta Entomologica Sinica    2021, 64 ( 2): 178-186.   DOI: 10.16380/j.kcxb.2021.02.005
Abstract202)      PDF(pc) (2224KB)(63)    PDF(mobile) (2224KB)(24)    Save
【Aim】 Odorant binding proteins (OBPs) play an important role in host localization, egg-laying site selection and other behaviors of insects. Clarifying the binding characteristics of AcerOBP14 with ligands helps to elucidate the molecular mechanism of olfactory recognition in Apis cerana cerana. 【Methods】 The expression levels of OBP14 in the antennae of the 20-day-old adult workers and drones of A. c. cerana and pollen foragers of A. c. cerana and Apis mellifera ligustica were detected by qRT-PCR. The prokaryotic expression vector pET28a/AcerOBP14 was constructed, and the recombinant AcerOBP14 protein was expressed and purified. The binding characteristics of AcerOBP14 with 37 odorant compounds were analyzed by fluorescence competition binding assay. 【Results】 The qRT-PCR analysis revealed that the expression level of OBP14 in the antennae of pollen foragers of A. c. cerana was significantly higher than those in the antennae of the 20-day-old adult workers and drones of A. c. cerana and pollen foragers of A. m. ligustica. Fluorescent competition binding assay results showed that AcerOBP14 had the binding ability with queen bee pheromone, alarm pheromone, Nasonov pheromone and a variety of plant volatiles, and showed the strongest binding ability to β-ocimene with the dissociation constant Ki value of 0.297 μmol/L. 【Conclusion】 AcerOBP14 has a broad ligand-binding spectrum, suggesting that it may be involved in a variety of physiological and behavioral responses of A. c. cerana, and play an important role in the pollen foraging behavior of A. c. cerana.
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Progress in the functional research of odorant receptors of agricultural insects
YOU Yin-Wei, ZHANG Long
Acta Entomologica Sinica    2021, 64 (5): 627-644.   DOI: 10.16380/j.kcxb.2021.05.010
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 Insects mainly rely on the olfactory system to seek foods, find mates, control mating, select oviposition sites and avoid natural enemies. Olfactory system is crucial for insect reproduction and survival. Odorant receptor (OR) is one of the key components of the olfactory system in insects. ORs can be activated by semiochemicals and then trigger special behaviors. With the development of sequencing technique, genomes and transcriptomes of plenty of agricultural insects have been sequenced, and OR gene families are analyzed and acquired from the sequencing data. Heterologous expression system and CRISPR/Cas9 system are frequently used in the research of OR functions nowadays. Heterologous expression system combined with recording system can be used to express target ORs and screen ligands. CRISPR/Cas9 system can be used to knock out target OR genes from insect chromosome, and then their functions are studied by electrophysiological techniques and behavioral experiments. In this article we systematically summaried the odorantresponse spectra and functions of the ORs of 30 agricultural insect species in six orders, Lepidoptera, Orthoptera, Hemiptera, Diptera, Hymenoptera and Coleoptera, particularly in Lepidoptera. Sex pheromones of agricultural insects are usually produced by females and consist of blends of two or more components at certain ratios, including behavioral antagonists involved in interspecific reproductive isolation. So several sex pheromone receptors in one species will be used to sense these pheromonal messages and then regulate intra- and inter-specific sexual behaviors. Some ORs are mainly tuned to plant volatiles including floral scent compounds, which play roles in host plant selection and oviposition site selection. Aggregation pheromone receptors can be activated by aggregation pheromones triggering aggregation behavior. Alarm pheromone receptors can be activated by alarm pheromones eliciting repellent behavior. Studies of odorant-response spectra and functions of ORs of agricultural insects will lay solid foundations for developing sex attractants, food attractants, antifeedants and aggregation pheromones used in pest control. At last we suggested the main research directions in the future for the agricultural insect ORs, including: (1) developing new heterologous expression systems for ORs; (2) investigating the functions of sex pheromone receptors in females specifically tuned to male-emitted sex pheromone components; and (3) exploring the molecular mechanisms of OR-odorant specific interactions.
