| The study of species complex of mosquito of medical importance is one of theimportant contents in molecular systematic.There are four species or subspeciesgenerally considered to be the members of the important Culex pipiens complex,namely, Culex pipiens pipiens, Cx. pipiens quinquefasciatus, Cx. pipiens pallens andCx. pipiens molestus.The former three are recorded in China,of which Cx. pipienspipiens is only found in Xingjiang, Cx. pipiens pallens and Cx. pipiensquinquefasciatus in north and south China respectively, Cx. pipiens molestus arefound in Beijing,Shenyang and Shanghai.As the important disease vectors throughout the world, the species of Cx. pipiensComplex, which are distributed all over the country, have the important medicalresearch value. Wherein the Cx. pipiens pallens and Cx. pipiens quinquefasciatus,which belong to Cx. pipiens pipiens, are the major vectors of the bancroftian filariasisin our country. This complex is the dominant mosquito species in Chinese urban andrural areas, among which the Cx. pipiens pallens acts as the common mosquitospecies in northern China and the main mosquito species responsible for the indoorhemophagia harassment in the cities and towns.The classification for this complex remains as the worldwide problem, which isrecognized in mosquito taxonomic study. International and domestic academics haveconducted a lot of studies on taxonomic status, morphology identification, andgeographical regionalization for this complex. The morphology is very similar amongvarious subspecies of this complex. Therefore it is difficult to distinguish from theappearance. The morphological character of the male genitalia is the important basisfor the infraspecific classification. And the DV/D ratio of male genitalia is themorphological identification character for this complex. The field acquisitiondifficulty, character information insufficiency, and identification slowness for themosquitoes of this complex cause the difficulty on rapid identification and the lack of system evolutionary data on mosquito; the above content, combined with thelimitation of morphological identification and the continuously diminished workforceof taxonomists, has formed enormous challenge on the taxonomy development forthis complex.Along with the rapid development of molecular biology techniques (especiallythe PCR technique and automatic nucleic acid sequencing technique), the maturegene amplified and sequenced technology, together with the relatively developednetworking and information technology, has provided the development of molecularphylogenetic taxonomy based on DNA. Compared with the traditional morphologicaltaxonomy, the molecular phylogenetic taxonomy could carry out the identificationusing the smallish slice of organism tissue, distinguish the species in the differentgrowth stages, and verify the traditional taxonomy system to compensate itsdeficiencies. Consequently, it has been widely applied in many aspects, such as theresearch of insect phylogeny, action evolution, population genetic variation anddifferentiation, the identification of distinguishing the species swarm, the verificationof infraspecific taxa, and so on. The advent of many kinds of molecular geneticmarkers provides the new thinking and method for researching the Cx. pipiensComplex mosquitoes in terms of system taxonomy, population genetics,populationecology, and so on, thus better illustrates the relationship between the vectormosquitoes with the disease transmission, and offers the essential reference for theprevention and treatment of Mosquito and the vectors diseases.Based on the external morphological classification and identification and theprevious work, this study focuses on the four subspecies of Cx. pipiens Complex inour country. After compositing multiple molecular genetic markers, we study the genesequences of the molecular genetic markers with the molecular biology techniques.By determining the genetic sequences, we analyze each molecular genetic markerdetailedly and establish the phylogenetic relationship of this complex at the molecularlevel.The results for this study are as follows:1. In the15Cx. pipiens Complex populations, Wherein the acquired COⅠgenesequence length is633bp, the COⅠand COⅡ gene sequences all show thetropism and the bias. and the COⅡgene sequence length is626bp. The formermeans that the A+T content is higher than C+G content, and the latter means thebias of amino acid. The difference degrees and genetic distances among their sequences are all of linear relationship, and the conversion is greater thantransversion. The base substitution does not achieve the level of saturation. Theabove information indicates that there is large evolutionary potential among theCx. pipiens Complex populations.2. In the15Cx. pipiens Complex populations, the COⅠand COⅡ gene sequencesgenetic distances all fall into the range from0to0.013, which indicates that thevariability of this complex is low and the genetic similarity is high. The result ofcluster analysis shows that the COⅠand COⅡgenes are very conservative