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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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Toxicity and risk assessment of eleven pesticides to workers of Bombus terrestris (Hymenoptera: Apidae)
WANG Hong-Dong, HAN Bing, WANG Yu-Sai, HAN Shuang, WANG Ru-Ming, LI Dong-Gang
Acta Entomologica Sinica    2021, 64 (11): 1350-1358.   DOI: 10.16380/j.kcxb.2021.11.012
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【Aim】 To assess the ecological risk of common pesticides to Bombus terrestris, so as to provide a scientific basis for the rational application of pesticides in greenhouses. 【Methods】 The acute oral toxicity and acute contact toxicity of eleven pesticides, including six insecticides (chlorfenapyr, beta-cyhalothrin, flupyradifurone, spirotetramat, isoprocarb, and diflubenzuron), three acaricides (cyflumetofen, fenpyroximate, and bifenazate), and two fungicides (kasugamycin and boscalid), to adult workers of B. terrestris were determined by feeding method and contact method, respectively, and their ecological risk was assessed. 【Results】 Among the 11 pesticides tested, beta-cyhalothrin, isoprocarb and chlorfenapyr showed high acute oral toxicity, flupyradifurone and fenpyroximate showed medium acute oral toxicity, and the others showed low acute oral toxicity to B. terrestris workers. In the acute contact toxicity test, beta-cyhalothrin and isoprocarb showed high toxicity, chlorfenapyr showed medium toxicity, and the others showed low toxicity to B. terrestris workers. The ecological risk assessment showed that isoprocarb and beta-cyhalothrin had medium risk, and flupyradifurone, boscalid, diflubenzuron, fenpyroximate, bifenazate, spirotetramat, kasugamycin and cyflumetofen had low risk to B. terrestris workers via oral and contact exposure, while chlorfenapyr had medium risk via oral exposure and low risk via contact exposure to B.terrestris workers. 【Conclusion】 It is suggested that isoprocarb, beta-cyhalothrin and chlorfenapyr with medium risk should be banned when using B. terrestris pollination in the flowering period of greenhouse crops, flupyradifurone and fenpyroximate should be used cautiously to avoid harm to B. terrestris, while the other six low toxic pesticides could be applied reasonably according to the field conditions, and the ecological risk of pesticides to bumblebees can be reduced by means of ventilation, air drying and interval setting.
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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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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
Abstract489)      PDF(pc) (8683KB)(236)    PDF(mobile) (8683KB)(87)    Save
【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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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
Abstract482)      PDF(pc) (1508KB)(79)    PDF(mobile) (1508KB)(38)    Save

【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 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, Ca 2+ 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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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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Research advances in the diversity of symbionts in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)
SHENTU Xu-Ping, SHI Jia-Teng, SONG Yang, YU Xiao-Ping
Acta Entomologica Sinica    2021, 64 (8): 998-1008.   DOI: 10.16380/j.kcxb.2021.08.011
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 There are a large number of symbionts in the brown planthopper (BPH), Nilaparvata lugens, and these symbionts exhibit the diversity not only in their species but also in their functions on hosts. Up to now, 19 and 53 genera of symbiotic fungi and bacteria (sequencing abundance>0.1%), respectively, have been identified by using molecular biological methods and high-throughput sequencing technology, but plenty of symbionts remain unknown in taxonomic status due to technical limitations and their unculturable characteristics. Symbionts play vital roles in the life activities of BPH including the growth, development, reproduction, nutritional metabolism, resistance variation and immune function, and various symbionts have different functions. The symbiotic fungi are mainly involved in the synthesis of sterols and essential amino acids, while the symbiotic bacteria mainly take part in the synthesis of vitamins. The symbionts have important influence on the virulence variation, the development of high resistance to insecticides and the reproduction of host BPH, but the molecular mechanisms have not yet been clarified. In this article we reviewed the diversity of symbionts of BPH and prospected the focal points of future research including the species identification of symbionts of BPH, the functional studies of specific and single species of symbionts, the diffusion pathway, diffusion species and regulatory mechanism of symbionts in different tissues of BPH, and the control of BPH using symbionts as targets.
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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
Abstract360)      PDF(pc) (4607KB)(54)    PDF(mobile) (4607KB)(13)    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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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
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【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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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
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 【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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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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Defensive alkaloids of myrmicine ants
BAI Ru, CHEN Li, WANG Wen-Kai
Acta Entomologica Sinica    2021, 64 (7): 875-886.   DOI: 10.16380/j.kcxb.2021.07.011
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Eusociality is a major characteristic of ants (Formicidae), with apparent division of labor among individuals. They use complex cooperative strategies to protect their nests from predators, pathogenic microorganisms, and ant competitors, and to capture preys. Myrmicinae is the largest group of Formicidae, with 147 living genera. They mainly spray alkaloid-rich venom secretions for defense and hunting. In this article, the composition of defensive alkaloids of Myrmicinae ants and their distribution characteristics in different genera and species were reviewed, whose chemical structure mainly includes piperidine, pyridine, pyrrole, indolizidine, pyrrolizidine and fatty amine, and their function and application were summarized and prospected. Piperidine alkaloids are the typical characteristics of the venom of Solenopsis species, while pyrrolidine, pyrroline, and pyrrolizidine alkaloids predominate in the venom of M onomorium species. Indolizidine alkaloids are the major components of the venom secretion of Myrmicaria ants and Solenopsis thief ants. In addition, fatty amines are also components of the venom of Monomorium species. These venom alkaloids possess a variety of biological activities and have great application value in development of pesticides and biomedicines.
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Regulatory mechanism of DOPA decarboxylase (DDC) on fecundity in Harmonia axyridis (Coleoptera: Coccinellidae)
CHEN Xu, TIAN Ren-Bin, XU Qing-Xuan, LI Shu, WANG Su, ZANG Lian-Sheng, XIAO Da
Acta Entomologica Sinica    2022, 65 (1): 1-9.   DOI: 10.16380/j.kcxb.2022.01.001
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【Aim】The objective of this research is to explore the effects of DOPA decarboxylase (DDC) on fecundity in the multicolored Asian beetle, Harmonia axyridis, and its regulatory mechanism. 【Methods】RNA interference (RNAi) was used to suppress the expression of DDC gene ( HaDDC) in the 4th instar larvae of H. axyridis, and the cumulative number of eggs laid in 20 d was counted from the 8th day after adult emergence and the egg hatching rate of offspring was recorded on the 3rd day after egg laying. Then the ovaries of female adults of H. axyridis were dissected, the ovarian tissue morphology and the number of ovarioles of the 8-day-old adults between the treatment group (injected with ds HaDDC) and the control group (injected with ds GFP), and the development of eggs in the ovarioles of the 14- and 20-day-old adults between the treatment group and the control group were observed and recorded.【Results】After the expression of HaDDC gene of both sexes of H. axyridis was inhibited by RNAi, the cumulative number of eggs laid by adults in 20 d was 38.67±7.80, which was significantly lower than that of the control group (371.33±84.31). After the expression of HaDDC gene in female and male adults was inhibited, the cumulative numbers of eggs laid in 20 d were 135.50±28.38 and 76.00±14.00, respectively, which were significantly different from that of the control group. The egg hatching rate of offspring was 0 when the expression of HaDDC gene in both sexes was inhibited. On the 8th day after emergence of H. axyridis, the ovarian tissue morphology and the number of ovarioles in the treatment group were not significantly different from the control. On the 14th day after adult emergence, the ovaries of H. axyridis were plump, and the oocytes were developed in the bilateral ovarioles. However, compared with the control group, no mature eggs were found in the treatment group.【Conclusion】Based on the above results, we can preliminarily conclude that DDC can regulate the fecundity of H. axyridis by participating in the development process from oocyte to mature eggs.
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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 Ni 2+-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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Cloning and expression profiling of odorant binding protein genes in Callosobruchus chinensis (Coleoptera: Bruchidae)
WANG Hong-Min, ZHANG Jing, ZHANG Chong, ZHUANG Guo-Dong, ZHENG Hai-Xia, ZHANG Xian-Hong
Acta Entomologica Sinica    2021, 64 (8): 920-928.   DOI: 10.16380/j.kcxb.2021.08.004
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 【Aim】This study aims to clone and identify odorant-binding protein (OBP) genes in the adzuki bean seed weevil, Callosobruchus chinensis and to assay their tissue-specific expression profiles in C. chinensis adults, so as to to lay a foundation for studying the function of OBPs in olfactory perception in C. chinensis. 【Methods】Based on the antenna transcriptome data of C. chinensis, six OBP genes in C. chinensis were cloned by RT-PCR and analyzed by bioinformatics. The expression levels of OBP genes in tissues including head (excluding antennae), antennae, abdomen, legs and wings of female and male adults of C. chinensis were analyzed by qRT-PCR. 【Results】 The open reading frames of six OBP genes from C. chinensis were obtained, and named as CchiOBP1-CchiOBP6 (GenBank accession number: MN832700-MN832703, MN901841-MN901842). CchiOBP5 is a segment of Minus-C OBP with incomplete C-terminus, and the others belong to complete classical OBPs. It was predicted that all the six CchiOBPs contain signal peptides. Phylogenetic analysis showed that CchiOBP1, CchiOBP2 and CchiOBP5 were closely related to OBPs of Chrysomelidae, and CchiOBP3, CchiOBP4 and CchiOBP6 were closely related to OBPs of Cerambycidae. The qRT-PCR results showed that six CchiOBP genes were differentially expressed in the antennae, head (excluding antennae), abdomen, wings and legs of C. chinensis adults. CchiOBP1-4 and CchiOBP6 were highly expressed in antennae of female and male adults of C. chinensis, and their expression levels in antennae were extremely significantly higher than those in the other adult tissues. CchiOBP5 was highly expressed in female antennae and head (excluding antennae), but in males it showed significantly higher expression level in legs than in other adult tissues, with the lowest expression level in antennae. 【Conclusion】 The nucleotide and amino acid sequences of six CchiOBP genes of C. chinensis have been determined, among which five CchiOBP genes are highly expressed in the antennae of female and male adults of C. chinensis, suggesting their important roles in olfactory recognition of host plants.

