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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
Table of Content
20 January 2018, Volume 61 Issue 1
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  • FORUM
    Vector biology and vector insects
    ZOU Zhen, KANG Le
    2018, 61(1):  1-10.  doi:10.16380/j.kcxb.2018.01.001
    Abstract ( 1432 )   PDF (1326KB) ( 590 )     
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    Vector biology is a rapidly developing branch of life science, and vector insects of medical and agricultural importance are important research targets. This article reviews the research progress in vector biology and vector insects, including the research background and significance, and research history and current status worldwide, and proposes the key areas and questions for further research, aiming to provide a basic information frame in vector biology and vector insects, to promote the research and technology development in the field, and to enhance the control level of vector organism and vector-borne diseases.
    RESEARCH PAPERS
    Screening of the deltamethrin-resistant strain of Aedes albopictus (Diptera: Culicidae) and its susceptibility to dengue virus
    JIA Zhi-Rong, LIU Zhuan-Zhuan, WANG Xiao-Ming, Tricia WILLIAMS, LIU Pei-Wen, LI Xiao-Cong, YAN Gui-Yun, CHEN Xiao-Guang
    2018, 61(1):  11-17.  doi:10.16380/j.kcxb.2018.01.002
    Abstract ( 921 )   PDF (1511KB) ( 368 )     
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    【Aims】 To establish a strain of the Asian tiger mosquito, Aedes albopictus,resistant to deltamethrin, and to compare the susceptibilities of the susceptible and resistant strains of Ae. Albopictus to dengue virus. 【Methods】 The lethal concentrationrequired to kill half of the sample (LC50 value) of deltamethrin against Ae. albopictus larvae was determined by impregnation method. Then, Ae. albopictus mosquitoes were selected at LC50 of deltamethrin for 11 generations. Deltamethrin resistance of each screened generation was tested by WHO tube assay. Female adults of the susceptible strain and the obtained resistant strain of Ae. albopictus were fed with defibrinated sheep blood mixed with dengue virus-2 (DENV-2). The midguts, ovaries and salivary glands from female adults of the two strains were dissected at 0, 4, 7 and 10 d post infection, and the infection rates and titers of DENV-2 in these tissues were detected by RT-PCR and RTqPCR, respectively. 【Results】 The resistance of Ae. albopictus to deltamethrin tended to be stable after selection for 9 generations. The LC50 value of deltamethrin against the 9th generation of this mosquito was 0.053 mg/L, the resistance ratio was 10.58, and the mortality rate of adults was 80%, showing that moderate resistance to deltamethrin was reached. DENV-2 was positive in all mosquitoes 0 d after infection and the virus titer in the resistant strain was higher than that in the susceptible strain. The infection rate in midguts of the susceptible and resistant strains by DENV-2 maintained between 92.75% and 97.18% at all the tested time points, without significant difference between both (P>0.05). The dissemination of DENV-2 in the ovaries started from 4 d. The infection rates in ovaries of the susceptible and resistant strains at 7 d and 10 d after infection by DENV-2 were significantly higher than those at 4 d (P<0.05). However, no significant difference existed between the two strains at the two time points of 7 and 10 d post infection (P>0.05). The average virus titer was higher in the resistant strain than in the susceptible strain at 4, 7 and 10 d post infection. The first positive detection of DENV-2 in salivary glands began at 7 d, and the infection rate showed no obvious increase at 10 d. Neither the infection rate nor the virus titer of salivary glands showed significant difference between the two strains (P>0.05). 【Conclusion】 The sequential administration of deltamethrin results in a gradual increase of the resistance level of Ae. albopictus larvae and adults. A laboratory strain of Ae. albopictus resistant to deltamethrin has been established. The susceptible and resistant strains infected with DENV-2 present similar infection rates but different virus titers, suggesting that the susceptibility of Ae. albopictus with moderate resistance to deltamethrin to dengue virus changes somewhat as compared to that of the susceptible strain.
