ABSTRACT
Objective To investigate the existence of genetic divergence of sympatric populations of Anopheles sinensis of different feeding preferences based on the rDNA-ITS2 sequence differences. Methods A large number of wild anopheles popu-lations were trapped all night by man-baited net and calf-baited net that had been set up between high-density natural villages of An. sinensis populations and vector-breeding sites,from which two groups of An. sinensis were separated by morphological iden-tification and brought back to the lab for conventional breeding. A large closed greenhouse which temperature and humidity was appropriate was selected as research settings of mark-release-recapture methods by female mosquitoes ,in the center of which above An. sinensis populations baited by man and calf and respectively correspondingly marked by red and yellow phosphors were released in together,in each side of which An. sinensis were recaptured simultaneously by man-baited net and calf-baited net. An. sinensis populations trapped by man twice were brought back to the lab and bred with man-blood,correspondingly ones trapped by calf with calf-blood. Man-preferring and calf-preferring strains were screened respectively from An. sinensis which had been baited by man and calf by the mark-release-recapture methods after parent and F1 mosquitoes,and sequencing and aligning of both rDNA-ITS2 were conducted via PCR amplification. Results The recapture ratios of wild parental mosquitoes An. sinensis of man-preferring group by man-baited net and calf-baited net were 54.07%(339/627)and 45.93%(288/627)re-spectively,and ones of calf-preferring group by man-baited net and calf-baited net were 58.01%(409/705)and 41.99%(296/705)respectively. Two groups of parental mosquitoes trended towards selecting the original blood hosts in host-seeking prefer-ence(χ2=19.42,P<0.01). The recapture ratios of F1 mosquitoes An. sinensis of man-preferring group by man-baited net and calf-baited net were 63.43%(765/1 206)and 36.57%(441/1 206),and ones of calf-preferring group by man-baited net and calf-baited net were 68.22%(1 039/1 523)and 31.78%(484/1 523). Two groups of F1 mosquitoes had more significant characteris-tics of selecting the original blood hosts in host-seeking preference(χ2=271.69,P<0.01)and showed the genetic differentia-tion phenomenon,but the results of sequencing and aligning of the rDNA-ITS2 via PCR amplification showed no difference in base sequence between the two strains and both were 469 bp. Conclusions The genetic divergence based on the rDNA-ITS2 se-quence does not happen in An. sinensis sympatric populations of different feeding preferences.
ABSTRACT
Background & objectives: Anopheles fluviatilis, which ranks second among the major malarial vectors in India occurs as a complex of three morphologically identical species (species S, T and U) of which only species S is a vector. Hence, it becomes pertinent to have a method for the detection of this vector species under field conditions to map the distribution of this vector. An rDNA-ITS2-PCR assay has been developed earlier for species S of this complex using female adult specimens. In order to widen the range of samples on which this technique can be employed, the utility of this PCR assay in detecting different life stages/gender/parts of the vector species was studied. Also, its reliability in detecting a single species S in pools of species T was studied. Methods: Mosquitoes were collected from Malkangiri and Koraput districts of Orissa State where species S and T of this complex are reported. The wild caught fed females, after egg laying were subjected to PCR assay for species identification. The F1 progeny of a few PCR identified specimens was raised and samples at larval, pupal and adult stages were used for PCR assay. Single adult specimen of species S was added to pools containing different numbers of adults of species T and the pools were subjected to DNA extraction and PCR assay. Results: The PCR assay could detect species S from pure DNA extracts of the immature stages and crude DNA extracts of parts of adult/whole adult mosquito of either gender. Crude DNA extracts of pools of mosquitoes had to be diluted and used in order to obtain the species diagnostic fragment. Interpretation & conclusions: The rDNA-ITS2-PCR assay producing an amplicon of 350 bp. diagnostic for species S, could detect all stages/gender. Any part of the adult can be used for species identification. Further, a single adult of species S in pools of as many as 99 adults of species T could be detected. Application of this PCR assay will be useful in mapping the distribution of species S, an important malarial vector.
ABSTRACT
Objective To establish the molecular identification of five members in Anopheles maculatus complex from China. Methods Different rDNA-ITS2 regions of An. maculatus complex were sequenced and analyzed. The species specific primers were designed, and PCR assay was used for the identification. Results The length and GC contents of ITS2 were 328 bp, 58.54% in An. pseudowillmori, 330 bp, 57.85% in An. maculatus, 337 bp, 59.05% in An. willmori, 334 bp, 58.68% in An. dravidicus, and 338 bp, 57.69% in An. sawadwongporni, respectively. The intra-species ITS2 sequences were conservative. The ranges of divergence level among five members were from 9.7% to 18.9% . Five distinct specific fragments were amplified by PCR assay using five species specific primers and 5. 8S primer. The length was 119, 186, 231, 327 and 406 bp respectively. Conclusion The diagnostic PCR assay based on ITS2 divergence to distinguish five members of An. maculatus complex was simple and reliable.