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Insect olfactory behavior assay system based on trajectory tracking in four-quadrant olfactometer
SUN Lin-Lin, ZHANG Xuan, SHI Jia-Min, CHANG Xue-Fei, YE Gong-Yin, WANG Fang
Acta Entomologica Sinica    2021, 64 (7): 828-839.   DOI: 10.16380/j.kcxb.2021.07.007
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【Aim】 The study aims to establish a suitable olfactory behavior assay system for small insects such as Drosophila melanogaster based on trajectory tracking in four-quadrant olfactometer by analyzing the olfactory behavior of D. melanogaster under different experimental conditions. 【Methods】 The movement trajectory of D. melanogaster in the four-quadrant olfactometer was tracked, and the residence time and moving speed within each field arena was analyzed using Ethovision XT software. Meanwhile, the effects of airflow velocity and ordor placement on the attraction of apple vinegar and the repellence of Eucalyptus globulus leaf oil to D. melanogaster were investigated. 【Results】 The airflow velocity suitable for olfactory behavior analysis of D. melanogaster was from 300 to 1 200 mL/min, and 300 mL/min airflow was recommended as the optimal airflow velocity at which the moving speed of D. melanogaster individuals was more uniform. Ordor placement had no effect on the olfactory behavior of D. melanogaster, but had some impact on the attraction index of ordor. Single ordor showed better efficiency. Meanwhile, the efficiency of single individual test was better than that of multiple individual test. 【Conclusion】 The olfactory behavior assay system based on trajectory tracking developed in this study can be used for screening and identifying effective components in semiochemical compounds of insects. Better efficiency can be obtained by testing each insect individually with a suitable airflow velocity and single ordor. Multiple individual test could be used in the primary screening of compounds.
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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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Electroporation can be used to explore the gene  function in the silkworm, Bombyx mori ( In English)
ZHOU Wen-Lin, Haruhiko FUJIWARA, Nozomi UEMURA, YE Ai-Hong, WU Xue-Hui, CHEN Xue-Dong, ZHANG Ting-Ting, CAO Jin-Ru
Acta Entomologica Sinica    2021, 64 (7): 809-816.   DOI: 10.16380/j.kcxb.2021.07.005
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【Aim】 To confirm the effectiveness of electroporation-mediated functional analysis system in the silkworm, Bombyx mori. 【Methods】 siRNAs were synthesized for the target gene Wnt1 (Wingless), which is known to be involved in larval melanin coloration in B. mori. The day-3 4th instar larvae of B. mori were injected with Wnt1 siRNAs and subjected to electroporation as the treatment group (ERFA-RNAi) and those injected with Wnt1 siRNAs but without subjected to electroporation were used as the negative control group, the epidermis of the corresponding speckled area of the 5th instar larvae was dissected, and the relative expression level of Wnt1 in the epidermis was detected with real-time quantitative RT-PCR (qRT-PCR) to verify the effect of electroporationmediated RNAi. The transposon vector pPIG-A3GR with the enhanced green fluorescent protein reporter gene (EGFP) and the red florescence protein (RFP) reporter gene (DsRed2) expression cassettes, was introduced into the 2nd instar larvae of B. mori by electroporation. After 72 h of normal rearing, the expression of EGFP and DsRed2 in the larvae was observed under a fluorescent stereo microscope, to verify the somatic transgenesis of the silkworm. 【Results】 After the introduction of Wnt1 siRNAs into the day-3 4th instar larvae of B. mori, the formation of a speckle pattern of the 5th instar larvae was prevented on the larval body surface, and the qRT-PCR analysis showed that the expression level of Wnt1 in the epidermis of the 5th instar larvae was significantly decreased. The positive rate of somatic transgenic silkworm was 56.60%, and two fluorescent reporter genes EGFP and DsRed2 were continuously expressed in larval, pupal and adult stages. 【Conclusion】 Electroporation is an efficient technology for exploration of gene function in vivo, by efficiently introducing exogenous RNA or DNA into silkworm.

