Genome-wide identification and characterization of odorant-binding protein （OBP） genes in the malaria vector Anopheles sinensis （Diptera： Culicidae）

Anopheles sinensis is a major malaria vector. Insect odorant-binding proteins （OBPs） may function in the reception of odorants in the olfactory system. The classification and characterization of the An. sinensis OBP genes have not been systematically studied. In this study, 64 putative OBP genes were identified at the whole-genome level of An. sinensis based on the comparison between OBP conserved motifs, PBP_GOBE and phylogenetic analysis with An. gambiae OBPs. The characterization of An. sinensis OBPs, including the motifs conservation, gene structure, genomic organization and classification, were investigated. A new gene, AsOBP73, belonging to the Plus-C subfamily, was identified with the support of transcript and conservative motifs. These An. sinensis OBP genes were classified into three subfamilies with 37, 15 and 12 genes in the subfamily Classic, Atypical and Plus-C, respectively. The genomic organization of An. sinensis OBPs suggests a clustered distribution across nine different scaffolds. Eight genes （0BP23-28, 0BP63- 64） might originate from a single gene through a series of historic duplication events at least before divergence of Anopheles, Culex and Aedes. The microsynteny analyses indicate a very high synteny between An. sinensis and An. gambiae OBPs. OBP70 and OBP71 earlier classified under Plus-C in An. gambiae are recognized as belonging to the group Obp59a of the Classic subfamily, and OBP69 earlier classified under Plus-C has been moved to the Atypical subfamily in this study. The study established a basic information frame for further study of the OBP genes in insects as well as in An. sinensis.

Genome-wide identification, characterization and evolution of cuticular protein genes in the malaria vector Anopheles sinensis （Diptera： Culicidae）

Thirteen cuticular protein （CP） families have been recognized in arthropods. In this study, 250 Anopheles sinensis CP genes were identified and named based on genome and transcriptome sequences. They were classified into 10 families based on mo- tifs and phylogenetic analyses. In 11 other insect species, nine had CP numbers 〉 150 while Apis mellifera and Tribolium castaneum had CP numbers less than 52. The CPs of eight species occupied 〉 1.4% of the total genomic gene number, whereas in three species the CPs occupied 〈 1%. The phylogenies for each CP family in An. sinensis were constructed and discussed. The 250 CPs each had 1-8 exons with 144 CPs （57.6%） having two exons. The intron length ranged from 66--3888 bp with 174 introns （54.0%） being 66--100 bp long. Except for two CPs on two contigs, 248 CPs were mapped onto 28 scaffolds with 136 genes （54.4%） restricted to five scaffolds. A total of 107 CPs were clustered and located at 27 loci. The CPR family had the conserved motif GSYS- LVEPDGTVRTV. The RR- 1 subfamily had an additional 21 amino acid （aa） motifs with the YVADENGF sequence that is common in insects. The RR-2 subfamily had an additional 50 aa motifs with two additional regions RDGDWKG and G-x（3）-VV. A comparison with 115 orthologous counterparts of An. gambiae CPs suggested purifying selection for all of these genes. This study provides basic information useful for further studies on biological functions of An. sinensis CPs as well as for comparative genomics of insect CPs.

Sialotranscriptome sequencing and analysis of Anopheles sinensis and comparison with Psorophora albipes sialotranscriptome （Diptera： Culicidae）

Most of adult female mosquitoes secrete saliva to facilitate blood sucking, digestion and nutrition, and mosquito-borne disease prevention. The knowledge of classification and characteristics of sialotranscriptome genes are still quite limited, Anopheles sinensis is a major malaria vector in China and southeast Asian countries. In this study, the An. sinensis sialotranscriptome was sequenced using Illumina sequencing technique with a total of 10 907 unigenes to be obtained and annotated in biological functions and pathways, and 10 470 tmigenes were mapped to An. sinensis reference genome with 70.46% of genes having 90%- 100% genome mapping through bioinformatics analysis. These mapped genes were classified into four categories： housekeeping （6632 genes）, secreted （1177）, protein-coding genes with function-unknown （2646） and transposable element （15）. The housekeeping genes were divided into 27 classes, and the secreted genes were divided into 11 classes and 96 families. The classification, characteristics and evolution of these classes/families of secreted genes are further described and discussed. The comparison of the 1177 secreted genes in An. sinensis in the Anophelinae subfamily with 811 in Psorophora albipes in the Culicinae subfamily show that six classes/subclasses have the gene number more than twice and two classes （uniquely found in anophelines, and Orphan proteins of unique standing） are unique in the former compared with the latter, whereas four classes/subclasses are much expanded and uniquely found in the Aedes class and is unique in the later. The An. sinensis sialotranscriptome sequence data is the most complete in mosquitoes to date, and the analyses provide a comprehensive information frame for further research of mosquito sialotranscriptome.

