Impact Of Temperature On Fecundity And Susceptibility Of Anopheles Stephensi To Plasmodium Yoelii And Its Molecular Mechanisms

As a kind of vectors,mosquitoes can transmit a variety of diseases such as malaria,Zika,dengue,yellow fever,filariasis,and Japanese encephalitis,etc,leading a great threat to human health.Among them,malaria is the most widespread disease with the largest number of people infected.Human malaria is mainly transmitted by anopheles mosquitoes.In recent years,with global warming,the habitat and living habits of mosquitoes were changing,and mosquito-transmitted malaria's epidemic area and time also changed,which brings great challenges to our efforts to fight mosquito-borne diseases such as malaria.Using An.stephensi as the animal model,we investigated the impact of temperature on the fecundity and susceptibility of An.stephensi to P.yoelii and the related molecular mechanisms as the following three parts.The aim of this study was to elucidate the changes of the reproductive ability,survival time,the difference of malaria transmission ability and part of the molecular mechanisms of An.stephensi at different temperatures.It will provide reference and scientific basis for early warning of mosquito population density changes and malaria prevalence under climate change and scientific formulation of control strategies.Methods:1.Impact of temperature on the fecundity of An.stephensi and its molecular mechanisms.An.stephensi were mantianed at five temperatures such as 16 ?,20 ?,24 ?,28 ? and 32 ?.After blood feeding,it was conducted to observe the differences of egg counts,oviposition rate,oviposition time and survival time of An.stephensi at different temperatures.Total RNA of mosquitoes was isolated at time points such as the day of blood feeding(before blood feeding)and the first day,the third day and the fifth day post blood feeding.RT-qPCR was used to detect the transcriptional levels of Vitellogenin(Vg)at 5 different temperatures.2.Impact of temperature on susceptibility of An.stephensi to P.yoelii and its molecular mechanisms.The adult An.stephensi were divided into 5 groups and raised at different temperatures including 16 ?,20 ?,24 ?,28 ? and 32 ?.The 3 days old anopheles were challenged with P.yoelii by blood feeding with mice infected with P.yoelii.Total RNA was isolated at 4 time points same with before.RT-qPCR was used to detect the trancriptional level of key molecules of signal pathways in innate immunity of anopheles.Mosquito midguts were dissected on day 9 post infection,and oocyst numbers were counted under fluorescence microscope.3.Impact of temperature on changes of gut microbiota of anopheles mosquitoes.An.stephensi with a same emergence day were divided into 5 groups and raised to 3 days age at 5 different temperatures as 16 ?,20 ?,24 ?,28 ? and 32 ?.Mosquito guts were dissected and put into EP tubes each containing 1ml anhydrous ethanol.Triplicates were set for each group.The samples were then sent to the Novogene company.The gut flora of An.stephensi from the 5 temperature groups were investigated,compared and analyzed by high-throughput sequencing of 16 S rDNA.Results:1.The ambient temperature had great influence on fecundity and survival time of anopheles mosquitoes.When the temperature was as low as 16 ?,An.stephensi had a low blood sucking rate and stopt ovipositing,and the median survival time was only 2.0 d.It indicates that the low ambient temperature as 16 ? is not suitable for the reproduction and survival of An.stephensi.With the increase of ambient temperature in the range of 20-28 ?,the gravid time of An.stephensi was gradually shortened,the number of oviposition was gradually increased and the survival time was gradually prolonged.When the ambient temperature rose to 32 ?,the gravid time was further shortened,but the number of oviposition decreased and the survival time was shortened.The results suggest that 28 ? may be the best ambient temperature for the fecundity and survival of An.stephensi.In order to further elucidate the molecular mechanisms of the impact of ambient temperature on fucundity of anopheles mosquitoes,the transcriptional levels of Vg,which is important for mosquito oviposition,were compared among the five temperature groups and analyzed by method of fluorescence quantitative PCR in this study.The results showed that the transcriptional level of Vg was always at basal low level before and after blood feeding at 16 ?,but increased very significantly under other temperatures in this study at 24 hours post blood feeding.Moreover,the difference of transcriptional level among temperature groups was consistent with the trend of the difference of oviposition numbers(with statistical difference).Three days after blood feeding the transcriptional level plummeted,and 5 days after blood feeding it was close to the base low level.The results suggest that Vg plays an important role in the oviposition of anopheles mosquitoes,and the ambient temperature might change the fecundity by affecting the transcriptional level of Vg in anopheles mosquitoes.2.Temperature had an obvious impact on susceptibility of An.stephensi to P.yoelii.The infection rate and oocyst density of An.stephensi infected by P.yoelii were both the highest at 24 ? followed by 20 ?.Too high or low ambient temperature was not suitable for the development of plasmodium in anopheles.Transcriptional levels of several key molecules of the innate immunity of the anopheles signaling pathways(Toll,Imd,and JAK-STAT)were significantly different among the 5 temperature groups.The innate immune response against P.yoelii was the weakest at 24 ? and enhanced at temperatures below or above 24 ?.It indicates that the innate immune mechanisms of anopheles may be involved in the impact of temperature on susceptibility of A.stephensi to P.yoelii.3.There was obvious difference of the composition of gut microbiota of An.stephensi among the temperature groups.The species richness among groups showed that the 28 ? group was significantly lower than the other temperature groups,and it was the highest at the 16 ?.There were 277 identical OTUs among the five temperature groups.From 16 ? to 32 ?,the numbers of unique OTUs at each temperature were 1062,595,1022,88,and 326,respectively.There were significantly less unique OTUs at 28 ? than the other temperatures.The relative abundance of of An.stephensi gut microbiota was significantly diverse among different temperature groups,suggesting that temperature could affect the composition proportion of different bacteria.For instance,the proportion of Gluconobacter in the gut microbiota of An.stephensi at 28 ? was significantly higher than that at other temperatures(literature suggests that such bacteria may be related to the immune response and development of mosquitoes).Besides,the 32 ? group had the highest Lactobacillus abundance.The comparative analysis of similarity(UPGMA cluster analysis)and difference coefficient(Distance index)of mosquito gut microbiota composition among different temperature groups showed that the difference between the 28 ? group and other groups was the most obvious.Conclusions:1.The changes of the fecundity and survival time of An.stephensi at different temperatures were determined.The most optimum ambient temperature was 28 ? for the fecundity and survival of An.stephensi.The molecular mechanism might be that temperature affected the fecundity by impact of temperature on the expression level of mosquito vitellogenin.2.Ambient temperature has a great impact on the susceptibility of An.stephensi to P.yoelii.24 ? is more suitable for the infection of An.Stephensi by p.yoelii than the other temperatures.It was found that Toll,Imd and JAK-STAT signaling pathways of An.stephensi were involved in the impact of temperature on susceptibility of Anopheles stephensi to P.yoelii.3.It was confirmed that the gut microbiota of An.stephensi was changed by temperature.The species richness and abundance were different under various temperatures.These changes of microbiota may be related to the fecundity and susceptibility of An.stephensi to P.yoelii.Therefore,it laid a foundation for further determining whether the change of gut microbiota was involved in the impact of temperature on the fecundity and vector capacity of anopheles mosquitoes.