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Molecular characterization and expression profiling of LY domain-containing trypsin genes in Anopheles sinensis (Diptera: Culicidae)#br#
ZHANG Xiao-Xiao, LEI Dan, LI Xiang-Ying, CHEN Bin
Acta Entomologica Sinica    2021, 64 (9): 1050-1060.   DOI: 10.16380/j.kcxb.2021.09.005
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【Aim】 To identify LY domain-containing trypsin (LY-trypsin) genes of Anopheles sinensis at the whole genome, and to explore their molecular characteristics, expression patterns and phylogentic relationships. 【Methods】 The amino acid sequences encoded by trypsin genes in An. gambiae, Aedes aegypti, Drosophila melanogaster, and Culex quinquefasciatus were downloaded from NCBI databases and used as queries to search for trypsin genes in An. sinensis genome using the local Blast program. LY-trypsin genes in An. sinensis were named based on domain characteristics and phylogenetic relationship. The structure, scaffold location, structural domain and phylogenetic relationships of LY-trypsin genes of An. sinensis, and their expression patterns at different developmental stages of An. sinensis, in different adult tissues of An. sinensis, and in female adults of An. sinensis before and after blood feeding were predicted using bioinformatics analysis. 【Results】 Twenty-seven LY-trypsin genes were identified in the whole genome of An. sinensis, while no LY-trypsin gene was identified in D. melanogaster, An. gambiae, C. quinquefasciatus, and A. aegypti. The 27 LY-trypsin genes of An. sinensis encode 329-1 125 amino acids, with the molecular weight of 36.8-125.5 kD, and pI of 4.73-8.94. Among them 20 LY-trypsin proteins have signal peptides, with their length ranging from 10 to 62 aa. The numbers of exons of the 27 LY-trypsin genes of An.sinensis range from one to five, and the length of introns ranges from 62 to 20 093 bp. The 27 LY-trypsin genes of An.sinensis are mapped on 11 scaffolds, their encoded proteins all have a conserved YWTD motif (LY motif), and the amino acid sequences encoded by 16 LY-trypsin genes of the 27 LY-trypsin genes have the active site containing the catalytic triad of serine, histidine and aspartic acid. Phylogenetic results showed that the 27 LY -trypsin genes of An. sinensis clustered into four branches. At different developmental stages, more than 50% of the LY-trypsin genes of An. sinensis showed similar expression patterns and were expressed at high levels during the larval stage. LY-trypsin genes were expressed in different adult tissues of An. sinensis. Only a few of LY-trypsin genes were expressed in female adults of An. sinensis before and after blood feeding without expression specificity. 【Conclusion】 In this study, LY-trypsin genes have been identified in the genome of An. sinensis for the first time, and the molecular characteristics and expression patterns of the LY-trypsin genes have been investigated. This study provides an information framework for the further study of LY-trypsin genes.
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Electrophysiological responses of Holotrichia oblita (Coleoptera: Melolonthidae) to peach tree volatiles 
ZHANG Nuo, CHEN Li, XIE Guang-Lin
Acta Entomologica Sinica    2021, 64 (9): 1112-1119.   DOI: 10.16380/j.kcxb.2021.09.011
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 【Aim】 Holotrichia oblita is an economically important agricultural pest with a wide range of distribution and various host plant species. This study aims to identify peach tree volatiles that have the electrophysiological activity to the antennae of the female adults of H. oblita, and to further explore the effect of doses of the identified volatiles on the electrophysiological response in female and male antennae. 【Methods】 Volatiles from peach tree leaves were collected by dynamic headspace adsorption. The electrophysiologically active compounds from peach tree volatiles were identified by gas chromatography-electroantennographic detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS) techniques, and the responses of the antennae of female and male adults of H. oblita to the identified volatile compounds at doses of 0.01, 0.1, 1, 10 and 100 μg were tested with electroantennogram (EAG) technique. 【Results】 There were seven compounds in the peach tree volatiles that could trigger the electrophysiological reaction in the antennae of female adults of H. oblita, including Z-β-ocimene, Z-3-hexenyl acetate, Z-3-hexanol, nonanal, methyl salicylate and two unknown compounds. All the five peach tree volatile compounds with the electrophysiological activity to the antennae of female adults of H. oblita could trigger distinct EAG responses at all test doses in both female and male adults of H. oblita. The EAG responses of females and males to the same compound tended to increase with the increase of dose. The EAG response of females was significantly higher than that of males.  【Conclusion】 H. oblita adults generally show significant dose-dependent electroantennographic responses to Z-β-ocimene, Z-3-hexenyl acetate, Z-3-hexanol, nonanal, and methyl salicylate. There exists significant sexual difference in the EAG response. These compounds can be used for behavioral response and field trapping experiments to further confirm their attractiveness to H. oblita.
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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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Gene cloning, expression and subcellular localization of a novel spore wall protein of Nosema ceranae (Microsporidia)
XIONG Liang, AO Tang-Yan, ZHANG Zhen, MA Zhen-Gang, ZHOU Ze-Yang
Acta Entomologica Sinica    2021, 64 (9): 1070-1079.   DOI: 10.16380/j.kcxb.2021.09.007
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【Aim】 The spore wall of Microsporidia plays an important role in the process of spore formation and host infection. The objective of this study is to identify a novel spore wall protein in Nosema ceranae, and to conduct the gene cloning and prokaryotic expression in order to clarify the subcellular localization of this protein. 【Methods】 Bioinformatics analysis of a novel spore wall protein AAJ76_1400036761 in N. ceranae was carried out using online software. The target DNA fragment was obtained by PCR and then cloned into the prokaryotic expression vector pCold II. The recombinant protein was induced by IPTG and purified by Ni 2+ affinity chromatography. Polyclonal antibody was prepared by immunizing mice with the recombinant protein as antigen. The subcellular localization of the protein was analyzed by indirect immunofluorescence and immuno-colloidal gold labelling. Western blotting was used to detect the interaction between the protein and the chitin spore coat of N. ceranae. 【Results】 The gene sequence of AAJ76_1400036761 was obtained from Microsporidia DB. It is 681 bp in length encoding 226 amino acids with the predicted isoelectric point of 6.84 and molecular weight of 26.19 kD. The results of SDS-PAGE and Western blotting showed that the recombinant protein could be highly expressed in Escherichia coli Rosetta cells. Western blotting result revealed that the prepared polyclonal antibody could specifically recognize AAJ76_140003676 in the total proteins of N. ceranae, suggesting that this protein is expressed in mature spores of N. ceranae. The subcellular localization analysis showed that AAJ76_140003676 is located at the spore wall of N. ceranae. The recombinant protein AAJ76_1400036761 could bind with the chitin spore coat of N. ceranae. 【Conclusion】 AAJ76_140003676 is expressed in mature spores of N. ceranae. It is a novel spore wall protein of N. ceranae located at the spore wall. This study lays a foundation for further studying the biological functions of this protein.
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Identification of bacterial proteins in the saliva of adults of the tea green leafhopper, Empoasca flavescens (Hemiptera: Cicadellidae)
PAN Cheng, NIU Yu-Qun, XIA Lu-Xia, WU Chun-Fang, JING Kai-Ting, CHENG Qiang-Yi, WANG Meng-Xin, HAN Bao-Yu
Acta Entomologica Sinica    2021, 64 (8): 929-942.   DOI: 10.16380/j.kcxb.2021.08.005
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 Abstract: 【Aim】 To understand the bacteria species in the watery saliva of adults of the tea green leafhopper, Empoasca flavescens, by identifying the bacterial proteins in their saliva. 【Methods】 Saliva of tea green leafhopper adults was collected by the self-made collection device with treching two layers of Parafilm with sucrose diet. After concentration by ultrafiltration and electrophoretic separation, saliva protein solution was hydrolysated by filter-aided sample preparation (FASP) method. Saliva proteins were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The bacterial proteins in saliva were searched against the UniProt database of bacterial protein by using Mascot 2.2. 【Results】 One hundred and forty-two proteins from 49 species of bacteria in 27 orders were identified. Bacterial proteins of Proteobacteria were the most abundant, with a total of 107 proteins, among which bacterial proteins of α-Proteobacteria and γ-Proteobacteria accounted for the most. Bacterial proteins of Firmicutes (28 proteins) were the second most abundant, belonging to Bacilli and Clostrida, respectively. These proteins are involved in amino acid, vitamin and energy metabolism. 【Conclusion】 The identified bacterial proteins may be important participants in the life cycle of leafhoppers. This study provides basic information for further study on the relationship among endosymbiotic bacteria, tea green leafhoppers and tea plants.
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Effects of knocking down neuropeptide F gene ( npf) on the feeding, growth and reproduction of Ostrinia furnacalis (Lepidoptera: Pyralididae)
SHI Jian, WANG Yuan, LIANG Jia, DU Juan, ZHAO Zhang-Wu
Acta Entomologica Sinica    2021, 64 (9): 1080-1091.   DOI: 10.16380/j.kcxb.2021.09.008
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【Aim】 The neuropeptide F (NPF), a type of neuropeptides in invertebrates named based on its C-terminal phenylalanine (F), participates in the regulation of many physiological functions in insects such as feeding, biological rhythm, learning and memory. The objective of this study is to clarify the effects of NPF on the growth and development of the Asian corn borer, Ostrinia furnacalis, so as to provide an important basis for pest control. 【Methods】 A method based on the efficient synthesis of dsRNA targeting genes by engineering bacteria was used to efficiently and economically knock down npf. Low concentration (0.01%) and high concentration (0.02%) of ds NPF and ds GFP (the control) were separately fed to the early 1st, 3rd and 5th instar larvae of O. furnacalis until pupation. The average feeding amount, body weight, body length, survival rate and pupation rate of the 5th instar larvae, as well as the emergence rate of pupae and the number of eggs laid by adults were detected. Besides, the developmental duration at different larval instars and pupal stage and adult life span were also detected. 【Results】 When the early 1st, 3rd and 5th instar larvae of O. furnacalis were fed with 0.01% and 0.02% dsNPF, the feeding amount, body weight, body length, survival rate and pupation rate of the 5th instar larvae, the emergence rate of pupae and the number of eggs laid per female adult significantly decreased compared with the control fed with the corresponding concentrations of ds GFP. The developmental duration of larvae and pupae in the treatment groups were significantly prolonged, and the life span of adults was significantly shorter. The earlier the larval instar was treated with ds NPF, the greater the impact on development. Among them, the 1st instar larvae treated with 0.01% dsNPF and the 3rd instar larvae treated with 0.02% ds NPF had 90% of their individuals died in the pupal stage, and the 1st instar larvae treated with 0.02% dsNPF had 90% of their individuals died in the larval stage. 【Conclusion】 The results suggest that NPF plays a regulation role in the development and feeding of O. furnacalis, providing a basis for the exploration of new green pest control.
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Effects of silencing of major royal jelly protein 1 (Mrjp1) gene by RNAi on learning and memory in worker bees of  Apis mellifera ligustica
YU Tian-Tian, QIU Yuan-Mei, HOU Meng-Shang, WANG Tian-Bao, SU Song-Kun, LI Zhi-Guo ​
Acta Entomologica Sinica    2021, 64 (10): 1145-1152.   DOI: 10.16380/j.kcxb.2021.10.003
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【Aim】 In the previous studies it was found that the learning ability of Apis mellifera ligustica worker bees treated with imidacloprid is decreased, and transcriptomic analyses showed that the expression of major royal jelly protein 1 gene Mrjp1 is significantly down-regulated in the brains of bees treated with imidacloprid, suggesting that MRJP1 may be involved in olfactory learning in honeybees. This study aims to verify the crucial role of MRJP1 in olfactory learning in A. mellifera ligustica workers through silencing Mrjp1 by RNA interference (RNAi). 【Methods】 The cDNA sequence of Mrjp1 was obtained by cloning technique and validated by sequencing, and then the sequence was used for designing primers for generating dsRNA for knockdown of Mrjp1 by RNAi. The worker bees of A. mellifera ligustica injected with dsMrjp1 were assigned to the treatment group (dsMrjp1-injected group), and those injected with dsEGFP were assigned to the control group (dsEGFP-injected group). Then, the olfactory learning and memory abilities of the two groups were compared based on the proboscis extension response (PER) assay. Finally, the relative expression level of Mrjp1 in the brain of A. mellifera ligustica workers after injection of dsMrjp1 was detected by quantitative realtime PCR (qRT-PCR). 【Results】There was a significant difference in the learning ability of A. mellifera ligustica workers between the dsMrjp1injected group and the dsEGFP-injected group, with the learning ability of the dsMrjp1-injected group significantly decreased. However, there was no significant difference in the faculty of memory of A. mellifera ligustica workers at 2 h after learning between the two groups. The qRT-PCR results showed that the expression level of Mrjp1 in the brain of A. mellifera ligustica workers in the dsMrjp1-injected group was significantly lower than that in the dsEGFP-injected group, indicating that A. mellifera ligustica workers with decreased learning ability correspondingly exhibit lower expression level of Mrjp1. 【Conclusion】 After knockdown of Mrjp1 by RNAi, the olfactory learning performance of A. mellifera ligustica workers is significantly decreased, while their memory performance is not significantly affected, suggesting that Mrjp1 is probably one of the key genes regulating learning in A. mellifera ligustica. The results of this study contribute to further understanding of the molecular mechanism related to olfactory learning in honey bees.