    Establishment of diagnostic doses of three pyrethroid insecticides for detecting resistance in Aedes albopictus (Diptera: Culicidae) in China
    GAO Jing-Peng, CHEN Han-Ming, MA Ya-Jun
    2018, 61(1):  18-24.  doi:10.16380/j.kcxb.2018.01.003
    Abstract ( 878 )   PDF (755KB) ( 365 )     
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    【Aim】 To establish the diagnostic doses of deltamethrin, permethrin and lambda-cyhalothrin for detecting resistance in Aedes albopictus in China.【Methods】Insecticide-impregnated papers were made with different concentrations of deltamethrin, permethrin and lambda-cyhalothrin. The susceptibilities of adults of A. albopictus laboratory strain were tested by the standard WHO tube bioassay, and the number of knockdowned individuals at 1 h post treatment and the number of dead individuals at 24 h post treatment were recorded. Log-probit regression, and the LC50 and LC99 values were calculated using Excel 2007 and SPSS20.0 software. The 2-fold LC99 value was used as the diagnostic dose to separate resistant individuals from susceptible individuals. The resistance of a field population of A. albopictus collected from Haikou city to the three pyrethroids was evaluated based on the diagnostic dose determined. 【Results】 The LC50 values of deltamethrin, lambda-cyhalothrin and permethrin to adult A. albopictus (laboratory strain) were 0.00619%, 0.01403% and 0.05009%, respectively, while the LC99 values were 0.05175%, 0.11859% and 0.53165%, respectively. Thus the corresponding diagnostic doses of the three pyrethroids were 0.1035%, 0.2372% and 1.0633%, respectively. Tested with the diagnostic doses of deltamethrin, permethrin and lambda-cyhalothrin, the mortalities of adults of the field population of Haikou were 22.58%, 36.29% and 40.83%, respectively, indicating that the Haikou population has developed resistance to all the three pyrethroid insecticides. 【Conclusion】 The diagnostic doses of three pyrethroids established in this study for A. albopictus can be used as the reference for monitoring insecticide resistance of adults of this mosiquito.
    Peptidoglycan recognition protein AaPGRP-LC is involved in immune responses against bacteria in Aedes aegypti (Diptera: Culicidae)
    YANG Qing-Tai, ZHAO Li-Lin, ZOU Zhen, WANG Yan-Hong, MA Rui-Yan
    2018, 61(1):  25-35.  doi:10.16380/j.kcxb.2018.01.004
    Abstract ( 1004 )   PDF (2965KB) ( 361 )     
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    【Aim】 The objective of this study is to elucidate the roles of peptidoglycan recognition protein LC (PGRP-LC) in response to bacterial infection in Aedes aegypti. 【Methods】 The mRNA abundance of genes of antimicrobial peptides (AMPs) in mosquitoes at different time after infection of Enterobacter cloacae was measured by quantitative real-time PCR (qPCR). After interference of PGRP-LC by RNA interference (RNAi), the expression profiles of immune-related genes were detected by qPCR. The recombinant protein AaPGRP-LC was expressed by prokaryotic expression system and purified through a Ni2+-NTA agarose column. The quality of the purified recombinant protein was tested by Western blotting. 【Results】 Gram-negative E. cloacae bacteria could activate the expression of genes of AMPs in Ae. aegypti at 6 h post infection. After depletion of AaPGRP-LC, the transcription levels of genes of AMPs were significantly decreased during bacterial infection. The expression levels of immune-related genes involved in Toll and IMD pathways were down-regulated after interference of AaPGRP-LC. Western blotting of the purified recombinant protein AaPGRP-LC showed a clear and unique target band. 【Conclusion】 AaPGRP-LC modulates the transcriptional expression of genes of many essential AMPs and plays an important regulatory role in response to bacterial infection. It is involved in the regulation of IMD pathway and may also regulate the Toll pathway. The obtained recombinant AaPGRP-LC can be used in the follow-up study.