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Fine structure of silk glands of Capnogryllacris nigromarginata (Orthoptera: Gryllacrididae)
DOU Yu-Jie, ZHAO Hui-Min, SHI Fu-Ming, CHANG Yan-Lin
Acta Entomologica Sinica    2021, 64 (7): 851-861.   DOI: 10.16380/j.kcxb.2021.07.009
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【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.
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Biological characteristics of adult Aphelinus maculatus (Hymenoptera: Aphelinidae)
LI Ke-Zhuo, FENG Shu-Jun, ZHAO Yan-Li, DUAN Li-Qing
Acta Entomologica Sinica    2021, 64 ( 2): 240-249.   DOI: 10.16380/j.kcxb.2021.02.011
Abstract198)      PDF(pc) (10023KB)(65)    PDF(mobile) (10023KB)(17)    Save
【Aim】 Aphelinus maculatus is one of the important parasitic wasps parasitizing on the cotton aphid, Aphis gossypii, discovered in recent years. There is little study on the biological characteristics including behavior and habits of Ap. maculatus so far. In order to explore and utilize this wasp effectively, it is important to ascertain its biological characteristics. 【Methods】 The emergence rhythm, mating behaviors, duration of various stages of mating, mating frequency, mating competition, sex ratio of offsprings reproduced by different reproductive modes, and longevity of adult Ap. maculatus were observed and recorded under microscope in the laboratory, and the parasitization process of this parasitoid on A. gossypii nymphs, the ovipositor insertion time of Ap. maculatus adults when parasitizing and feeding on A. gossypii, and the parasitization and feeding selection of Ap. maculatus adults to different day-old nympyhs of A. gossypii were also observed. 【Results】 Ap. maculatus adults emerge mainly at 6∶00-10∶00 am. Female and male adults mate on the day of emergence. The mating process can be divided into four phases: courtship, precopulation, copulation and postcopulation. The copulation duration is short, only lasting for 5.5±0.2 s. One male can copulate with 2-4 unmated females within 3 h, while the female only copulate once. Mating competition occurs among females and also among males. Ap. maculatus can reproduce offspring by parthenogenesis and amphigenesis. Only male offsprings are reproduced by parthenogenesis, but both female and male offsprings by amphigenesis. The parasitization process can be divided into such steps as searching host, examining host, probing insertion site, parasitic oviposition or feeding host, and cleaning and carding. The decision of parasitic oviposition or host feeding is related to the ovipositor insertion time into aphid body. When the ovipositor insertion time is longer than 4.8 min, adult wasps are more likely to feed host. Ap. maculatus wasps prefer to feed the early instar nymphs of A. gossypii, rarely feed 4 d or 5 dold nymphs, but can parasitize different dayold nymphs of A. gossypii. One female wasp can parasitize 14.8 A. gossypii nymphs and feed 3.7 A. gossypii nymphs on the average within 24 h. The adult longevity of Ap. maculatus is related to nutrient supplement after adult emergence. Adult wasps fed 10% sucrose and 10% honey could live 24±2.5 d and 28±1.6 d, respectively. 【Conclusion】 Ap. maculatus can reproduce by parthenogenesis and amphigenesis. Mating competition occurs among both females and males. The adult longevity of Ap. maculatus can be prolonged by supplying sucrose or honey.