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DNA methylation affects wing development of Bombyx mori through regulating autophagy
GONG Cheng-Cheng, LÜ Hao, ZHENG Si-Chun, XU Guan-Feng
Acta Entomologica Sinica    2022, 65 (2): 119-129.   DOI: 10.16380/j.kcxb.2022.02.001
Abstract294)      PDF(pc) (12458KB)(159)    PDF(mobile) (12458KB)(180)    Save
【Aim】This study aims to investigate whether DNA methylation affects wing development of Bombyx mori through regulating autophagy. 【Methods】B. mori ovarian Bm12 cells and B. mori prepupae were treated with 5azadC, a DNA methylation inhibitor, at the doses of 1 and 2 μg, respectively, the number of Bm12 cells was observed under fluorescence microscopy, the methylation level in Bm12 cells was detected by dot blotting, the autophagy intensity was detected by lysosome staining, the expression levels of autophagyrelated protein (Atg) genes were detected by RT-qPCR, and the typing of the autophagy marker LC3 protein in Bm12 cells was detected by Western blotting and immunohistochemistry. Meanwhile, the wing phenotype of adult was observed, and the abnormal wing rate and wing area were calculated. After B. mori prepupae were treated with the autophagy activator SMER28 (2 μg), and using autophagy inhibitor Spautin-1 (2 μg) to rescue 5-aza-dC-treated prepupae, the autophagy intensity in the wing cells was detected by lysosome staining, the wing phenotype was observed, and the abnormal wing rate and wing area were calculated. 【Results】Treatment with 1 μg of 5-aza-dC inhibited the growth and decreased the methylation level of Bm12 cells, increased the autophagy level in Bm12 cells at 12, 24 and 48 h after treatment, and up-regulated the expression of Atg genes in Bm12 cells at 48 h after treatment. Injection of 2 μg of 5-aza-dC into B. mori prepupae increased the autophagy level, up-regulated the expression of Atg genes in adult wing cells, and resulted in a large proportion of deformed wings at 24, 48 and 72 h after treatment, with the increased abnormal wing rate (increased by 72.62%) and the decreased wing area (decreased by 66%) of adult. Injection of 2 μg of SMER28 into B. mori prepupae increased the autophagy level in the adult wing cells and resulted in a large proportion of deformed wings at 24, 48 and 72 h after treatment, with the increased abnormal wing rate (increased by 75.13%) and the decreased wing area (decreased by 48.79%) of adult. In addition, rescue experiment using Spautin-1 to rescue the 5-aza-dC-treated prepupae revealed that the autophagy inhibition could alleviate the effect of DNA demethylation on the adult wing development. 【Conclusion】The results of this study demonstrate that DNA methylation plays a role in the wing development of B. mori by regulating autophagy. Our results provide the experimental evidence for the regulation of DNA methylation on the insect development.
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Effects of antimicrobial peptide HI-3 from Hermetia illucens (Diptera: Stratiomyidae) on the glutamine and glutamic acid metabolic pathway of human colon carcinoma HCT-8 cells
GAO Jia-Min, XU Xiao-Yan, HU Zi-Yuan, CHEN Ying-Dan, SUN Hong-Xia, YANG Yu-Feng, XIA Qiang
Acta Entomologica Sinica    2021, 64 (9): 1041-1049.   DOI: 10.16380/j.kcxb.2021.09.004
Abstract291)      PDF(pc) (1437KB)(58)    PDF(mobile) (1437KB)(27)    Save