    Genome-wide identification, characterization and evolutionary analysis of genes of the heme peroxidase family in Anopheles sinensis (Diptera: Culicidae)
    YIN Hua-Chun, Zhang-Li-Juan, Chen-Bin
    2018, 61(1):  36-47.  doi:10.16380/j.kcxb.2018.01.005
    Abstract ( 974 )   PDF (7204KB) ( 379 )     
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    【Aim】 To identify genes of the heme peroxidase (HPX) family of Anopheles sinensis at the whole-genome level, to predict the basic features of these HPX members, and to explore the phylogenetics and evolution of the HPX members of five representativedipterans. 【Methods】 The amino acid sequences encoded by HPX genes in Drosophila melanogaster, Bombyx mori and other insects were downloaded from NCBI databases and used as queries to search for the HPX genes in An. sinensis genome using the local Blast program. The HPX genes in Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus on the whole genome were also identified by using the same method. The HPX genes identified in An. sinensis were named following the nomenclature system established for An. gambiae HPX genes. The characteristics of these HPX genes in An. sinensis, including the structure and scaffold location of these genes, the substitution rate and conserved domain of their amino acids, and the 3D structure of their proteins, were predicted using bioinformatics analysis. These An. sinensis HPX genes were also located on chromosome through the synteny analysis with An. gambiae HPX genes. The phylogenetic relationships of the HPX genes of five representative species of Diptera were constructed based on nucleotide sequences using maximum likelihood method with PAUP 4.0 and MEGA 6.0. 【Results】 The An. sinensis genome contains 20 HPX genes, while those of An. gambiae, Ae. aegypti and Cx. quinquefasciatus contain 18, 14 and 12 HPX genes, respectively. The HPX proteins of the four mosquito species were all classified into Peroxinectin, Peroxidasin, DBLPX and DUOX subfamilies, with the molecular weights of their amino acids ranging from 61.6-186.6 kD, except AsHPX8 (29.6 kD). The 20 HPX genes of An. sinensis contain 98 exons and 75 introns, and the distribution patterns of their exons and introns are quite diverse among different genes. These HPX genes of An. sinensis are mapped on 10 scaffolds, and syntenied to the chromosomes 2R, 3R, 2L, 3L and X in reference of An. gambiae genome. All the amino acid sequences of HPX genes in An. sinensis (except DUOX) each has one heme and five Ca2+ binding sites, and contains two cysteine sites in each of N-terminus and C-terminus, which define two disulfide bonds. The ω values of An. sinensis and An. gambiae orthologousgene pairs were all less than 1, suggesting that HPX genes has no obvious environmental selective pressure during their evolution. The HPX genes of the five representative dipteran species were divided into 16 groups on the phylogenetic tree, 11 of which showed to be monophyletic with >83% bootstrap support and homologous genes clustered together. 【Conclusion】 This study provides the basic information frame for the HPX genes of An. sinensis. The HPX proteins in different mosquito species have similar molecular weights, and this is associated with a high degree of structure conservation of the HPX family proteins. The DUOX of mosquitoes gradually lost its function with their deletion of the main functional loci, and this is related to its specific environmental adaptation. The peroxidase domain in the DBLPX subfamily is the most conserved in all subfamilies of the HPX family. 
    Heterologous expression, purification and crystallization of α-carboxylesterase AsAe7 from Anopheles sinensis (Diptera: Culicidae)
    ZHANG Tian-Tian, MIAO Ya, XU Bai-Ying, CHEN Bin
    2018, 61(1):  48-58.  doi:10.16380/j.kcxb.2018.01.006
    Abstract ( 905 )   PDF (5855KB) ( 370 )     
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     【Aim】 Carboxylesterase is one kind of the important detoxifying enzymes in insects, and is associated with insecticide resistance. The aim of this study is to explore the structure and the function of the carboxylesterase Ae7 from Anopheles sinensis (AsAe7) based on its preliminary crystallography. 【Methods】 The Asae7 gene was bioinformatically analyzed, cloned, and expressed in prokaryotic expression system. The recombinant protein was purified with nickel chelate affinity chromatography and gel filtration chromatography. The polymerization of AsAe7 was detected by gel filtration chromatography and chemical crosslinking analysis, and the crystal screening was performed by sitting drop vapor diffusion technique. 【Results】 Bioinformatic analysis revealed that AsAe7 is a hydrophilic protein of 61.053 kD without transmembrane regions and signal peptide. The 3D structural prediction showed that AsAe7 adopts an α/β-hydrolase superfamily fold. Multiple sequence alignment result demonstrated that Ae7 proteins are highly conserved in different insects. The 1 626 bp coding sequence of Asae7 was cloned, and the recombinant plasmid pET28a-Asae7 was constructed correctly. The SDS-PAGE analysis illustrated that the fusion protein AsAe7 expressed in Escherichia coli mainly existed in the supernatant. The highly purified and stable protein was then obtained with two-step affinity chromatography. Gel filtration chromatography and chemical crosslinking analysis showed that AsAe7 mainly exists as monomer in vitro. Finally, protein crystals of AsAe7 were obtained by crystal screening. 【Conclusion】 Crystals of the recombinant AsAe7 have been obtained by crystallography, which lays the foundation for illustrating the crystal structure of AsAe7 and the molecular mechanisms of AsAe7-mediated metabolic resistance at atomic resolution.