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Classification, phylogeny and evolution of the Calyptratae (Insecta: Diptera) 
YAN Li-Ping, PEI Wen-Ya, ZHANG Dong
Acta Entomologica Sinica    2021, 64 (6): 757-768.   DOI: 10.16380/j.kcxb.2021.06.011
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 The Calyptratae (Diptera: Calyptratae) comprise 20% of the diversity of Diptera, one of the four superradiations of insects. The Calyptratae are distributed widely in the world, exhibit enormously diverse living habits, play vital roles in maintaining the stability of ecosystem, and are not only the hotspot groups in the studies of vectors, forensic medicine, pollinators and natural enemies of insects, but also key groups for exploring the phylogeny of Diptera and its successful adaptive radiation. To trace the evolutionary history of the Calyptratae, numerous dipterologists have conducted studies at various taxonomiclevels. TheCalyptratae arewellsupportedasa monophyletic group, and divided into three
superfamilies, i.e., Hippoboscoidea, Oestroidea, and Muscoidea, with monophyletic Oestroidea nested within paraphyletic Muscoidea, which are a sister group of Hippoboscoidea. At the family level, the Streblidae (Hippoboscoidea), Anthomyiidae (Muscoidea), Calliphoridae (Oestroidea), and Rhinophoridae (Oestroidea) are paraphyletic, and new families have been established, e.g., Polleniidae (Oestroidea) and Ulurumyiidae (Oestroidea). Therefore, the family level relationship of Calyptratae is still insufficiently resolved. Studies have been performed to reconstruct the evolutionary history of Hippoboscidae (Hippoboscoidea), Streblidae, Nycteribiidae (Hippoboscoidea), Muscidae (Muscoidea), Scathophagidae (Muscoidea), Sarcophagidae (Oestroidea), Gasterophilinae (Oestroidea: Oestridae), in terms of the origin and dispersal, host shift, and feeding habit. However, due to the lack of the biology information of some key groups and a well-resolved phylogeny, the evolutionary history of Calyptratae remains open. In this article we reviewed the research progress of classification, phylogeny and evolution of calyptrate flies, being the first review of the progress of the related research subjects of this group in the phylogenomic era.
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Effects of a sublethal dose of imidacloprid on the olfactory learning behavior of Apis mellifera ligustica workers and an analysis of their brain transcriptomes
HOU Meng-Shang, QIU Yuan-Mei, ZHAO Bi-An, YU Tian-Tian, LIANG Li-Qiang, SU Song-Kun, LI Zhi-Guo
Acta Entomologica Sinica    2021, 64 (7): 817-827.   DOI: 10.16380/j.kcxb.2021.07.006
Abstract198)      PDF(pc) (3819KB)(55)    PDF(mobile) (3819KB)(16)    Save
【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.
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Effects of the serine protease homologue SgSPH from the venom of Scleroderma guani (Hymenoptera: Bethylidae) on the phenoloxidase activity in the host hemolymph
LI Li-Fang, WU Chao-Yan, HAN Kai-Jian, WU Guo-Xing, ZHU Jia-Ying
Acta Entomologica Sinica    2021, 64 ( 2): 170-177.   DOI: 10.16380/j.kcxb.2021.02.004
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【Aim】 The aim of this study is to clone and express the venom serine protease homologue (SPH) gene of Sclerotium guani (SgSPH), and to investigate the effect of the venom protein encoded by this gene on the phenoloxidase activity in the host hemolymph. 【Methods】 The open reading frame (ORF) of venom SgSPH gene was cloned from S. guani by RT-PCR. Its sequence features were analyzed using bioinformatic software. The relative expression levels of the venom SgSPH gene at different developmental stages (egg, early instar larva, late instar larva, mature larva, spinning larva, pupa in yellow cocoon, pupa in black cocoon, and 1-5-day-old adults) and in different female adult tissues (head, thorax, abdomen without venom apparatus and venom apparatus) of S. guani were determined by qPCR. This venom gene was expressed with prokaryotic expression vector pSUMO-Mut. The expressed recombinant protein was purified using Ni-chelating affinity chromatography, and examined by SDS-PAGE and Western blot analysis. The inhibitory effect of the recombinant SgSPH on the phenoloxidase activity in the pupal haemolymph of Tenebrio molitor was measured by enzymatic activity assay. 【Results】 The ORF of the venom SgSPH gene (GenBank accession number: MT920663) of S. guani was cloned. It is 798 bp in length, encoding 265 amino acids, with the signal peptide consisting of amino acids 1-20, and the predicted protein molecular mass of 30.53 kD and pI of 9.59. Multiple sequence alignment results showed that SgSPH of S. guani shares low amino acid sequence identity (9%-17%) with the serine proteases and SPHs from venoms of other parasitoid wasps, and lacks a conservative catalytic triad. The qPCR results indicated that SgSPH gene was abundantly expressed at the adult stage and in the venom apparatus of S. guani. SDSPAGE and Western blot analyses showed that the recombinant SgSPH was successfully expressed and the highly purified recombinant SgSPH was obtained. Enzymatic activity assay results showed that the recombinant SgSPH was able to inhibit the phenoloxidase activity in the pupal hemolymph of the host T. molitor. 【Conclusion】 The results suggest that the venom SgSPH of S. guani can interfere with the host phenoloxidase cascade.