【Aim】 To enrich the knowledge of the anticancer mechanism of antimicrobial peptide HI-3 extracted from Hermetia illucens by studying the effects of HI-3 on the amino acid metabolism of human colon carcinoma HCT-8 cells. 【Methods】 CCK-8 assay was used to determine the inhibition rates of different concentrations (80, 160, and 320 μg/mL) of antimicrobial peptide HI-3 on HCT-8 cells. The metabolites in HCT-8 cells were determined by GS-MS, and then the metabolic pathway with the most significant difference in amino acid contents and the target enzyme in this pathway were screened by pathway analysis based on R software. After the HCT-8 cells were treated with 320 μg/mL HI-3, the activity of the selected target enzyme glutaminase (GLS) was assayed by enzyme activity kit, its mRNA and protein expression levels were detected by RT-qPCR and Western blot, respectively, and the changes in the contents of important metabolites including glutamine (Gln), glutamic acid (Glu), glutathione (GSH), α-ketoglutaric acid (α-KG) and ATP involved in the glutamine and glutamic acid metabolic pathway in HCT-8 cells were detected by biochemical kits and ELISA kit. 【Results】 The inhibition rates of HCT-8 cells treated by HI-3 at the concentrations of 80, 160, and 320 μg/mL were 33.85%  ±3.50%, 46.26%±0.90%, and 55.53%±1.70%, respectively, and the inhibition rates increased with the increase of the HI-3 concentration. HI-3 at the concentration of 320 μg/mL showed the greatest effects on the glutamine and glutamic acid pathway with the contents of amino acid metabolites in this pathway most significantly different from those in the negative control group (0 μg/mL HI-3). The activity of GLS (the target enzyme in this pathway) and the mRNA and protein expression levels of its gene GLS in the 320 μg/mL HI-3 treatment group were extremely significantly lower than those in the negative control group. Moreover, the contents of important metabolites in this pathway, including Gln, Glu, GSH, α-KG and ATP, in the 320 μg/mL HI-3 treatment group were also significantly reduced as compared with those in the negative control group. 【Conclusion】 Antimicrobial peptide HI-3 from H. illucens at the concentration of 320 μg/mL has the most significant effect on the glutamine and glutamic acid metabolic pathway in HCT-8 cells, and can significantly inhibit the proliferation of HCT-8 cells by blocking this pathway.

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Effect of piwi knock-down on hemocyte proliferation and differentiation in Drosophila melanogaster
LIU Xiao-Nan, ZHAO Su-Juan, WANG Bo, WANG Hong-Xin, HAO Yang-Guang
Acta Entomologica Sinica    2021, 64 (12): 1359-1366.   DOI: 10.16380/j.kcxb.2021.12.001
Abstract290)      PDF(pc) (17680KB)(145)    PDF(mobile) (17680KB)(27)    Save
【Aim】 To explore the effect of piwi knock-down on hemocyte proliferation and differentiation in Drosophila melanogaster. 【Methods】 The D. melanogaster strains e33C-Gal4 and Hml-Gal4-UAS-2×EGFP were crossed with the wild strain w 1118 and the strain UAS-piwi RNAi, respectively, to reduce the expression of piwi gene in circulating hemocytes or lymph glands of D. melanogaster. The localization of Piwi protein in hemocytes and its effect on hemocyte proliferation and differentiation in D. melanogaster were detected by immunofluorescence staining. 【Results】 Piwi protein was expressed in circulating hemocytes and the whole lymph glands of D. melanogaster, and was mainly localized in the cytoplasm. The knock-down of piwi gene resulted in significant increase in the number of circulating hemocytes and the number of cells in the M phase of mitosis, but the differentiation of plasmatocytes and lamellocytes in circulating hemocytes were not affected. The knock-down of piwi gene had no effect on the proliferation of lymph gland hemocytes, but resulted in the excessive differentiation of plasmatocytes and the generation of lamellocytes. 【Conclusion】 The knock-down of piwi gene in circulating hemocytes in Drosophila can induce the overproliferation of hemocytes, while the knockdown of piwi gene in lymph glands can induce abnormal differentiation of hemocytes.
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Niche and interspecific association of darkling beetles in a desert grassland of alluvial fans in Helan Mountain, northwestern China
YANG Gui-Jun, WANG Yuan, WANG Min, JIA Long
Acta Entomologica Sinica    2021, 64 (7): 840-850.   DOI: 10.16380/j.kcxb.2021.07.008
Abstract285)      PDF(pc) (1713KB)(57)    PDF(mobile) (1713KB)(13)    Save
 【Aim】 The darkling beetle plays an important role in maintaining biodiversity and ecosystem function in desert semi-desert habitats. This study aims to reveal the niche and interspecific associations of darkling beetles so as to lay a basis for studying the mechanism of community construction in desert grasslands of alluvial fans. 【Methods】 A field survey was carried out to investigate darkling beetle communities using pitfall in a desert grassland of alluvial fans in Helan Mountain, northwestern China from May to October 2019. The sampling transect of about 200 m×200 m was divided equally into 100 square plots. Based on 2×2 contingency table analysis, niche breadth, niche overlap, variance ratio test, Chi-square test, association coefficient (AC), percentage of cooccurrence (PC), Ochiai index (OI), Dice index (DI) and Spearman rank correlation coefficient, we analyzed the niche and interspecific association of darkling beetle species at a small scale. 【Results】 A total of 1 086 individuals of adult darkling beetles belonging to 10 species from 7 genera were collected. Microdera kraatzi kraatzi, Anatolica pandaroides and Anatolica planata were the dominant species in the whole community. The spatial niche breadth of A. planata was the largest, the temporal niche breadth of Scytosoma pygmaeum was the largest and the temporal-spatial niche breadth of M. kraatzi kraatzi was the largest. The temporal, spatial and temporal-spatial niche breadth of Opatrum subaratum was the smallest, which was at a weak competitive position. The results of niche breadth clustering showed that M. kraatzi kraatzi, A. pandaroides, S. pygmaeum and S. dissilimarginis are wide niche species. The temporal niche overlaps of species pairs of darkling beetles were more significant than their spatial niche overlaps. The variance ratio and W test results showed that there existed a significantly positive interspecific association. The results of the Chi-square test, association coefficient (AC), and the Spearman rank correlation coefficient showed that the ratio of species pairs of the positive and negative associations was greater than 1 and the overall correlation tended to be positive. The percentage of co-occurrence (PC), Ochiai index and Dice index indicated that there was a significant positive correlation among the dominant species M. kraatzi kraatzi, A. pandaroides and A. planata. According to constellation diagram of the Spearman rank correlation coefficient network, the 10 species were divided into three ecological species groups. 【Conclusion】 The dominant species have a wide niche, the temporal niche and spatial niche of species vary inconsistently, and the niche overlaps of species pairs with significant positive connection are also relatively higher. Clustering of ecological species groups reflects the differences in the ecological adaptability of species pairs. The results provide references for studying the succession of darkling beetle community in desert grasslands of alluvial fans.
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Analysis of the transcriptomes of Galeruca daurica  (Coleoptera: Chrysomelidae) adults at different summer diapause stages ( In English)
LI Yan-Yan, CHEN Long, LI Ling, TAN Yao, PANG Bao-Ping
Acta Entomologica Sinica    2021, 64 (9): 1020-1030.   DOI: 10.16380/j.kcxb.2021.09.002
Abstract284)      PDF(pc) (2355KB)(68)    PDF(mobile) (2355KB)(19)    Save

【Aim】 This study aims to explore the crucial genes and metabolic pathways involved in obligatory summer diapause in Galeruca daurica, a new pest with great outbreak in the Inner Mongolia grasslands, northern China. 【Methods】 Using RNA-Seq, we performed sequencing, transcriptional profiling and functional prediction for G. daurica adults at different summer diapause stages, i.e. pre-diapause (PD), diapause (D) and post-iapause (TD), and screened differentially expressed genes (DEGs) at different summer diapause stages based on the RNA-Seq data. The expression levels of ten DEGs screened based on the RNA-Seq data were verified by qPCR. 【Results】 A total of 202 770 198 clean reads from nine libraries were filtered, and 12 078 060 transcripts were assembled into 82 292 unigenes with an average length of 783.59 bp and a N50 of 1 545 bp. The 2 395 (2 119 up-regulated and 277 down-regulated) and 62 (59 up-regulated and 3 down-regulated) DEGs were identified in the D vs PD and TD vs D omparison groups, respectively. The KEGG analysis revealed that the glycolysis/gluconeogenesis and fatty acid biosynthesis pathways were significantly enriched in the D vs PD and TD vs D comparison groups, respectively. Additionally, many DEGs related to the Ca2+ signaling were differentially expressed during diapause. Finally, the expressional analysis result by qPCR for ten DEGs showed a high consistency between qPCR and RNA-Seq results. 【Conclusion】 The glycolysis/gluconeogenesis, fatty acid biosynthesis, and Ca2+signaling pathways may play an important role in diapause regulation of G. daurica. This study establishes a foundation for future studies on the molecular mechanism underlying obligatory summer diapause in G. daurica.