    Expression, purification and crystallization of the glutathione-S-transferase E6(AsGSTE6) from Anopheles sinensis (Diptera: Culicidae)
    MIAO Ya, ZHANG Tian-Tian, XU Bai-Ying, CHEN Bin
    2018, 61(1):  59-67.  doi:10.16380/j.kcxb.2018.01.007
    Abstract ( 838 )   PDF (5234KB) ( 406 )     
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    【Aim】 This study aims to explore the structure of the glutathione-S-transferase E6 (AsGSTE6) from Anopheles sinensis based on its preliminary crystallography. 【Methods】 AsGSTE6 was predicted and analyzed by bioinformatics method. AsGSTe6 was cloned, and the recombinant plasmid pET28a-AsGSTe6 was constructed and expressed in prokaryotic expression system. The recombinant protein was purified with nickel chelate affinity chromatography and gel filtration chromatography. The polymerization state of AsGSTE6 was detected by gel filtration chromatography and chemical crosslinking analysis. Crystal screening was performed by sitting drop vapor diffusion techniques. 【Results】 Bioinformatic analysis revealed that AsGSTE6 is a hydrophilic protein with the calculated molecular weight of 25.28 kD, without transmembrane regions and signal peptides in the amino acid sequence. The 3D structural prediction showed that AsGSTE6 contains a small N-terminal domain following a βαβαββα pattern and a large C-terminal all-α domain. The result of multiple sequence alignment demonstrated that GSTE6 proteins are highly conserved in insects. The 672 bp coding sequence of AsGSTe6 was cloned, and the recombinant plasmid pET28a-AsGSTe6 was constructed correctly. The recombinant protein AsGSTE6 was expressed in soluble form in Escherichia coli. And then the highly purified and stable protein was obtained with two step affinity chromatography. Gel filtration chromatography and chemical crosslinking analysis showed that AsGSTE6 mainly existed as dimer in vitro. Finally, the protein crystals of AsGSTE6 were obtained by crystal screening. 【Conclusion】 Crystals of AsGSTE6 have been obtained by methods of crystallography, which lays the foundation for 3D structure determination of AsGSTE6.
    Virtual screening of inhibitors for kynurenine formamidase of Anopheles gambiae (Diptera: Culicidae)
    SONG Shuai-Bao, AI Shang-Jie, GUAN Huai, HAN Qian
    2018, 61(1):  68-78.  doi:10.16380/j.kcxb.2018.01.008
    Abstract ( 1008 )   PDF (7645KB) ( 291 )     
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    【Aim】 To screen potential inhibitors targeting kynurenine formamidase (KFase) of Anopheles gambiae, which could be a candidate insecticide to reduce malaria transmission, by virtual screening. 【Methods】 Protein sequences homologous with KFase of An. gambiae were searched and downloaded from NCBI using BLASTP web server. Phylogenetic tree of homologous proteins was constructed by MEGA6 using maximum likelihood method. Homology modeling of KFase of An. gambiae was performed by SWISS-MODEL web server using KFase of Drosophila melanogaster (PDB ID: 4E14) as a template. The small-molecule compounds were downloaded from ZINC database and then screened by the method of random forest. Docking analysis of the homology model and selected small molecule compounds was carried out, and the screening results were further validated using molecular dynamics simulation. 【Results】 Three small-molecule compounds, i.e., N-(2,4-diketo-1H-pyrimidin-6-yl)-2-fluoro-benzamide, 3-(4-fluorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid, and N-(2-oxo-2, 3-dihydro-1H-imidazo[4, 5-b] pyridin-5-yl)-succinamic acid, have the lowest docking energy to KFase of An. gambiae with the affinity energy of -9.0, -8.7 and -8.9 kcal/mol, respectively. 【Conclusion】 The three small-molecule compounds, i.e., N-(2,4-diketo-1H-pyrimidin-6-yl)-2-fluoro-benzamide, 3-(4-fluorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid, and N-(2-oxo-2, 3-dihydro-1H-imidazo[4, 5-b] pyridin-5-yl)-succinamic acid, could be candidate competitive inhibitors of KFase of An. gambiae. Whether these compounds can be used as candidate mosquitocides needs further experimental validation.