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Effects of exogenous juvenile hormone on the diapause termination and post-diapause development of Chrysoperla sinica (Neuroptera: Chrysopidae)
HUANG Hai-Yi, ZHAO Yue-Ming, WU Xiao-Liang, CHEN Zhen-Zhen, XU Yong-Yu
Acta Entomologica Sinica    2021, 64 (3): 392-399.   DOI: 10.16380/j.kcxb.2021.03.011
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 【Aim】 The aim of this study is to clarify the effective doses of exogenous juvenile hormone (JH) and the optimal treatment period for rapid diapause termination in the green lacewing, Chrysoperla sinica. 【Methods】 The pre-oviposition period, oviposition duration, female longevity and number of eggs laid per female of diapause adults of C. sinica exposed to different doses of exogenous JH (0, 5, 15, 25 and 35 μg/adult) by topical application and the changes of these four indexes of diapause C. sinica adults treated at different day-old age (0, 5, 10, 20, 30 and 40 day-old) with 15 μg/adult exogenous JH were determined. 【Results】 The pre-oviposition period of the diapause adults of C. sinica exposed to 15 and 25 μg/adult exogenous JH was 6.82 and 6.29 d, respectively, significantly shorter than that (10.55 d) in the control group (only treated with acetone). The oviposition duration, female longevity and the number of eggs laid per female of the diapause adults of C. sinica exposed to 15 μg/adult exogenous JH were all the highest, significantly higher than those of the control group. After diapause C. sinica adults were exposed to 15 μg/adult exogenous JH at different day-old age, the pre-oviposition period of adults treated at 0, 5,10 and 20 day-old age was significantly shorter than that of the control group non-subjected to exogenous JH treatment, the oviposition duration and female longevity of the adults treated at 10, 20 and 30 day-old age showed no significant difference from those of the control group non-subjected to exogenous JH treatment, while the numbers of eggs laid per female of the adults treated at 10, 20 and 30 day-old age were significantly reduced as compared to the control group non-subjected to exogenous JH treatment. Both adults subjected and non-subjected to exogenous JH treatment at 20 day-old age had higher fecundity. 【Conclusion】 Considering all the four indexes of pre-oviposition period, oviposition duration, oviposition amount and female longevity, we recommend 15 μg/adult exogenous JH as the optimal dose for the rapid diapause termination of C. sinica. The optimal treatment period for rapid diapause termination is the adult diapaused for 20 d, which has the least effect on the reproductive ability of C. sinica adults. The bottleneck problem of long time needed for diapause termination of C. sinica has been solved in this study, providing a new idea for the efficient storage and utilization of natural enemies with adult diapause and the fast diapause termination.

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Effects of Ascosphaera apis infection on the expression of immune- and detoxification-related genes and contents of proteins, lipids and glucoses in the gut of Apis mellifera ligustica larvae
HOU Meng-Shang, ZHAO Bi-An, QIU Yuan-Mei, WAN Kun-Lin, LIANG Li-Qiang, LI Zhi-Guo, SU Song-Kun
Acta Entomologica Sinica    2021, 64 (5): 574-584.   DOI: 10.16380/j.kcxb.2021.05.004
Abstract195)      PDF(pc) (1423KB)(72)    PDF(mobile) (1423KB)(14)    Save
【Aim】 Ascosphaera apis is a fungal pathogen that infects Apis mellifera ligustica larvae and causes chalkbrood in honeybee colonies. This study aims to investigate the effects of A. apis infection on the expression of genes related to intestinal immunity and detoxification and the contents of proteins, lipids and carbohydrates in the gut of A. m. ligustica larvae, and to analyze the relationship between the immune response and nutrients of A. m. ligustica under A. apis infection. 【Methods】 The A. m. ligustica larvae were raised in the laboratory. Each 3-day-old larva was inoculated with 2×106 spores/mL of A. apis in the treatment group, and that in the control group was fed with the normal food. Then, the gut samples of 6-day-old larvae were collected. The expression levels of genes related to immune, detoxification and development and genes of pathogens in the gut samples were detected by qRT-PCR, and the contents of proteins, lipids, glucose, and glycogen in the gut samples were determined by biochemical methods. 