Correlation between adult pyrethroid resistance and knockdown resistance(kdr)mutations in Aedes albopictus(Diptera:Culicidae)field populations in China

Background:Arboviral disease transmitted by Aedes albopictus such as dengue fever is an important threat to human health.Pyrethroid resistance raises a great challenge for mosquito control.A systematic assessment of Ae.albopictus resistance status in China is urgently needed,and the study of correlation between pyrethroid resistance and knockdown resistance(kdr)mutations would provide information to guide the control of the Ae.albopictus vector.Methods:Five field populations of Ae.albopictus were collected from Jinan(JN),Hangzhou(HZ),Baoshan(BS),Yangpu(YP)and Haikou(HK),China in 2017.Insecticide-impregnated papers were prepared with four pyrethroid chemicals,deltamethrin,permethrin,beta-cypermethrin and lambda-cyhalothrin.The susceptibility of Ae.albopictus to pyrethroids was tested by the WHO tube assay.Kdr mutations were identified by PCR and sequencing.Moreover,the correlation analysis between kdr alleles and pyrethroid resistance was performed.Results:All five populations of Ae.albopictus showed resistance to four pyrethroid insecticides.One kdr mutant allele at codon 1532 and three at 1534 were detected with frequency of 5.33%(I1532T),44.20%(F1534S),1.83%(F1534 L)and 0.87%(F1534C),respectively.Both 1532 and 1534 mutation mosquitoes were found in the BS and YP populations.Allele I1532T was negatively correlated with deltamethrin resistance phenotype(OR<1),while F1534S mutation was positively correlated with deltamethrin and permethrin resistance(OR>1).Conclusions:The five field populations of Ae.albopictus adults were all resistant to deltamethrin,permethrin,betacypermethrin and lambda-cyhalothrin.Mutant F1534S was clearly associated with pyrethroid resistance phenotype in Ae.albopictus and this could be developed as a molecular marker to monitor the pyrethroid resistance problem in China.

Leishmania infection and blood sources analysis in Phlebotomus chinensis (Diptera:Psychodidae) along extension region of the loess plateau, China

Background:Visceral leishmaniasis(VL)was one of the most important parasitic diseases in China,caused by Leishmania protozoans and transmitted by sand flies.Recently VL cases have reappeared in China,including the extension region of the Loess Plateau.The purpose of this study was to collect fundamental data on the host-vector VL system in the Loess Plateau to assist in the development of prevention and control measures.Methods:Sand flies were collected by light traps from rural areas in Shanxian,Henan,China in 2015,as well as in Wuxiang and Yangquan,Shanxi,China in 2017.The blood sources of sand flies were analyzed by PCR detecting the host-specific mitochondrial cytochrome b(mtDNA cyt b)gene fragments.Leishmania infection in sand flies was detected by amplifying and sequencing ribosomal DNA internal transcribed spacer 1(ITS1).The Leishmania specific antibodies in the sera of local dogs were detected by ELISA kit.Results:Blood sources showed diversity in the extension region of the Loess Plateau,including human,chicken,dog,cattle,pig and goat.Multiple blood sources within a sand fly were observed in samples from Yangquan(17/118,14.4%)and Wuxiang(12/108,11.1%).Leishmania DNA was detected in sand flies collected from Yangquan with minimum infection rate of 1.00%.The ITS1 sequences were conserved with the Leishmania donowni complex.The positive rate of Leishmania specific antibodies in dogs was 5.97%.Conclusions:This study detected the blood sources and Leishmania parasites infection of sand flies by molecular methods in the extension region of Loess Plateau,China.A high epidemic risk of leishmaniasis is currently indicated by the results as the infection of Leishmania in sand flies,the extensive blood sources of sand flies including humans,and positive antibody of Leishmania in local dog sera.Given the recent increase of VL cases,asymptomatic patients,dogs and other potential infected animals should be screened and treated.Furthermore,the density of sand flies needs to be controlled and personal protection should be strengthened.

Phylogeny of certain members of Hyrcanus group(Diptera:Culicidae)in China based on mitochondrial genome fragments

Background:Species of the A nopheles hyrcanus group are widely distributed in Palearctic and Oriental regions and some of them are important malaria vectors.The cryptic species of An.hyrcanus group was almost impossible to identify based only on their morphology.The phylogenetic relationship of An.hyrcanus group was also not clear.Methods:Five members of An.hyrcanus group were identified by rDNA ITS2 sequencing as An.yatsushiroensis,An.belenrae,An.kleini,An.lesteri and An.sineroides.The mitochondrial genome fragments were sequenced and annotated using the mitochondrial genome of An.sinensis as reference.Based on the four segments and Joint Data sequences of these species,and other four anopheline species downloaded from GenBank,intraspecific as well as interspecific genetic distances were calculated and the phylogenetic trees were reconstructed by the methods of neighbor joining,maximum parsimony,minimum evolution and maximum likelihood.Findings:Four parts of mitochondrial genomes,which were partial fragments COI+tRNA+COII(F5),ATP6+CO///(F7+F8),A/D7(F19)and IrRNA(F21),were obtained.All fragments were connected as one sequence(referred as Joint Data),which had a total length of 3393 bp.All fragment sequences were highly conservative within species,with the maximum p distance(0.026)calculated by F19 of An.belenroe.The pairwise interspecific p distance calculated by each fragment showed minor or even no difference among An.sinensis,An.kleini and An.belenrae.However,interspecific p distances calculated by the Joint Data sequence ranged from 0.004 (An.belenrae vs An.kleini)to 0.089(An.sineroides vs An.minimus),and the p distances of the six members o f An.hyrcanus group were all less than 0.029.The phylogenetic tree showed two major clades:all subgenus A nopheles species(including six members of An.hyrcanus group,An.otroparvus and An.quadrim aculatus A)and subgenus Cellia(including An.dirus and An.minimus).The An.hyrcanus group was divided into two clusters as{(An.lesteri,An.sineroides)An.yatsushiroensis)and({An.belenrae,An.sinensis)An.kleini)).Conclusions:The An.hyrcanus group in this study could be divided into two clusters,in one of which An.belenrae,An.sinensis and An.kleini were most closely related.More molecular markers would make greater contribution to phylogenetic analysis.