| Mosquitoes are important vectors of many human diseases, such as dengue, yellow fever, encephalitis, malaria and filariasis etc. It has been the working emphasis for the World Health Organization to reduce the disease mortality and effectively control mosquito vectors. At present, the prevention of mosquito relies mainly on the biological control, chemical prevention and environmental control, on the basis principle of comprehensive prevention or control. However, the widely use of chemical pesticides not only cause the mosquito resistant to pesticides to some extent, but also accelerate the evolution speed of resistant gene. In this paper, three detoxification gene families were studied to reveal the resistance mechanism, mainly from the following several aspects.1. Three detoxification enzymes families, cytochorme P450 monooxygenase, glutathione-S-transferase and carboxyl/cholinesterase are important parts of defense system against xenobiotics for insect, they were implicated in resistance metabolism to insecticides. These enzymes perform various of different biological functions, for example, carboxylesterase could hydrolysis. Their function diversity depends on their sequence difference. In this paper, all detoxification genes from Culex pipiens quinquefasciatus were used to create phylogenetic tree through Mega 3.0 clustering software. As a result,80 carboxylesterase genes were classified into A-N 14 clusters,35 GST genes were classified into Delta, Epsilon, Omega, Sigma Theta, Zeta six clusters,198 cytochrome P450s genes were classified into CYP3, CYP4, CYP6, CYP9, CYP2 and mitochondrial six clusters. The specific classification and evolutionary information were summaried for all detoxification genes, which not only helps to illuminate the function of each gene, but also provides theoretical basis for exploration of resistance mechanism exsited in mosquito.2. With the rapidly development of New Generation Sequencing technology, it become possible to sequence a species' transcriptome with low cost, high throughput, faster and precise. In this paper, four different development stages of organophosphorus resistance strain Culex pipiens quinquefasciatus:egg, larve, pupa, adult were sampled for total RNA extraction, sequencing and comparitive analysis, many stage-specific expressed genes (1076 genes for egg; 545 for 3rd instar larva; 465 for pupa; 580 for adult) and differentially expressed genes (larva/egg:3217 bar; pupa/egg:2885 bar; adult/egg:3491 bar) were screened and filtered. For further understanding of functions and metabolic pathways invovled for these stages-specific genes and differentially expressed genes, GO analysis and pathway enrichment analysis were performed. Finally, we monitored and constructed gene expression patterns for 80 carboxylesterase genes,35 GST and 198 cytochorme P450 monooxygenase genes,respectively, we found that only 50 carboxylesterase genes,29 GST,138 cytochorme P450s genes were expressed during four different developmental stages. The reason for remaining genes that didn't express may due to the low expression amount or too low to be detected.3. In situ hybridization (ISH) is one of the classical technical used to localize the aim gene in organisms. Through in situ hybrdization between DIG-labelled nucleic acid probe and paraffin embedding mosquito tissue, which contains the mRNA information of carboxylesterase, glutathione-S-transferase and cytochrome P450s, we can localize these genes into exact sites in the body of mosquitoes. The ISH technology were adopted in this paper and 2 representative carboxyesterase genes were clearly positioned in ovary, brain, ganglia, midgut etc.. It provides reliable experimental basis for the study of carboxylesterase gene expression patterns, function and resistant mechanism.4. The knowledge of mosquitoes' histological structures is the basis of various researches. In this study, improved consecutive paraffin tissue sections, HE staining and organ dissection were performed to study the histological structures of Culex pipiens quinquefasciatus, on the levels of morphology and histology. The digestive, emunctory, reproductive, nervous and respiratory systems of Culex pipiens quinquefasciatus were demonstrated clearly. Detailed and improved protocols of parraffin tissue section preparation and HE staining were discussed. This study provides the information of histological structures of Culex pipiens quinquefasciatus, which will shed light on the exploration of gene functions of mosquito using in situ hybridization, immunohistochemistry, etc. |
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