【Results】 After A. apis infection, the expression levels of immune-related genes LOC406144, Apid1, Def1, Def2, LOC406142, PGRP-SA, Pgrp-s2, PGRP-S3, PGRP-lc, GNBP-1, GNBP3, PPOact, LOC552247, domeless, KAT2A and bsk in the larval gut of A. m. ligustica were significantly up-regulated and that of cactus was significantly down-regulated. The expression levels of detoxification-related genes Cat, GSTS3, Cyp4g11, LOC725294, Ampka-r1, LOC411223, LOC409791, AmNOS and Sod2 in the larval gut of A. m. ligustica infected by A. apis were significantly up-regulated and those of CYP6AS14, CPR14, CYP306A1 and LOC100576555 were significantly down-regulated. The expression level of development-related gene usp in the larval gut of A. m. ligustica infected by A. apis was significantly up-regulated and those of VGMC, HEX70b and Vg were significantly down-regulated. The expression levels of pathogen genes BQCV capsid protein and Ascosphaera apis 28S rRNA in the larval gut of A. m. ligustica infected by A. apis were significantly up-regulated. The contents of proteins and lipids in the larval gut of A. m. ligustica infected by A. apis were significantly down-regulated, and those of glucose and glycogen were significantly up-regulated. 【Conclusion】 The A. apis infection influences the expression of genes related to immune, detoxification and development and genes of pathogens in the gut of A. m. ligustica larvae, affecting the immune response and energy stress response and accelerating the loss of proteins and lipids and the energy reservation of glucose and glycogen, and these may result in abnormal physiological activities of A. m. ligustica larvae.
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 Biology and management of the litchi stink bug, Tessaratoma papillosa (Hemiptera: Tessaratomidae): Progress and prospects (In English)
YAO Qiong, QUAN Lin-Fa, XU Shu, DONG Yi-Zhi, LI Wen-Jing, CHEN Bing-Xu
Acta Entomologica Sinica    2021, 64 (5): 645-654.   DOI: 10.16380/j.kcxb.2021.05.011
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 Litchi stink bug, Tessaratoma papillosa (Hemiptera: Pentatomidae), is one of the most widespread and destructive pest species on litchi (Litchi chinensis) and longan (Euphoria longan) in South China and Southeast Asia. T. papillosa feeds on the buds, tender branchlets, flowers, and fruits of host plants. Furthermore, the nymph over the 3rd instar and adult of T. papillosa are the vectors of witches’ broom pathogen on longan tree. In this article, we provide a detailed review on T. papillosa based on the research over the past 60 years in China, in order to provide references for the further study and development of green control technology of this pest species. T. papillosa is a hemimetabola insect which occurs one generation a year. Its nymphal duration and adult longevity are about 80 d and 203-371 d, respectively. Moreover, averagely one female adult of T. papillosa can deposit 190 eggs in its lifetime. Regarding the life habits, T. papillosa has the aggregation behavior, possesses phototaxis and chromatics tropism, and prefers tender branchlets. By the classification and distribution observation of antennal sensilla, RNA-seq analysis of antennae, dissection and observation of scent gland and comparative analysis of secretary components from scent gland, the two organs of antennae and scent gland of T. papillosa have been more deeply studied and understood. To date, the occurrence of T. papillosa is mainly forecasted through its ovarian development of female adults. Together with the pest forecasting technology, management of T. papillosa mainly depends on chemical control, supplemented by cultural, physical and biological control measures so far. The biological control technology has been reported the most, with aspects of the protection and utilization of natural enemies and the utilization of botanic pesticide. Owing to the seasonal restriction of experimental insect source and geographical limitation of infestation, the research progress of T. papillosa is relatively slow, the in-depth studies are limited and the research fields are relatively narrow. In future study, we can explore the selection mechanism of host plants, the interaction among host plants, natural enemies and symbiotic bacteria, and pesticide resistance from the perspectives of omics, molecular biology, cell biology and other aspects, to provide new clues for green pest control technology of T. papillosa.

