目的 对约氏疟原虫感染后8、11和14 d的斯氏按蚊进行转录组测序,分析约氏疟原虫血淋巴子孢子诱导斯氏按蚊的免疫信号通路及效应机制。方法 约氏疟原虫BY265株按常规腹腔接种感染健康昆明小鼠,3 d后选择血液内有配子体的小鼠供斯氏按蚊雌蚊吸血,于吸血感染后8、11和14 d各取斯氏按蚊20只,提取总RNA进行高通量转录组测序,分析按蚊基因表达情况;使用统计算法Bray Curtis进行层级聚类,分析各样本间基因表达的差异;根据每百万转录本（TPM）值进行差异基因分析;使用gplots package软件绘制聚类热图,分析感染后8、11和14 d,Toll信号通路（Toll）、免疫缺陷信号通路（Imd）、信号转导和转录激活因子（STAT）和c-Jun氨基末端蛋白激酶（JNK）通路,效应分子如含硫酯蛋白家族分子（TEP）、抗菌肽、一氧化氮合酶（NOS）、硝化反应相关分子等的表达情况;利用实时荧光定量PCR（qPCR）扩增Rel1、Rel2转录因子、含硫脂蛋白分子（TEP1）和血红素过氧化物酶（HPX8）进行验证。结果 层级聚类分析显示,斯氏按蚊感染后11 d与8 d和14 d的基因表达变化差异较大。差异基因分析结果显示,斯氏按蚊感染后11 d与8 d相比,有1 109个基因上调,257个基因下调;感染后14 d与8 d相比,有62个基因上调,136个基因下调;感染后14 d与11 d相比,有174个基因上调,196个基因下调。聚类热图分析结果显示,在感染后11 d与8 d和14 d的比较中,斯氏按蚊Rel1转录因子、Toll5A和Toll1A受体分别上调约1.9、1.8和2.1倍;双过氧化物酶和双氧化酶均上调约2倍;HPX8上调约1.5倍。qPCR验证结果显示,感染后11 d Rel1、Rel2和HPX8的相对表达量为3.43、3.95和4.01,与对照组的0.82、0.88和1.01差异有统计学意义（P < 0.01）。结论 约氏疟原虫血淋巴子孢子可诱导斯氏按蚊Toll信号通路通过直接或协调其他通路调控硝化反应相关分子表达。

Objective To investigate the possible immune signaling pathways in Anopheles stephensi induced by hemolymph sporozoites and the underlying mechanisms, through transcriptome sequencing of the mosquitoes at 8, 11 and 14 days after infection with Plasmodium yoelii. Methods The BY265 strain of P. yoelii was inoculated into healthy Kunming mice through routine intraperitoneal inoculation. After 3 days, mice with gametophytes in the blood were selected for blood sucking by female An. stephensi mosquitoes. At 8, 11 and 14 days after blood-sucking infection, 20 mosquitoes were collected to extract total RNA. Gene expressionsin Anopheles were analyzed by high-throughput transcriptome sequencing. The statistical algorithm Bray Curtis dissimilarity was used to perform hierarchical clustering analysis of the difference of gene expression between samples. Differential gene analysis based on the value of transcripts per million was performed; the gplots package software was used to draw cluster heat maps to analyze the expression changes of important immune signal pathways (Toll-like receptor signaling, immune deficiency signaling pathway, signal transducer and activator of transcription, c-Jun N-terminal protein kinse) and effector molecules (thioester-containing proteins, antimicrobial peptide, nitric oxide synthase, nitrification-related molecules) in An. stephensi infected with P. yoelii at 8, 11 and 14 days. Based on the transcription sequencing data, four immune genes were screened out, which were thioester-containing protein 1 (TEP1), transcription factor Rel1 and Rel2, and heme peroxidase (HPX8). Real-time fluorescent quantitative PCR (qPCR) was performed to examine the expression level of these genes in An. stephensi 8, 11 and 14 d post-infection. Results The hierarchical cluster analysis of the transcriptome sequencing samples indicated that the gene expression at 11 days after infection with P. yoelii was quite different from that at 8 and 14 days. Differential gene analysis showed that there were 1 109 genes up-regulated and 257 gene down-regulated at 11 days versus 8 days after infection; 62 genes up-regulated and 136 gene down-regulated at 14 days versus 8 days after infection; and 174 genes up-regulated and 196 gene down-regulated at 14 days versus 11 days after infection. Clustering heat map analysis showed that the Rel1 transcription factor, Toll5A and Toll1A receptors of Anopheles Toll-like receptor signaling pathway were up-regulated by 1.9 times, 1.8 times and 2.1 times at 11 days versus 8 and 14 days after infection; double peroxidase and dual oxidase up-regulated by about 2 times; and HPX8 up-regulated by 1.5 times. qPCR verified that the relative expression levels of Rel1, Rel2 and HPX8 were 3.43, 3.95 and 4.01 at 11 days after infection, which had significant statistically difference with that of the control group (0.82, 0.88 and 1.01). Conclusion The hemolymph sporozoites of P. yoelii can induce the Toll signaling pathway in An. stephensi through directly or coordinately with other pathways regulating the expression of nitrification-related molecules.