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Light and dark adaptation of adult compound eyes of Spodoptera frugiperda (Lepidoptera: Noctuidae) and their transformation rate to light-adapted state under yellow light
JIANG Yue-Li, WU Yu-Qing, LI Tong, MIAO Jin, GONG Zhong-Jun, DUAN Yun, MEI Shi-Qiong, WANG Xue-Qin, LIU Qi-Hang
Acta Entomologica Sinica    2021, 64 (9): 1120-1126.   DOI: 10.16380/j.kcxb.2021.09.012
Abstract282)      PDF(pc) (2943KB)(65)    PDF(mobile) (2943KB)(32)    Save
【Aim】 The phototaxis of moths is directly related to the transformation of light and dark-adapted states of compound eyes. This study aims to clarify the relationship between light and transformation of light- and dark-adapted states of compound eyes of Spodoptera frugiperda. 【Methods】 Quickly taking photos at different time, the light- or dark-adapted state of compound eyes, light and dark-adapted transformation rate of S . frugiperda under light- or dark-adapted state and exposed to yellow light of different light intensities were statistically investigated. 【Results】 The maintenance rate of light-adapted state of compound eyes of S. frugiperda adults gradually increased as the light intensity increased after exposure to yellow light for 1 h under light-adapted state, those of males under yellow light at 0.1-0.5 and 4-6 lx were 67.77% (32.23% into dark-adapted and middle states) and 100%, respectively, while those of females under yellow light at 7-10 lx reached 98.90%. When S. frugiperda adults were exposed to yellow light for 3 h under light-adapted state, the maintenance rate of light-adapted state of compound eyes also gradually increased as the light intensity increased, those of males and females under yellow light at 0.1-0.5 lx were 50.00% and 32.23%, and those of females and males under yellow light at 7-10 lx were 90.00% and 100%, respectively. When S. frugiperda adults were exposed to yellow light at different intensities for 30 min under dark-adapted state, the compound eyes gradually transformed into light-adapted state, the light-adapted transformation rates of compound eyes of both females and males under yellow light at 0.1-0.5 lx were 93.33%, and those of females under yellow light at 0.6-0.9 lx and males under yellow light at 1-2 lx were 100%. 【Conclusion】 These results suggest that S. frugiperda adults have strong photosensitivity, and females are slightly more photosensitive than males.
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Discovery of the small cocoon mutant sc of Bombyx mori traveled onboard Tiangong-2 space laboratory in space and its gene mapping#br#
SHEN Guang-Sheng, SHEN Xing-Jia, ZHANG Long, ZHAO Qiao-Ling, GAO Meng-Jie, TANG Shun-Ming, HUANG Jing-Yi, CHEN Yan-Hua, JIANG Tao, ZHU Juan, WANG Mei-Xian
Acta Entomologica Sinica    2021, 64 (9): 1031-1040.   DOI: 10.16380/j.kcxb.2021.09.003
Abstract280)      PDF(pc) (3029KB)(73)    PDF(mobile) (3029KB)(13)    Save
【Aim】 The hybrid offspring of an alive female silkworm, Bombyx mori “Qiufeng× Baiyu” brought back to the earth after travelling onboard Tiangong-2 space laboratory in space for 33 d in 2016 was mated with the male moth of “Baiyu” strain reared on the earth ’s surface for producing eggs. Small cocoon individuals were found from the offspring, and then the normal cocoon strain TG and small cocoon mutant strain sc of ‘space silkworm’ were isolated and established. The objective of this study is to reveal the gene that leads to this mutant by analyzing its inherited characters and mapping the mutant gene. 【Methods】 Phenotypic analysis of TG and sc was performed. The sc, TG and normal large cocoon strain 0223V1 were served as experimental materials for preparation of ( sc♀×0223V1♂) F 1 and its backcross types, ( sc♀×0223V1♂) F 1♀×sc♂ and sc♀×( sc♀×0223V1♂) F 1♂, which were designated as BC1F and BC 1M, respectively. To screen polymorphic SSR markers, the genomic DNAs of sc, 0223V1 and F 1 were used as templates, and 10 SSR primers were randomly selected from each linkage group of B. mori for PCR amplification. By utilizing the characteristic of non-crossing-over between chromosomes in female B. mori, BC 1F population was used to identify the linkage group of sc gene. According to the SSR linkage map of B. mori, BC 1M population was used for gene mapping. 【Results】 Phenotypic analysis showed that the body size of sc larvae was smaller than that of TG larvae, and the cocoon weight of sc was about half that of TG. Genetic analysis showed that the mutant was controlled by a pair of recessive gene sc. Gene mapping results showed that sc is located between S2930-363 and S2930-289 SSR markers in the 3rd linkage group of the B. mori genome, with a physical distance of 684 kb containing 33 candidate genes. 【Conclusion】 This small cocoon mutant of space silkworm is controlled by a pair of recessive gene named sc located in the 3rd linkage group of the B. mori genome.
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Research progress of endosymbionts in lepidopteran insects
LIU Yuan, WANG Hao, WANG Zhi-Peng, CHEN Li-Min, WANG Ya-Ru, HOU You-Ming
Acta Entomologica Sinica    2021, 64 (12): 1465-1477.   DOI: 10.16380/j.kcxb.2021.12.012
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The symbiotic relationship between endosymbionts and their insect hosts is ubiquitous in nature, and they are interdependent, interacting and coevolving. In recent years, studies on insect endosymbionts mainly focus on hemipteran and dipteran insects. However, a growing number of studies show that the interaction mode and mechanism between lepidopteran insects and their endosymbionts are also attracting more and more attention. Lepidopteran insects are widely distributed and play important roles in the ecosystem as herbivores and pollinators, and most of their larvae can cause great economic losses to the agricultural and forestry production. The diversity of endosymbiont community in lepidopteran insects is relatively low, which is mainly dominated by secondary symbiont Wolbachia. A few species are also infected with Spiroplasma, Arsenophonus and Rickettsia. These endosymbionts are mainly transmitted maternally from mother to offspring, whereas their horizontal transmission may also be occurring in nature. And they play important roles in the growth and development, reproductive manipulation, environmental adaptation and genetic evolution of hosts. Nowadays, diagnostic polymerase chain reaction (PCR), high-throughput amplicon sequencing, and metagenomic sequencing are generally used to detect endosymbionts. However, there are still some difficulties in the research of endosymbionts in lepidopteran insects, including that most endosymbionts can not be cultured in vitro, and the biological functions of endosymbionts with low abundance are difficult to be determined. Considering the distribution of endosymbionts and difficulties in lepidopteran insects, it is suggested that future research should focus on secondary symbionts and their biological functions.
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Cloning of Spodoptera exigua (Lepidoptera: Noctuidae) caspase genes and their expression in response to apoptosis inducers and pathogenic microorganism infection
SONG Xiao-Hui, YANG Chang-Jin, LI Ni, HUANG Guo-Hua, YU Huan
Acta Entomologica Sinica    2021, 64 (7): 800-808.   DOI: 10.16380/j.kcxb.2021.07.004
Abstract277)      PDF(pc) (4177KB)(54)    PDF(mobile) (4177KB)(11)    Save
【Aim】 To identify the expression patterns of caspase in Spodoptera exigua induced by apoptosis inducer and under pathogenic microorganism stress, so as to lay a foundation for further study on the mechanism of apoptosis in Lepidoptera insects. 【Methods】 RT-PCR was used to amplify the full-length coding regions of two caspase genes ( SeCasp-3 and SeCasp-4) from the 3rd instar larvae of S. exigua. qPCR was used to detect the expression patterns of the two SeCasp genes in the fat body cells of S. exigua after induction by apoptosis inducers hydrogen peroxide (H 2O 2) (100 μmol/L), actinomycin D (ActD) (10 μg/mL) and dexamethasone (DEX) (50 μg/mL), and in the 3rd instar larvae of S. exigua infected by pathogens Bacillus thuringiensis kurstaki (10 8 colonies/mL), Escherichia coli TG1 (10 8 colonies/mL), Heliothis virescens ascovirus 3h (HvAV-3h) (1.16×10 11genome copies/mL) and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (5 000 OBs/μL). 【Results】 The coding regions of SeCasp-3 (GenBank accession no.: MW183334) and SeCasp-4 (GenBank accession no.: MW183335) are 942 and 843 bp in length, encoding 313 and 280 amino acids, respectively. The putative protein sequences of SeCasp-3 and SeCasp-4 show 45.54% and 58.46% amino acid sequence identities with Dronc of Bombyx mori, respectively, and SeCasp-3 and SeCasp-4 show high homology. SeCasp-3 and SeCasp-4 exhibited different expression patterns in the fat body cells of S. exigua induced by different chemicals. The relative expression levels of both SeCasp-3 and SeCasp-4 in the fat body cells treated by 100 μmol/L H 2O 2 and 10 μg/mL ActD for 24 and 48 h were significantly up-regulated. In the fat body cells of S. exigua treated by 50 μg/mL DEX for 24 and 48 h, the relative expression level of SeCasp-3 showed no significant change, but that of SeCasp-4 was significantly enhanced by several thousand folds as compared to the control. The expression patterns of SeCasp-3 and SeCasp-4 in the 3rd instar larvae of S. exigua infected by different pathogens were basically similar. The general linear model analysis showed that the infection of B. thuringiensis kurstaki and E. coli TG1 caused no significant change in the expression levels of SeCasp-3 and SeCasp-4 in the 3rd instar larvae of S. exigua, while the infection of HvAV-3h and AcMNPV significantly inhibited the expression of the two genes. 【Conclusion】 Two S. exigua caspase genes were identified and their expression responses to apoptosis inducers and pathogenic microorganism infection were assayed in this study, laying a foundation for further exploring the function of caspase and process of insect apoptosis.
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Expression of Kazal-type serine protease inhibitor gene AgKaSPI in Aphis glycines (Hemiptera: Aphididae) in response to Akanthomyces lecanii infection  
CHEN Ya-Ru, YANG Hong-Jia, LI Ze, CHE Jin-Ming, WANG Ze-Qun, FAN Dong
Acta Entomologica Sinica    2021, 64 (8): 908-919.   DOI: 10.16380/j.kcxb.2021.08.003
Abstract275)      PDF(pc) (1905KB)(56)    PDF(mobile) (1905KB)(12)    Save