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Binding mode of bisphenol A (BPA) with Drosophila melanogaster estrogen-related receptor (dERR) and its effect on the expression of dERR gene
WANG Li-Chao, LI Jia-Peng, ZHENG Xiang-Xiang, WANG Juan, LIAO Yan-Feng, OUYANG Xia-Hui
Acta Entomologica Sinica    2021, 64 (10): 1127-1135.   DOI: 10.16380/j.kcxb.2021.10.001
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【Aim】 Estrogen-related receptors (ERRs) belong to the nuclear receptor (NR) superfamily and play an important role in regulating metabolism and energy conversion in insects. Bisphenol A (BPA) is related to insect reproduction and neurological diseases. This study aims to explore the mechanism of BPA affecting Drosophila melanogaster ERR (dERR). 【Methods】 The docking of small molecule BPA with dERR protein constructed by Modeller 9.25 was simulated by AutoDock Vina, the molecular dynamics simulation was carried out using Gromacs 5.1.9, and the binding mode of BPA and dERR was explored in combination with the calculation of binding free energy. qRT-PCR method was used to detect the effects of exposure of BPA at three concentrations (0.1, 1 and 10 μg/L) for 6, 12 and 24 h on the transcription levels of dERR gene in adults and the 2nd instar larvae of D. melanogaster. 【Results】 Based on molecular docking and polar solvation energy, it is found that side chain amino acids such as Phe370 and Leu334 are the key amino acids for the binding of BPA to dERR. qRT-PCR analysis showed that the transcription levels of dERR gene in adults and the 2nd instar larvae of D. melanogaster changed significantly at 6 and 12 h after treatment with BPA of different concentrations, but those of 0.1 μg/L BPA treatment were almost equal to those of the control at 24 h. 【Conclusion】 BPA can affect the expression of dERR in D. melanogaster, and the mechanism may be related to the potential specific binding of BPA and dERR. 

Key words: Drosophila melanogaster; BPA; estrogenrelated receptor; molecular docking; molecular dynamics simulation; qRT-PCR

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Heterogeneity and bamboo lignocellulose degradation ability of microbiota in different sections of the alimentary canal of Cyrtotrachelus buqueti (Coleoptera: Curculionidae)
TANG Hao, WANG Ming-Jun, YANG Xiao-Wen, WU Min, LUO Wen, LUO Chao-Bing
Acta Entomologica Sinica    2021, 64 (4): 449-459.   DOI: 10.16380/j.kcxb.2021.04.004
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【Aim】 Symbiotic microbiota in the alimentary canal of Cyrtotrachelus buqueti participate in the degradation of bamboo lignocellulose. This study aims to reveal the heterogeneity and lignocellulose degradation ability of symbiotic microbiota in different sections of the alimentary canal of larval C. buqueti. 【Methods】 The composition analysis and functional prediction of microbiota in different sections of the alimentary canal including mouthpart (YB), foregut (YFG), midgut (YMG) and hindgut (YHG) of larval C. buqueti were carried out using 16S rRNA sequencing. The carbohydrate active enzyme (CAZy) genes in the genomes of bacteria belonging to core genera in each alimentary canal section were analyzed to predict the lignocellulose degradation ability. The liquid suspensions of mouthpart mixed bacteria (MPJ), foregut mixed bacteria (FJ), midgut mixed bacteria (MJ) and hindgut mixed bacteria (HJ) of C. buqueti larva were used to degrade bamboo shoot particles in vitro and to verify their lignocellulose degradation ability. 【Results】 The diversity analysis of microbiota in the alimentary canal of larval C. buqueti showed that the diversity of microbiota from YFG, YMG and YHG were higher than that from YB, and the species diversity of microbiota from YFG was the highest, while that of YB was the lowest. In YFG, YMG and YHG samples, Firmicutes, Bacteroidetes and Proteobacteria showed the highest relative abundance, while Proteobacteria, Firmicutes and Actinobacteria were the most abundant in YB. The analysis of CAZy genes in the genomes of bacteria belonging to core genera showed that most microbial genomes in the alimentary canal of larval C. buqueti contain abundant CAZy genes, especially in that of the genus Bacteroides, indicating their relationship with lignocellulose degradation. The experiment results of in vitro bamboo shoot particle degradation by MPJ, FJ, MJ and HJ of C. buqueti larva showed that their cellulose degradation efficiencies were 21.7%, 39.9%, 44.2% and 21.0%, respectively, their hemicellulose degradation efficiencies were 72.7%, 52.3%, 65.7% and 61.5%, respectively, and their lignin degradation efficiencies were 20.5%, 41.3%, 39.9% and 37.9%, respectively. 【Conclusion】 The heterogeneity of microbiota in the alimentary canal of larval C. buqueti can affect the lignocellulose degradation ability, so the microbiota can be used as one of the important sources for the separation of high.efficiency lignocellulose.degrading bacteria. This study provides some reference information for the industrial transformation and utilization of bamboo biomass.