withinthe Cx. pipiens Complex population thus unfit for serving as the molecularcharacteristic index of subspecies categories in molecular systematics study. Itindicates from another perspective that the COⅠand COⅡ gene sequences arerelatively similar, thus it can be seen that they still belong to the same population.3. In the30Cx. pipiens Complex populations, wherein the acquired AChE2genesequence length is925bp, the AChE2gene sequences also show the tropism andthe bias. The former means that the A+T content are higher than C+G content, andthe latter means the bias of amino acid. The difference degrees and geneticdistances among their sequences are also all of the linear relationship, and theconversion is greater than transversion. The base substitution does not achieve thelevel of saturation.4. In the30Cx. pipiens Complex populations, the AChE2gene sequences geneticdistances all fall into the range from0to1.292, which indicates that for thiscomplex the variability, is high. The result of cluster analysis shows that theAChE2gene sequences has the crossing and overlapping phenomenons in termsof Cx. pipiens Complex mosquito subspecies categories classification thus cannotbe unified with the traditional morphological classification. The aboveinformation indicates that for the AChE2gene, the Cx. pipiens Complex mosquitodifferentiation in our country has not reached the degree in which it can bedistinguished by this gene sequence.5. The study for34geographic strains has been conducted. Except the strain ofHuocheng County in Xinjiang, the Cx. pipiens Complex mosquitoes of other33geographic strains all have the Wolbachia infection, and the infection rate is24%～100%, which indicates that the Wolbachia infection phenomenons withinthe Cx. pipiens Complex geographical populations are widespread and unevenlydistributed, and that the infectious status is diversified. Among32geographic strains we have measured the wsp gene sequences of the Wolbachia strain andfound that the diversity is relatively low. After the sequence comparison andalignment they are exactly consistent. The above information indicates that in ourcountry there is the closed relationship and high homology among the Cx. pipiensComplex.6. After comprehensively analyzing the male genitalia morphology, DV/D ratiomosquitoes living environment, and the autogeny in22geographical field sites,we conclude that the Cx. pipiens Complex mosquitoes of the following geographicstrains are Cx. pipiens pipiens: the strain of Heixiazi Island in Heilongjiang, thestrain of Jiayuguan in Gansu, the strain of Zhangye in Gansu, the strain of Minqinin Gansu, the strain of Wuwei in Gansu. It is confirmed the discovery of the newrecord of Cx. pipipens molestus in Manzhouli, Inner Mongolia.7. For the22geographic strains, the DV/D ratio of male genitalia appears strongnegative correlation with the latitude. Utilizing its regression equation, we haveverified that the theoretical dividing line for the geographical distribution of Cx.pipiens pallens and Cx. pipiens quinquefasciatus in our country is about30°. Theabove information indicates that the male genitalia DV/D ratio of Cx. pipiensComplex changes with the variation of geographic latitude.8. For25Cx. pipiens Complex geographical populations, the average number ofallelic genes for each site is5.92, the average polymorphism information contentis0.697and the average Shannon’s diversity index is1.236and the averageheterozygosity observed of the25Cx. pipiens Complex populations is0.471. Theabove information indicates that in our country the genetic variation within the Cx.pipiens Complex populations is rich. All highly polymorphic with high geneticpotential and it could reflect the genetic diversity information among thepopulations.9. The Hardy-Weinberg equilibrium inspection is conducted at9microsatellite sitesfor the25Cx. pipiens Complex populations. Except the Cx2site, Cp3site, andCx7site, other sites all deviate from the HWE. The average fixation index of the9microsatellite sites among the25Cx. pipiens Complex populations is0.224, andthe average genetic differentiation coefficient is0.228, which indicates that thereis no random mating among the populations and the high degree of geneticdifferentiation. The average value of the gene flow is1.008, which indicates thatthe genetic similarity among the populations is low, and that there is the gene exchange, which may also occurred during some period in the past, among theselected Cx. pipiens Complex populations.10. The genetic distances of the25Cx. pipiens Complex populations fall into therange from0.089to1.775, which indicates that the genetic diversity among thepopulations is large. The result of the cluster analysis shows that themicrosatellite markers at the9sites can basically reflect the genetic relationshipsamong the Cx. pipiens Complex populations properly and describe therelationships among the populations accurately, thus indicate that thegeographical distribution has certain connection with this complex. |
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