Study On The Sugar Feeding And The Olfaction Associated Genes In Culex Pipiens Pallens

Culex pipiens pallens Coquillett, the most prevalent Culex species in China, is the primary vector of lymphatic filariasis and epidemic encephalitis. The control of this mosquito species heavily depends on the application of large quantities of chemical insecticides for a long time, which has resulted in the environmental pollution, insecticide resistance and threat to the health of human being and livestocks. Sugar feeding is a common behaviour of adult mosquitoes, and most mosquito species need to obtain sugar to replenish their energy reserves. Meanwhile, based on the sugar feeding of mosquitoes, several methods have been widely applied in controlling a variety of mosquito species. However, how mosquito adults successfully locate their host plants remains unclear. In addition, the relationship between sugar feeding, fitness values, physiological conditions and the preferences of mosquitoes to host plants deserves further research. This dissertation investigated the sugar feeding behaviour of Cx pipiens pallens, the relationship between mosquitoes’fitness and physiological conditions and their preferences to various host plant species, the behavioural responses of adult mosquitoes to common host plant volatiles, the volatile profile of its host plant (Abelia chinensis) and the olfaction asscociated genes in Cx. pipiens pallens. The following results were obtained:(1) The wing length, body weight and physiological conditions of newly emerged adults fed with different amounts of food in their larval periods was measured, and the temporal variations of proportions of female and male mosquitoes that ingested the colour-dyed sugar solution were determined. The results revealed that the larval diet regimes strongly affected the body size, physiological conditions and energy reserves of mosquito adults. The proportions of sugar-fed mosquitoes from rich larval diet regimes were higher than the ones from other regimes when they were newly emerged. The proportions of sugar-fed females were consistently higher than the ones of sugar-fed males from the same larval diet regimes.(2) Newly emerged Cx. pipiens pallens were provided with various flowering plant and seed pod species with differential preferences under laboratory condition, and the effect of feeding on different sugar sources on the survivorship and fecundity of mosquitoes was investigated. The results showed that the survival times and numbers of eggs laid by mosquitoes fed with their preferred sugar sources were significantly higher than those from other groups in most cases; the proportions of sugar-fed mosquitoes when provided with preferred sugar sources were higher than those from other groups, and the differences in survival times might be attributed to the amounts of ingested sugar from the tested flowering plants and seed pods.(3) The behavioural response of Cx. pipiens pallens to the volatiles of three flowering plant species(Ligustrum quihoui, Abelia chinensis, Nerium indicum) was determined in the Y-shape olfactometer. In addition, the physiological conditions and energy reserves of female and male mosquitoes were measured when they were provided with these three flowering plant species as their nectar host plants. The results indicated that mosquitoes could discriminate and respond to the volatiles of different flowering plant species, and the preferences of mosquitoes to the tested flowering plants were consistent with previous reported results; there were significant differences between the physiological conditions of mosquitoes from different groups; the energy reserves of mosquitoes fed with preferred plant species were higher than those fed with less preferred sugar sources.(4) Behavioural response of female Cx. pipiens pallens to 18 kinds of common plant volatile compounds at different concentrations was determined in the Y-shape olfactometer. A synthetic full-component blend composed of behaviourally active compounds and a reduced blend composed of six most attractive compounds were formulated, and their attractiveness to mosquitoes was tested againt the solvent and each other, respectively. The results showed that 15 individual compounds which included (E)-β-ocimene, benzaldehyde, hexanol, phenylethyl alcohol, (Z)-3-hexen-1-ol and linalool as well as the full blend and the reduced blend were attractive to Cx. pipiens pallens; there was no significant differences between the attractiveness of the full blend and the one of the reduced blend.(5) The volatile compounds of A. chinensis florets were collected by solid phase microextraction, and they were analyzed the constituents by gas chromatography-mass spectrometry. The major volatile compounds released by A. chinensis florets included benzaldehyde, a-pinene, phenyl acetaldehyde, benzyl alcohol and phenylethyl alcohol; the pentane extract and the synthetic compound blend were both attractive to female Cx. pipiens pallens in the Y olfactometer assays.(6) Ten genes encoding putative odorant binding proteins (OBPs) and one gene encoding putative olfactory co-receptor (Orco) were identified from the antenna of Cx. pipiens pallens adults, and their full-length cDNA sequences were obtained. The results of quantitative real-time PCR indicated that the genes encoding OBPs were mainly expressed in the antenna of males and females; the gene encoding Orco was mainly expressed in the antenna of adults and the head (without antenna) of females. DNA sequences encoding mature CpalOBP2 and CpalOBP3 proteins were ligated into pET-22b plasmids and transferred into Escherichia coli BL21 (DE3) pLysS cells, respectively. The recombinant proteins were successfully induced and expressed in the E. coli strains.On the whole, this dissertation investigated the effect of larval diet regimes on the body size of Cx. pipiens pallens adults and the proportions of sugar-fed mosquitoes; it explored the effect of feeding on various flowering plants and seed pods with differential preferences on the proportions of sugar-fed mosquitoes, survivorship, fecundity and physiological conditions of Cx. pipiens pallens; it determined the behavioural response of female Cx. pipiens pallens to common host plant volatile compounds and their synthetic blends and analyzed the volatile profile of A. chinensis florets; the preliminary researches on the olfaction associated genes in Cx. pipiens pallens were conducted. The results revealed that the body size, physiological conditions and the proportions of sugar-fed mosquitoes were affected by their larval diet regimes; feeding on different nectar host plants significantly affected mosquitoes’ survival times, fecundity, energy reserves and the proportions of sugar-fed mosquitoes; female Cx. pipiens pallens could be differentially attracted by a variety of compounds, whose attractiveness was closely related to their concentrations; the volatile profile of A. chinensis florets was maily composed of aromatic compounds, whose pentane extract and synthetic blend was attractive to Cx. pipiens pallens as well; ten genes encoding OBPs and one gene encoding Orco were identified in the dissertation. The results would provide novel insights into exploiting the "mosquito-plant" relationships; the researches regarding the species of mosquito-preferred host plants and their behaviourally active compounds could provide valuable information for developing novel environmentally friendly and safe phytochemical attractants as well as attractive toxic sugar baits (ATSB) for mosquitoes on basis of their sugar feeding behaviour.