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目的研究不同日龄斯氏按蚊(Anopheles stephensi)传播伯氏疟原虫(Plasmodium berghei)能力的变化以及可能机制。方法选取4日龄和25日龄雌性斯氏按蚊,用原虫血疟为4%~8%的伯氏疟原虫感染BALB/c小鼠饲喂蚊虫,感染后8 d解剖蚊虫肠道,镜检计数蚊虫肠道中疟原虫卵囊数,比较4日龄和25日龄蚊虫对疟原虫易感性的变化。选取感染前4日龄和25日龄雌性斯氏按蚊,采用LB平板培养法检测其体内可培养共生菌的量,用实时定量PCR(real-time quantitative PCR,qPCR)方法检测其体内的共生菌总量。选取感染前4日龄和25日龄雌性斯氏按蚊,采用qPCR检测其体内天蚕抗菌肽1(cecropin,CEC1)、CEC3、防御素(defensin,DEF;gambicin,GAM)、攻击素(attacin,ATT)以及一氧化氮合成酶(nitric oxide synthase,NOS)、双氧化酶(dual oxidase,DUOX)和含硫酯蛋白1(thioester protein 1,TEP1)等主要免疫效应因子的表达水平。结果感染伯氏疟原虫后8 d,4日龄斯氏按蚊中肠道疟原虫卵囊中位数是139,25日龄按蚊卵囊数中位数是3,4日龄按蚊伯氏疟原虫的感染密度是25日龄的46倍(P<0.01)。qPCR结果显示,25日龄蚊虫体内共生菌总量为4日龄蚊虫的1.5倍,差异有统计学意义(P<0.05);LB平板培养法结果显示,25日龄肠道可培养共生菌平均为28 889个菌落形成单位(colony forming units,cfu),是4日龄(3 200 cfu)的9倍,差异有统计学意义(P<0.01)。25日龄斯氏按蚊体内NOS基因表达量是4日龄的2.4倍(P<0.01),抗菌肽ATT、DEF、CEC3、CEC1的表达量分别为4日龄的27%、48%、14%、61%,差异均有统计学意义(P<0.05);4日龄与25日龄斯氏按蚊体内GAM、DUOX、TEP1的表达量均未发生显著变化。结论随斯氏按蚊日龄增加,斯氏按蚊抗菌肽水平发生显著下调,体内共生菌增多,NOS表达量上调,按蚊抵抗疟原虫的能力增强。
Objective To study the changes of Plasmodium berghei (Plasmodium berghei) and their possible mechanism in Anopheles stephensi at different ages. Methods Female mosquitoes of 4-day-old and 25-day-old were used to infect BALB / c mice infected with P. berghei with 4% -8% Plasmodium falciparum. Mosquitoes were dissected on the 8th day after infection, The number of malaria parasite oocysts in the intestine of mosquitoes was counted, and the susceptibility of malaria parasite to 4-day-old and 25-day-old mosquitoes was compared. Anopheles sinensis was collected at 4 days and 25 days before infection. The amount of viable bacterial cells cultured in vivo was detected by LB plate culture method. The symbiosis in vivo was detected by real-time quantitative PCR (qPCR) Total bacteria. Anopheles sinensis was collected at 4 days and 25 days before infection. The expression of cecropin (CEC1), CEC3, DEF (gambicin, DEF) and attacin ATT and the expression of major immune effector factors such as nitric oxide synthase (NOS), dual oxidase (DUOX) and thioester protein 1 (TEP1). Results The median length of oocysts in the midgut of the 4-day-old Anopheles stephensi was 139 days after infection with P. berghei. The median number of oocysts in the 25-day-old mosquito was 3 and 4 days The infection density of P. berghei was 46 times greater than that at 25 days (P <0.01). The results of qPCR showed that the total amount of commensal bacteria in 25-day-old mosquitoes was 1.5 times higher than that of 4-day-old mosquitoes, the difference was statistically significant (P <0.05). The results of LB plate culture showed that the average number of 25- There were 28 889 colony forming units (cfu), which were 9 times of 4 day old (3 200 cfu), the difference was statistically significant (P <0.01). The expression of NOS gene in Anopheles stephensi was 2.4-fold (P <0.01) 4-day-old on the 25th day. The expression of ATT, DEF, CEC3 and CEC1 was 27%, 48%, 14% %, 61%, respectively. There was no significant difference in the expression of GAM, DUOX and TEP1 between 4 days old and 25 days old Anopheles stephensi. Conclusion With the increase of the age of Anopheles stephensi, the antibacterial peptide level of Anopheles stephensi was significantly down-regulated, the amount of symbionts increased, the expression of NOS was up-regulated, and Anopheles mosquito resistance to Plasmodium increased.