Emergence of Zika Virus in Culex tritaeniorhynchus and Anopheles sinensis Mosquitoes in China

Zika virus(ZIKV)has been isolated from mosquitoes such as Aedes,Mansonia uniformis,and Culex perfuscus;However,the isolation of ZIKV from Anopheles sinensis and Culex tritaeniorhynchus has not yet been reported.In June and July2018,22,985 mosquitoes and 57,500 midges were collected in Jiangxi Province in southeastern China.Among them,six strains of ZIKV were isolated from mosquitoes:four from An.sinensis and two from Cx.tritaeniorhynchus.Molecular genetic analysis showed that the ZIKV isolated from An.sinensis and Cx.tritaeniorhynchus belonged to genotype 2 in the Asian evolutionary branch of ZIKV.In addition,the ZIKV strains isolated from An.sinensis and Cx.tritaeniorhynchus had amino acid substitutions identical to ZIKV strains prevalent in South America since 2015.This study is the first to isolate ZIKV from mosquito specimens collected in the wild of Jiangxi Province,China;This is also the first time that ZIKV has been isolated from An.sinensis and Cx.tritaeniorhynchus.Given that An.sinensis and Cx.tritaeniorhynchus have a very wide geographical distribution in China and even in eastern and southern Asia,the isolation of several strains of ZIKV from these two mosquitoes poses new challenges for the prevention and control of ZIKV infection in the mainland of China and countries and regions with the same distribution of mosquitoes.

Sequencing and analysis of the complete mitochondrial genome in Anopheles sinensis(Diptera:Culicidae)

Background:Anopheles sinensis(Diptera:Culicidae)is a primary vector of Plasmodium vivax and Brugia malayi in most regions of China.In addition,its phylogenetic relationship with the cryptic species of the Hyrcanus Group is complex and remains unresolved.Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes,of which mitochondrial genome data of An.sinensis is not available.Methods:An.sinensis samples was collected from Shandong,China,and identified by molecular marker.Genomic DNA was extracted,followed by the Illumina sequencing.Two complete mitochondrial genomes were assembled and annotated using the mitochondrial genome of An.gambiae as reference.The mitochondrial genomes sequences of the 28 known Anopheles species were aligned and reconstructed phylogenetic tree by Maximum Likelihood(ML)method.Findings:The length of complete mitochondrial genomes of An.sinensis was 15,076 bp and 15,138 bp,consisting of 13 protein-coding genes,22 transfer RNA(tRNA)genes,2 ribosomal RNA(rRNA)genes,and an AT-rich control region.As in other insects,most mitochondrial genes are encoded on the J strand,except for ND5,ND4,ND4L,ND1,two rRNA and eight tRNA genes,which are encoded on the N strand.The bootstrap value was set as 1000 in ML analyses.The topologies restored phylogenetic affinity within subfamily Anophelinae.The ML tree showed four major clades,corresponding to the subgenera Cellia,Anopheles,Nyssorhynchus and Kerteszia of the genus Anopheles.Conclusions:The complete mitochondrial genomes of An.sinensis were obtained.The number,order and transcription direction of An.sinensis mitochondrial genes were the same as in other species of family Culicidae.

Experimental infections of mosquitoes with severe fever with thrombocytopenia syndrome virus

Background:Severe fever with thrombocytopenia syndrome(SFTS)is a newly identified emerging infectious disease,which is caused by a novel bunyavirus(termed SFTSV)in Asia.Although mosquitoes have not been identified as the primary vectors,as revealed by epidemiological surveys,their role in transmitting this SFTSV as a suspicious vector has not been validated.Findings:In this study,we conducted experimental infections of mosquitoes with SFTSV to examine the role of mosquitoes in the transmission of the virus.We did not detect viral replication in Culex pipiens pallens,Aedes aegyptis and Anopheles sinensis as revealed by qRT-PCR assay.In addition,we failed to isolate SFTSV from the Vero cells cultured with suspensions of SFTSV-infected mosquitoes.Conclusion:The results of the present study demonstrate little possibility that mosquitoes act as vectors for the emerging pathogen SFTSV.