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Effects of three microbes, Lactobacillus plantarum, Acetobacter malorum, and Saccharomyces cerevisiae, on the behavior and development of Drosophila melanogaster
WANG Lu, WEI Bo-Fan, LI Miao-Miao, LI Xiao-Zhe, WANG Bo, KAN Yun-Chao, QIAO Hui-Li
Acta Entomologica Sinica    2021, 64 (4): 460-470.   DOI: 10.16380/j.kcxb.2021.04.005
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【Aim】 Gut microbes play very important roles in various life processes of host. This study aims to further clarify the mechanism of interaction between gut microbes and host by exploring the effects of three microbes, Lactobacillus plantarum, Acetobacter malorum, and Saccharomyces cerevisiae, on the foraging, oviposition and development of Drosophila melanogaster. 【Methods】 Attraction assays were performed to detect the attractiveness of L. plantarum, A. malorum or S. cerevisiae in the culture to the unmated and mated adults of D. melanogaster, and the attractiveness of the single microbe or the mixture of the two or three microbes to the non-virgin D. melanogaster. Oviposition assays were performed to detect the oviposition preference of non-virgin D. melanogaster to the single microbe or the mixture of the two or three microbes. The eggs of D. melanogaster were transferred to the media inoculated with L. plantarum, A. malorum and S. cerevisiae, respectively, and the normal medium (control), and the larval body weight was measured; meanwhile, the eggs of D. melanogaster were transferred to the media inoculated with live and inactivated single microbe of the three microbes, respectively, and the normal medium, and the number of pupae was counted to ascertain the effects of different microbes on the development of larvae of D. melanogaster. The relative expression levels of InR, a key gene of insulin signaling pathway, in D. melanogaster larvae raised in the media containing L. plantarum, A. malorum and S. cerevisiae, respectively, and the normal medium for 72 h were detected by qRT-PCR. 【Results】 A. malorum mainly affected the oviposition of D. melanogaster, while L. plantarum and S. cerevisiae could affect both its foraging and oviposition. Compared with single microbe, the mixture of the two or three microbes had stronger attractiveness to non-virgin D. melanogaster. The oviposition preference assays showed that L. plantarum, A. malorum, S. cerevisiae and the mixture of the two or three microbes had significant attractiveness to D. melanogaster for oviposition in the order of mixture>A. malorum>S. cerevisiae>L. plantarum. In addition, L. plantarum, A. malorum and S. cerevisiae could promote the development of D. melanogaster larvae. The live microbes accelerated the development of D. melanogaster from larvae to pupae in the early stage of inoculation, but the promotion effect of the inactivated microbes lagged behind. The expression level of InR in the larvae of D. melanogaster cultured on the medium inoculated with A. malorum decreased significantly as compared to that in the control, while those in the larvae cultured on the media inoculated with L. plantarum and S. cerevisiae, respectively, significantly increased. 【Conclusion】 A. malorum can induce D. melanogaster to lay eggs, and L. plantarum and S. cerevisiae are attractive to D. melanogaster for foraging and oviposition. Meanwhile, microbial diversity can increase the foraging and oviposition preference of D. melanogaster. L. plantarum, A. malorum and S. cerevisiae may affect the growth and development of D. melanogaster larvae via different regulatory mechanisms.
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