    Establishment and identification of a new cell line from Culex pipiens quinquefasciatus (Diptera: Culicidae)
    LUO Lan, CUI Feng
    2018, 61(1):  79-85.  doi:10.16380/j.kcxb.2018.01.009
    Abstract ( 790 )   PDF (3239KB) ( 278 )     
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    【Aim】 This study aims to establish and molecularly identify a cell line based on tissues from the newly hatched larvae of the mosquito Culex pipiens quinquefasciatus (Diptera: Culicidae). 【Methods】 The shredded tissues of the newly hatched larvae of the parathion-resistant strain Shengui of C. pipens quinquefasciatus were cultured in M199 complete medium supplemented with 20% heatinactivated fetal bovine serum, and the primary cells were obtained. The primary cells were treated with trypsin and subcultured into a cell line. The tissue origin of the cell line was identified by observing its biological characteristics under a microscope and comparing DNA amplification fingerprinting and sequencing the ribosome DNA internal transcribed spacer. 【Results】 The cell line Cxq-1 was well maintained in M199 complete medium and subcultured continually for 12 months. After being frozen in liquid nitrogen, Cxq-1 was successfully resuscitated for several times. DNA amplification fingerprinting polymerase chain reaction clearly distinguished Cxq-1 from two other insect cell lines. Sequencing of ribosome DNA internal transcribed spacer of Cxq-1 indicated that its rDNA-ITS1 and rDNA-ITS2 sequences has higher than 99% identity with those of C. pipens quinquefasciatus registered in the GenBank, confirming the origin of Cxq-1. 【Conclusion】 The newly established cell line Cxq-1 from newly hatched larvae of C. pipens quinquefasciatus has been confirmed to be a cell line of this mosquito. This cell line provides an important platform for studying the molecular mechanisms of insecticide resistance, mosquito-borne viruses and parasites in the future.
    Evaluation of fumigation activity of thirty-six essential oils against Culex pipiens pallens (Diptera: Culicidae)
    CHEN Wei, WU Hua, MA Zhi-Qing, FENG Jun-Tao, ZHANG Xing
    2018, 61(1):  86-93.  doi:10.16380/j.kcxb.2018.01.010
    Abstract ( 916 )   PDF (1124KB) ( 358 )     
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    【Aim】 This study aims to obtain essential oils with higher bioactivities so as to provide more valuable materials for mosquito prevention and control. 【Methods】 The fumigation activity of thirty-six essential oils against Culex pipiens pallens adults was evaluated by airtight fumigation in sealed conical flasks or hermetic round canisters. 【Results】 The results of fumigation assay in sealed flasks showed that 10 essential oils including Pinus massoniana leaf oil, Magnolia oil, red moor besom leaf oil, dai-dai leaf oil, Forsythia oil, Perilla leaf oil, Elsholtzia oil, tea tree oil, vetiver oil, and Angelica oil possessed high toxicity against C. pipiens pallens adults with low KT50 values (<10 min) at 12 μL/L, and P. massoniana leaf oil exhibited the highest fumigation activity with the KT50 value of 3.40 min. The fumigation of red moor besom leaf oil and grapefruit oil caused higher mortality rates at 24 h after treatment, with the LC50 values of 0.95 and 1.11 μL/L, respectively. The results of fumigation assay in hermetic round canisters revealed that the KT50 value of P. massoniana leaf oil against C. pipiens pallens adults was 13.79 min at the concentration of 12 μL/L, and the mortality rate of C. pipiens pallens adults caused by red moor besom leaf oil at the concentration of 1.67 μL/L was 100%. 【Conclusion】 P. massoniana leaf oil and red moor besom leaf oil have strong fumigation activity against C. pipiens pallens adults, and both oils thus hold great potential to be developed as mosquito control agents.