 【Aim】 The objective of this study is to explore the role of Kazal-type serine protease inhibitor KaSPI in the development, digestion, and immune defense processes of the soybean aphid, Aphis glycines. 【Methods】 The cDNA sequence of Kazal-type serine protease inhibitor gene from A. glycines was cloned by PCR based on the transcriptome data of A. glycines. qRT-PCR was used to detect the expression levels of AgKaSPI in the 1st-4thinstar nymphs and adults of A. glycines, and in A. glycines adults at 3, 6, 12, 24, 48 and 72 h after infection by Akanthomyces lecanii. After RNAi of AgKaSPI with siRNA for 3, 6, 12, 24 and 48 h, the RNAi efficiency was detected by qRT-PCR, and the mortality and fecundity of A. glycines adults at 12, 24, 48 and 96 h post RNAi were recorded. The AgKaSPI content and the activities of serine protease, trypsin and chymotrypsin in A. glycines adults were detected by double antibody sandwich method after infection by A. lecanii and RNAi of AgKaSPI. The mortality of A. glycines adults at 12, 24, 48 and 96 h after infection by A. lecanii following RNAi of AgKaSPI for 6 h was observed and recorded. 【Results】 The cDNA sequence of a serine protease inhibitor gene AgKaSPI (GenBank accession no. MK440557) was cloned from A. glycines. AgKaSPI is 1 019 bp in length, with the open reading frame of 324 bp, encoding 107 amino acids. AgKaSPI has the Kazal domain, with the molecular weight of 11.43 kD and isoelectric point of 9.24. AgKaSPI was expressed at different developmental stages of A. glycines. At 24 h after infection by A. lecanii, the expression level of AgKaSPI and the corresponding AgKaSPI content in A. glycines adults were significantly up-regulated, being 4.31-fold and 1.69-fold as high as those of the control group, respectively, while the activities of serine protease, trypsin and chymotrypsin were inhibited. At 6 h after RNAi of AgKaSPI, the expression level of AgKaSPI in A. glycines adults decreased by 71.05%. The KaSPI content decreased by 51.11% and the activities of serine protease and chymotrypsin increased significantly at 12 h after RNAi of AgKaSPI. The fecundity per one hundred individuals decreased by 49 born aphids and the mortality of A. glycines adults increased by 10.12% at 96 h after RNAi of AgKaSPI. 【Conclusion】 AgKaSPI is expressed at different developmental stages of A. glycines, and the expression level of AgKaSPI is significantly up-regulated at 24 h after infection by A. lecanii. AgKaSPI may participate in the immune response of A. glycines to A. lecanii infection by regulating the serine protease activity.

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Adaptability of different geographical populations of Atractomorpha sinensis (Orthoptera: Pyrgomorphidae) to environmental temperature
LI Wen-Bo, GAO Yu, CUI Juan, TANG Jia-Wei, SHI Shu-Sen
Acta Entomologica Sinica    2021, 64 (8): 956-966.   DOI: 10.16380/j.kcxb.2021.08.007
Abstract266)      PDF(pc) (1919KB)(95)    PDF(mobile) (1919KB)(25)    Save
【Aim】 This study aims to ascertain the adaptability of different geographical populations of Atractomorpha sinensis in response to environmental temperature. 【Methods】By using soybean leaves as the food source, we observed the growth and development process of various developmental stages of five geographical populations of A. sinensis from Yan′an, Shaanxi (YA), Zhengzhou, Henan (ZZ), Chengdu, Sichuan (CD), Qujing, Yunnan (QJ) and Guangzhou, Guangdong (GZ) under the conditions of different constant temperatures (16, 20, 24, 28 and 32℃), RH 70% and a photoperiod of 16L∶8D, evaluated the variations in the developmental duration, developmental rates, developmental threshold temperatures and effective accumulated temperatures of different geographical populations, and analyzed the acorrelation between population biological indices and habitat environmental temperature. 【Results】 The developmental rate of different geographical populations of A. sinensis increased with the increase in temperature. However, the CD and GZ populations could not complete their generation at 16℃. As the habitat latitude and altitude increased, the developmental threshold temperatures of generation and various developmental stages of the populations gradually decreased, while their effective accumulated temperatures exhibited an increasing trend. The developmental threshold temperatures of the GZ, QJ, CD, ZZ and YA populations were 15.61, 10.76, 14.93, 11.46 and 10.51℃, respectively. The developmental threshold temperatures of generation of the GZ and CD populations were significantly higher than those of the other populations. The effective accumulated temperature of the GZ, QJ, CD, ZZ and YA populations were 828.41, 1 482.94, 963.13, 1 295.80, and 1 430.98 degree-days, respectively. The effective accumulated temperatures of the QJ and YA populations were significantly higher than those of the other populations. The temperature 24℃ is the optimal environmental temperature for the five geographical populations. The effective accumulated temperature of each developmental stage of different geographical populations is significantly negatively correlated with the annual average temperature of their habitats, while the developmental threshold temperature is significantly positively correlated with the annual average temperature of their habitats. 【Conclusion】 The results suggest that there exist differences in the adaptability of different geographical populations of A. sinensis to environmental temperature, and their adaptability to low environmental temperature increases with the increase in habitat latitude and altitude, while that to high environmental temperature show the opposite trend.
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Scanning electron microscopic observation of the external morphology of Lardoglyphus konoi (Acariformes: Acaridida: Lardoglyphidae) at different developmental stages
YAO Run, JIANG Feng, WANG Mei-Qing, ZHANG Qin, YUAN Ya-Ping, ZHU Zhi-Wei, LI Chao-Pin, ZHAN Xiao-Dong
Acta Entomologica Sinica    2021, 64 (8): 976-981.   DOI: 10.16380/j.kcxb.2021.08.009
Abstract265)      PDF(pc) (10027KB)(60)    PDF(mobile) (10027KB)(14)    Save

