Survey Of Mosquito Population In China-myanmar Bordering Area, Molecular Identification Of Mosquito Species And Bioassay Of Bacillus Sphaericus Insecticidal Effect

Mosquito-borne disease is a kind of infectious disease transmitted by mosquito biting and is seriously threatening human health. The mosquito population structure is complex with high species richness in the bordering area of China and Myanmar because of the special climate, geographical environment and frequent contact cross the border. So mosquitoes transmitted disease such as malaria is prevalent throughout the year. And, this area has become an important way of imported malaria to China. Study on mosquito population around the bordering area of China and Myanmar will provide an important reference for prevention and control of mosquito-borne diseases in this area.Firstly, the survey of mosquito population was conducted in the Kachin state of Myanmar around the bordering area of China and Myanmar to understand the local mosquito population composition and density. Then, the anopheles mosquito species found in this area was molecularly identified using the molecular biology and bioinformatics technologies. Finally, the bioassay of insecticidal effect of Bacillus sphaericus(Bs) on Anopheles sinensis was carried out using Probit analysis method.Methods:1. Mosquitoes were captured using light trap. CDC miniature light traps were placed in houses at dusk and then collected in the following morning. Traps were placed in the same house for two consecutive nights each week. Once collected, mosquitoes were morphologically separated as Anopheles, Culex, Aedes, Armigeres, and other subfamilies or genera. Anopheles mosquitoes were further morphologically identified to species or species complex in the field when possible.2. The genomic DNA was extracted from a single anopheles mosquito. Then the second internal transcription spacer of ribosomal DNA(ITS2) segments was amplified by PCR. And the PCR product was analysed by electrophoresis and sequencing. Finally, the anopheles species were molecularly identified by ITS2 sequences analysis and the construction of evolutionary tree.3. Engorged Anopheles sinensis were captured from the bordering area of China and Myanmar and maintained in the insectary to lay eggs and hatch the larvae. Then, the third instar larvae were used to conduct the bioassay of Bs insecticidal effect by Probit analysis. According to the results of preliminary experiment, different concentrations from low to high of Bs were added to 500 ml dechlorination water in enamel bowls containing larvae respectively. The blank control group was set up at the same time. The deaths of larvae were counted at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h post treatment.Results:1. In total 8056 mosquitoes including 1536 Anopheles, 6260 Culex, 45 Aedes and 215 other mosquitoes were captured in JHK, L1, JYH, MSY around the bordering area of China and Myanmar. Among the captured mosquitoes, culex was the most dominant genus followed by anopheles. According to the anopheles species identification, 13 anopheles species were identified including An. minimus, An. maculatus, An. kochi, An. splendidus, An. sinensis, An. barbirostris, An. vagus, An. tessellates, An. peditaeniatus, An. crawfordi, An. maculipennis, An. culicifacies and An. lesteri. Among them, An. minimus was most dominant, followed by An. culicifacies, An. sinensis, An. vagus, An. peditaeniatus and An. maculatus, etc. Anopheles distributions are different among the study sites.2. Ten of the above 13 anopheles species were further determined by DNA extraction, ITS2 PCR amplification, sequencing and sequence alignment. The molecular identification result was consistent with the morphological identification for An. minimus, An. maculates, An. kochi, An. splendidus, An. sinensis, An. vagus, An. peditaeniatus and An. culicifacies. However, the molecular identification was inconsistent with the morphological identification for the rest of the 2 anopheles species, An. barbirostris and An. lesteri.3. The accumulated mortality of the third instar larvae of Anopheles sinensis was increased gradually with the increase of Bs concentration among the groups. The log value of the concentrations and the probability value of mortality had a good linear relationship.Conclusions:1. This study indicates that culex is most dominant followed by anopheles in the bordering area of China and Myanmar according to the mosquito population composition and density. The mosquito general composition varied significantly among the study sites due to different natural environment. Anopheles proportion in village environment is much higher than that in urban environment. Anopheles mosquito species identification results suggest that the anopheles species richness is high in the region and An. minimus is most dominant.2. Molecular biology and bioinformatics technology is an effective and accurate method for the identification of mosquito species, and can be used as a good supplement to the traditional identification based on morphology. This study identified anopheles mosquito species in the bordering area of China and Myanmar more accurately using the nucleic acid molecular identification techniques.3. Bacillus sphaericus can be used as effective bio-larvacide in the control of Anopheles sinensis in the bordering area of China and Myanmar. Reasonable dosage should be considered in the application of Bs. The results of this study provide an important reference for the reasonable application of Bs and accurate chose of the dosage in this area.