    Screening of plant-derived synergistic agents for mosquito coil based on the bioefficacy against Culex pipiens pallens (Diptera: Culicidae)
    ZHANG Jie, LIU Hui, YANG Xiao-Wei, ZHANG Xing
    2018, 61(1):  94-102.  doi:10.16380/j.kcxb.2018.01.011
    Abstract ( 828 )   PDF (931KB) ( 283 )     
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    【Aim】 This study aims to compare the synergism of 12 essential oils such as wintergreen oil to solid mosquito repellent in the bioassay of solid mosquito coil against Culex pipiens pallens, so as to provide a theoretical basis for the development and application of solid mosquito coil. 【Methods】 With the 3-5 day-old adults of Cx. pipiens pallens as the test insects, the synergistic effects of 12 essential oils including wintergreen oil, octagonal leaf oil, sassafras oil, camphor oil, citronella oil, litseacubeba oil, white camphor oil, lemon grass oil, peppermint oil, star anise oil, linaloe wood oil and lavender oil on allethrin in solid mosquito coil were determined using closed cylinder. Mixtures of essential oils with obvious synergistic effects screened above were made, the best mixture was screened using closed cylinder and the 5.8 m3 cabin, and its quality was tested. 【Results】 The results showed that five essential oils including wintergreen oil, linaloe wood oil, sassafras oil, white camphor oil and camphor oil had good synergistic effects on allethrin, with the synergistic ratios of 1.292-1.364, and the mixture of wintergreen oil and camphor oil (1∶1) had an obvious synergistic effect on allethrin. After selection of the solvent and emulsifier, the formula of synergists was determined as 30%-84% plant extracts, 10%-53.5% solvent and 6.5% emulsifier (NP-2), and the most economic formula of synergists was 30% wintergreen oil+30% camphor oil+3.35% ethyl acetate+30.15% butyl acetate+6.5% NP-2. 【Conclusion】 The formulated synergist (30% wintergreen oil+30% camphor oil+3.35% ethyl acetate+30.15% butyl acetate+6.5% NP-2)has an obvious effect on allyl permethrin in solid mosquito coil with a promising future of commercialization.
    Molecular phylogenetic relationships among forty species (subspecies) of the genus Culex (Diptera: Culicidae) from China
    SUN Ling, FU Wen-Bo, YAN Zhen-Tian, CHEN Bin
    2018, 61(1):  103-113.  doi:10.16380/j.kcxb.2018.01.012
    Abstract ( 980 )   PDF (2824KB) ( 345 )     
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    【Aim】 To sequence the COⅠ and ITS2 sequences of the Culex species (subspecies) with distribution record in China, and to construct and discuss the molecular phylogenetic relationships among these species (subspecies). 【Methods】 COⅠ and ITS2 sequences of a total of 20 Culex species (subspecies) were newly sequenced, and COⅠ sequences of other 20 Culex species (subspecies) and ITS2 sequences of other three Culex species (subspecies) were downloaded from NCBI databases. The base composition, interspecific genetic distance and saturation analysis of COⅠ of 40 species (subspecies) and ITS2 sequences of 23 species (subspecies) were analyzed, and the incongruence length difference (ILD) test was conducted for COⅠ+ITS2 sequences. The phylogenetic relationships of these Culex species (subspecies) were inferred using maximum likelihood (ML), Bayesian interference (BI), neighbor joining (NJ) and maximum parsimony (MP) based on the nucleotide sequences of two molecular data sets (COⅠ and ITS2), respectively. Kishino-Hasegawa (KH) and Shimodaira-Hasegawa (SH) tests were used to evaluate the differences of these four phylogenetic trees constructed, and the most reasonable phylogenetic tree was selected. 【Results】 The newly sequenced COⅠ and ITS2 sequences are 625-685 and 300-559 bp in length, respectively. The pairwise genetic distances among Culex mosquito species (subspecies) range from 0.002 to 0.198 for COⅠ and from 0.006 to 1.807 for ITS2. The ILD test of COⅠ+ITS2 sequences of 23 Culex species (subspecies) showed that the data set was incompatible, so COⅠ+ITS2 sequences were inappropriate to construct the phylogenetic trees of these species (subspecies). By KH and SH tests among these four phylogenetic trees, the BI tree based on COⅠ sequence was the most reasonable tree, while the MP tree based on ITS2 sequence was the most reasonable tree. The BI tree constructed based on the COⅠ nucleotide sequences showed that all members, except for Cx. infantulus and Cx. brevipalpis of the same subgenus, are clustered together, which is consistent with the traditional morphological classification. Both the subgenus Lutiza and Barraudius are grouped into the subgenus Culex. The subgenus Maillotia and subgenus Nexoculex are grouped together. The subgenus Culiciomyia, subgenus Eumelanomyia and subgenus Lophoceramyia are monophyletic. The MP tree based on the ITS2 sequences showed that the relationships between subgenera and species (subspecies) are chaotic. 【Conclusion】 The molecular phylogenetic tree reconstructed shows that the subgenus Culex is not a monophyletic group. Compared with ITS2 and COⅠ+ITS2, COⅠ is a more desirable molecular marker for the reconstruction of the phylogeny of Culex species. The molecular phylogenetic relationships reconstructed in this study lay a foundation for studying the phylogenetic relationships among subgenera and species (subspecies) of the genus Culex in China.