 【Aim】 To observe the external morphology and ultrastructure characteristics of Lardoglyphus konoi at different developmental stages. 【Methods】 Based on the individual samples of different developmental stages of L. konoi cultured in the laboratory, the external morphology and ultrastructure of various developmental stages of L. konoi, including egg, larva, nymph and adult, were observed under scanning electron microscope (SEM). 【Results】 The egg of L. konoi is oval. The larva has three pairs of legs, but has no genital organs, genital setae and anal setae. The nymph has four pairs of legs, and the genital area, genital setae and anal setae still look under-developed. The hypopus has a tapering front end and a round and pear-shaped rear end. Sucker plate appears in the end idiosoma, and there are 13 suckers on the sucker plate with 2-2-4-5 distribution. The chelicera of adult mite is slender, scissor-like, and there are several small teeth on its fixed digit and movable digit. The external vertical seta (ve) is about half the length of the internal vertical seta (vi), and situated at the same horizon as the internal vertical seta. Tarsus end of the 3rd leg in the male adult is replaced by two thick, blunt teeth. At the ventral idiosoma, there is a genital region and an anal region with a pair of anal suckers. All legs in the female adult have forked claws. The ventral gonopore is a longitudinal crack. The anus does not reach the posterior margin of idiosoma. 【Conclusion】 The external morphology and ultrastructure of L. konoi at different developmental stages are observed by SEM, and the morphological characteristics of the mite are understood in more details, providing a theoretical basis for further study of its taxonomy and biology.

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Comparative microRNA microarray and transcriptome analyses of the testis and ovary of the 5th instar larvae of the silkworm, Bombyx mori
ZHANG Bing, LI Na, KAN Yun-Chao
Acta Entomologica Sinica    2021, 64 (11): 1235-1243.   DOI: 10.16380/j.kcxb.2021.11.001
Abstract263)      PDF(pc) (2033KB)(134)    PDF(mobile) (2033KB)(19)    Save
【Aim】 This study aims to identify microRNA (miRNA) and possible target genes involved in the gonadal development of Bombyx mori through miRNA microarray and transcriptome analyses of the testis and ovary of the 5th instar larvae of B. mori. 【Methods】 A new generation of highthroughput sequencing platform was used to perform miRNA microarray analysis and transcriptome sequencing of the testis and ovary (defined as test and control, respectively) of the 5th instar larvae of B. mori. According to the criteria of P<0.05 and log 2(fold change, FC)≥2, the differentially expressed miRNAs of test vs control were screened. According to the criteria of q≤0.05 and [log 2 (fold change)]≥1, the differentially expressed genes (DEGs) of test vs control were screened by comparative analysis. Eight up-regulated and 12 down-regulated differentially expressed miRNAs were randomly selected,and their expression and their predicted five target genes were verified by qRT-PCR. The DEGs and target genes of the differentially expressed miRNAs were enriched by KEGG pathway analysis. 【Results】 Sixty-eight differentially expressed miRNAs and 3 991 DEGs were identified from the testis and ovary (test vs control). Among them, 36 and 32 miRNAs were up-regulated and down-regulated, respectively, and 2 033 and 1 958 DEGs were up-regulated and down-regulated, respectively. qRT-PCR verification results of differentially expressed miRNAs were consistent with the microarray data. KEGG pathway enrichment analysis showed that the DEGs were significantly enriched in the signal pathways involved in metabolism and ribosome. The possible target genes of differentially expressed miRNAs in the DEGs were predicted. Four groups of miRNAs and their target genes showing the opposite expression trend were found, including bmo-miR-2774a and LOC101745556, bmo-miR-92b and LOC101735954, bmo-miR-3266 and LOC733130 and LOC778467, respectively. One group of miRNA and its target gene showing the consistent expression trend was bmo-miR-3321 and LOC101744895. The results of qRT-PCR of five target genes were consistent with the results of transcriptome sequencing. 【Conclusion】 In this study, the transcriptome and miRNA microarray data of the testis and ovary of the 5th instar larvae of B. mori have been obtained, and four groups of miRNAs and their target genes showing the opposite expression trend and one group of miRNA and its target gene showing the consistent expression trend screened and verified, laying a foundation for exploring the development differences between the testis and ovary of B. mori.
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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
Abstract260)      PDF(pc) (5419KB)(105)    PDF(mobile) (5419KB)(38)    Save

【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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Influence of foliar fertilizers on the development and  reproduction of the citrus red mite, Panonychus citri (Acari: Tetranychidae), and the growth of citrus seedlings ( In English)
LIU Zhe, George Andrew Charles BEATTIE, CEN Yi-Jing, XU Chang-Bao
Acta Entomologica Sinica    2021, 64 (9): 1104-1111.   DOI: 10.16380/j.kcxb.2021.09.010
Abstract254)      PDF(pc) (961KB)(61)    PDF(mobile) (961KB)(11)    Save

【Aim】 The citrus red mite, Panonychus citri, is an important pest of citrus in China where foliar fertilizers are commonly used in citrus orchards. The objective of this research is to assess the influence of two fertilizers, urea and compound amino acid, on the development and reproduction of this mite, and on the growth of citrus seedlings. 【Methods】 Two foliar fertilizers, urea (0.50%) and compound amino acid (0.17%), were applied separately to potted citrus (Citrus reticulata Blanco cv. Shatangju) seedlings in the laboratory, and the seedlings sprayed with water were used as the control. The life-table parameters [the net reproductive rate (R0), mean generation time (T), intrinsic rate of increase (rm), finite rate of increase (λ) and population trend index (I)] of P. citri, the growth parameters (leaf length, width and area; stem length; plant height; number and length of new shoot leaves) of citrus seedlings and the nutrient (N, P and K) contents in leaves of citrus seedlings were investigated under controlled environment conditions. 【Results】 The duration of immature stages of P. citri was not influenced by the fertilizers, but the survival rate of  deutonymphs on citrus seedlings applied with 0.50% urea (95.40%) was significantly higher than those on the control citrus seedlings (applied with water) (78.26%) and on citrus seedlings applied with 0.17% compound amino acid (75.61%). The fecundity of female mites reared on urea treated citrus seedlings (42.1/♀) was also significantly higher than that of females reared on the control citrus seedlings (33.1/♀). The longevity of females reared on compound amino acid treated citrus seedlings (19.5 d) was significantly longer than that of females reared on urea treated citrus seedlings (14.8 d) and the control citrus seedlings (14.5 d), and the longevity of males reared on compound amino acid treated citrus seedlings (17.6 d) was significantly longer than that of males reared on the control citrus seedlings (13.1 d). Overall, P. citri reared on urea treated citrus seedlings had the highest net reproduction rate (Ro) (17.88) and population trend index (I)(18.08), both significantly higher than that reared on the control citrus seedlings (10.08 and 11.17, respectively). Leaf growth (leaf length, width and area) of citrus seedlings was significantly promoted by both fertilizers. Both fertilizers also significantly increased N, P and K contents, and the ratio of N/K in leaves of citrus seedlings. 【Conclusion】 Urea and compound amino acid as foliar fertilizer both promote the growth of citrus seedlings. Compound amino acid should be recommended in preference to urea because spraying of urea increases P. citri populations in contrast to compound amino acid. However, P. citri population should be also monitored because that although compound amino acid does not facilitate population growth, it does increase the longevity of adult mites.

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