Molecular Identification And Control Of Biting Midges(Diptera:Ceratopogonidae)

Biting midges(Diptera,Ceratopogonidae)are tiny flies,whose body size rarely exceed 3 mm,which is one of the most strikingly diverse groups of Insecta.In recent years,biting midges have attracted great interest because of their agricultural,ecological,medical,and veterinary importance.Most midges feed on invertebrates but three genera have evolved the habit feeding on warm-blooded mammals,such as human,cattle and swine.They are Culicoides,Lasiohelea and Leptoconops,which are namely blood-sucking midges.However,the ecological habits of three genera show great difference in their control.They are greatly different in activity time and larval breeding sites.Therefore,the exact identification of biting midges could provide an important reference to their control.Up to now,most of studies have been focused on the identification of biting midges by single gene and much attection was paid to a small number of Palaearctic species within the Culicoides associated with Bluetongue virus(BTV)transmission,but molecular identification based on different genes and control studies of biting midges from Oriental region are poorly documented.In this study,a molecular identification system of Culicoides and the genetic structure of Culicoides arakawai from the districts cross Taiwan Strait based on mitochondrial and nuclear genes were established and studied.Meanwhile,the phylogeny of the subfamily Forcipomuiinae based on molecular data was analysised.In addition,the response of field-collected midges to five commonly used insecticides was evaluated to clarify the susceptibility of Asia species of biting midges.The results are as follows:1.Barcoding in the genus CulicoidesThe genus Culicoides is the largest among the three genera of blood-sucking midges,and their number also comes first in Ceratopogonidae.Some species of Culicoides can cause significant economic losses and transmit arbovirus diseases to both animals and humans,such as African horse sickness,Bluetongue,Oropouche.However,it is difficult to identify the Culicoides species based on the morphology.The sequencing of mitochondrial and nuclear genes have been proposed as an ideal method for rapid identification to species.To establish DNA barcodes for Culicoides species,a total of 284 specimens of Culicoides representing 77 different species which were collected from 13 provinces in mainland China and Taiwan district in the past 6 years were used.As a result,a total of 264 sequences of COI,175 sequences of cyt b,260 sequences of 16S rDNA and 190 sequences of ITS gene were obtained in the present paper.The clusters generated from the concatenated dataset of COI+16S rDNA+cyt b+ ITS were in agreement with the morphological determination,which consist of subgenus Oecacta Poey,subgenus Beltranmyia Vargas,subgenus Culicoides Latreille,subgenus Avaritia Fox,subgenus Haemophoructus Macfie,subgenus Trithecoides Wirth et Hubert and subgenus Monoculicoides Khalaf.The use of COI barcodes,combined with 16S rDNA,cyt b,ITS as a tool for the identification of biting midges could not only differentiate most studied species,but also identify the complex and cryptic species.2.Genetic diversity and phylogeographic studies of Culicoides arakawaiCulicoides arakawai Arakawa,1910 is one of the most widely distributed biting midges in the southeast China,which transmits fatal diseases to chicken,swine,and other livestock.Leucocytozoon caulleryi and many different pathogens have been frequently isolated from C.arakawai,indicating that it might be responsible for several epidemics of Culicoides-borne diseases.However,its genetic structure and phylogeographic relationships remain disputed.In this study,the mitochondrial gene(COI,16S rDNA)and nuclear gene(ITS1-ITS2)were used as genetic markers to examine the genetic diversity and population structure of C.arakawai throughout its range in Mainland China and Taiwan district.According to the geographic zoning,10 populations of C.arakawai were divided into Central China,the Southeast,the Southwest and South China.Molecular variance(AMOVA)analysis based on the concatenated dataset indicated that the genetic variance was attributable to variation within populations with the proportion of it in mitochondrial gene and nuclear gene were 96.75%and 91.44%,respectively.Phylogenetic analysis based on different molecular dataset clustered into three well supported lineages.Amongest them,lineage Ⅰ was formed with haplotype AH5 and AH7,lineage Ⅲ was formed with haplotype YN1 and GX9,other haplotypes formed lineageⅡ.It was suggested that there were no significant genetic differences among populations.Airflow and human activities might be the main reason of its diffuse.3.Phylogeny of the subfamily Forcipomuiinae based on molecular analysisSubfamily Forcipomyiinae Lenz,1934 is the second large group of Ceratopogonidae with 1671 described species widely distributed in the world.This subfamily includes the genera viz.Atrichopogon,Forcipomyia,Lasiohelea,and Atriculicoides.However,the taxonomic status of Lasiohelea within the subfamily Forcipomyiinae has led to many arguments and confusion for several decades.In this study,mitochondrial genes(COI,16S rDNA)and nuclear genes(18S rDNA,28S rDNA)were employed to infer the phylogeny of subfamily Forcipomyiinae.Phylogenetic analysis showed that Lasiohelea as a monophyletic clade was well supported.Lasiohelea should be regarded as a separate genus.4.The efficacy of five insecticides in controlling Biting midgesFive commonly used insecticides in Asia,including 2 types of pyrethroids,2 types of organophosphates,and 1 combined pesticide mix,based on WHO standards were tested and evaluated.Field-collected adult midges were exposed to different concentrations of deltamethrin,dichlorvos,chlorpyrifos,beta-cypermethrin,and beta-cypermethrin combined with emamectin benzoate.After exposure to each insecticide for 1 hour(h),the midges were transferred to a recovery chamber and observed for 24 h post-exposure.The knockdown time,the number of deaths after 20,40,60 min of exposure,and the LC50 were determined.Significant differences in toxicity were observed between the various insecticides.The LC50 ranged from 0.061±0.003 mg/L for deltamethrin to 599.932±16.621 mg/L for dichlorvos.The toxity was the highest with deltamethrin,followed by chlorpyrifos,beta-cypermethrin,emamectin benzoate,and dichlorvos.These results revealed that deltamethrin should be the first line of treatment for Culicoides.