    Sequencing and analysis of the complete mitochondrial genome of Armigeres subalbatus (Diptera: Culicidae)
    LI Xu-Dong, Chen-Bin
    2018, 61(1):  114-121.  doi:10.16380/j.kcxb.2018.01.013
    Abstract ( 906 )   PDF (3592KB) ( 439 )     
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     【Aim】 To sequence and analyze the complete mitochondrial genome of Armigeres subalbatus, and to explore the phylogenetic position of Armigeres in Culicinae based on mitochondrial genome sequences. 【Methods】 The nucleotide sequence of the mitochondrial genome of Ar. subalbatus was determined by using PCR amplification and DNA sequencing. The general features and base composition of Ar. subalbatus mitochondrial genome were analyzed, and the phylogenetic relationship of Ar. subalbatus with seven other species of Culicinae was constructed by using maximum likelihood method (ML) and Bayesian inference (BI) based on the nucleotide sequences of concatenated protein-coding genes (PCGs). 【Results】 The complete mitogenome of Ar. subalbatus is 14 891 bp in length (GenBank accession no.: KY978578), and contains 37 genes, including 13 PCGs, 22 tRNAs and 2 rRNAs. Its gene organization is consistent with those of the known mitogenomes of Culicidae. The mitogenome has a clear bias in nucleotide composition with a positive AT-skew and a negative GC-skew. All 13 PCGs use the ATN as the initiation codon except COI with TCG, and TAA as the termination codon except COII with the incomplete T. All tRNAs could form the typical cloverleaf structure except tRNASer (AGN), whose DHU-arm is lacking. The phylogenetic relationship of eight species within Culicinae based on the mitochondrial genomes presents as Culex+(Armigeres+Aedes). 【Conclusion】 The phylogenetic relationship of Culicinae constructed based on the mitochondrial genomes shows that Armigeres is closer related to Aedes than to Culex, which is consistent with the result of the traditional morphology-based taxonomy.
    REVIEW ARTICLES
    Taxonomy and fauna of Culicidae: history and current knowledge
    FU Wen-Bo, CHEN Bin
    2018, 61(1):  122-138.  doi:10.16380/j.kcxb.2018.01.014
    Abstract ( 1336 )   PDF (1692KB) ( 503 )     
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    The family Culicidae (all true mosquitoes) is the most important medical insect group and thought to be the deadliest animal killer. Accurate classification and identification of mosquitoes are the basis of the basic research and control of vector mosquitoes. Modern taxonomy of mosquitoes originated from the species description of thegenus Culex by Carl Linnaeus in 1758, and since then the taxonomic system of the Culicidae has basically formed with named species gradually increasing. In this article we reviewed the taxonomic research history of the Culicidae in the world, and the taxonomic system, species and biology at the genus level and above with some preliminary discussions and perspectives, on the basis of compiling worldwide species name list and geographical distribution records. Up to date, there are 41 mosquito genera, 201 subgenera and 3 573 species known in the world, among which 20 genera, 63 subgenera and 419 species recorded in China. The subfamily Anophelinae is believed to be a monophylum, and contains three genera (Anopheles, Bironella and Chagasia), 11 subgenera  and 489 species. There are 476 known species in the genus Anopheles, accounting for 97% of the total species in Anophelinae, which contains all malaria vectors. The subfamily Culicinae is believed to be polyphyletic with a total of 11 known tribes, 38 genera, 190 subgenera and 3 048 species, and its phylogeny is still not settled. The tribes Aedini, Culicini, Sabethini and Uranotaeniini have 1 262, 800, 432 and 270 known species, respectively, ranking as the four largest tribes in Culicinae. Many species especially in the genera Culex, Aedes and Mansonia are serious vectors of infectious diseases. Among all these mosquito species, the highest species number (1 075 species) is recorded for the Oriental Region, followed in species number by Neotropical Region (951 species), Ethiopian Region (798), Australian Region (542 species) and Palaearctic Region (251), and Nearctic Region has the least number of species (196). However, a large number of species exist across different regions. The information will provide a basis for the understanding of the taxonomy and distribution of Culicidae. Due to the wide existence of cryptic species, the actual number of species should be 3-5 times as high as the established species number. The systematics of Culicidae is of important theoretical and application value for the accurate identification of mosquito species, the research of basic biology and mosquito-borne disease transmission mechanism, and the control of vector mosquitoes and mosquito-borne diseases. The taxonomy, phylogeny and fauna of mosquitoes are still urgent and important research subjects.
    SHORT COMMUNICATIONS
    Binding characteristics of the odorant binding protein AsinOBP1 of Anopheles sinensis (Diptera: Culicidae) with the mosiquito repellent DEET
    SHI Zong-Pan, RAN Yong-Hong, ZHANG Jing-Jing, ZHANG Jing, YAN Zhen-Tian, CHEN Bin, HE Zheng-Bo
    2018, 61(1):  139-148.  doi:10.16380/j.kcxb.2018.01.015
    Abstract ( 805 )   PDF (3713KB) ( 387 )     
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     【Aim】 To study the binding characteristics of the odorant binding protein AsinOBP1 from Anopheles sinensis with the mosiquito repellent N,N-diethyl-m-toluamide (DEET) in comparison with Aedes aegypti OBP1 (AaegOBP1) and Culex quinquefasciatus OBP1 (CquiOBP1), and to identify the key residues responsible for the binding to DEET by OBP1s from different mosquito species. 【Methods】 The recombinant protein AsinOBP1 was expressed using a prokaryotic expression system and then purified. The DEET binding properties of AsinOBP1 were evaluated by fluorescence competitive binding assays using N-phenyl-1-naphthylamine (1-NPN) as the fluorescence probe. The binding affinities of AsinOBP1, AaegOBP1 and CquiOBP1 to DEET were compared, and the key residues responsible for this binding were identified using molecular docking. 【Results】 AsinOBP1 was able to bind to DEET, with a dissociation constant of 29.55 μmol/L. Under the same experimental conditions, CquiOBP1 and AaegOBP1 were also able to bind to DEET, with their dissociation constants of 17.15 and 12.81 μmol/L, respectively. It was shown that AaegOBP1 had the strongest binding affinity to DEET, followed by CquiOBP1 and AsinOBP1. The molecular docking of AsinOBP1 revealed that one DEET molecule is bound to each subunit at a site located near the interface between the two monomers of AsinOBP1. The DEET binding pocket is formed by residues belonging to helices α4, α5 and α6 (Leu-92, Leu-95, His-96, Leu-99, Ala-107, Met-108, Met-110, Gly-110, Cys-114, Leu-115, Trp-133, Met-108′, Lys-112′ and Leu-115′). The residues interacting with the tolyl group and ketonic oxygen of DEET are identical in the three recombinant proteins. However, among the five key residues interacting with the diethyl group of DEET, one residue is different: in AaegOBP1 the residue is Leu, whereas in CquiOBP1 and AsinOBP1 it is Met. Given that the residue Leu is more hydrophobic than Met, it was speculated that this might contribute to a higher affinity of AaegOBP1 to DEET. 【Conclusion】 AsinOBP1, AaegOBP1 and CquiOBP1 are all able to bind to DEET, but differ in affinity. Further study should be focused on the causes of their difference in affinity so as to better understand the mechanisms by which OBP1s bind to DEET.
    CONTENTS
    Contents of Vol. 61 Issue 1
    2018, 61(1):  149. 
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