JNK signaling regulates oviposition in the malaria vector Anopheles gambiae

This study addressed the role of the stress and immune signaling pathway, c-Jun N-terminal kinase (JNK) in the egg laying behaviour of female Anopheles gambiae mosquitoes that is licensed by mating. The study originated from a microarray analysis of heads of mated or virgin females which identified only a small number (24 genes) of significant expression changes 16 of which were connected to processes linked to wounding and coagulation responses such as melanization which can, in turn be linked to JNK signalling. To explain this gene signature we hypothesised that perhaps mating might increase JNK signalling in the heads of females. Much of the data here are western blots which attempt to document changes in the levels of phospho-JNK in different tissues and following different stimuli. In Figure 1 we document increases in pJNK in the head after mating and use RNAi to deplete JNK pathway components which reduce the number of mated blood fed females who oviposit their eggs. The data shown (Figure 1 d) reflect the number of females in multiple experiments who oviposited eggs by day 4 after mating. In Figure 2 we show that using RNAi to deplete puckered (dsp5) a JNK phosphatase, the levels of pJNK are artificially raised in the heads of virgin females and not strikingly in the reproductive tracts of the same females, and that this is enough to cause oviposition in a fraction of blood-fed virgin females that can be blocked by concomitant depletion of JNK itself, revealing JNK as the dominant target of puckered in driving this phenotype. Again the western blot data document changes in pJNK levels in head or reproductive tract after dspuc injection and the numbers of females ovipositing after differing dsRNA treatments and multiple experiments. In Figure 3 we mimic mating by injecting a steroid hormone 20 hydroxyecdysone (20E), normally transferred to females from the male during mating and previously shown to be sufficient to induce oviposition in blood fed virgin females, and find that this too increases pJNK levels selectively in the head - we saw no consistent effect in the reproductive tract - as well as oviposition in blood fed virgins that is inhibited by JNK pathway depletion. Again data show western blots for pJNK after 20E or control (solvent) injection and number of females ovipositing after differing RNAi treatments. Supplementary data support the main conclusions of the paper by: (S1) showing tissue and pathway selectivity of pJNK activation in the head after mating; (S2) comparing relative transcript levels of the two JNK genes known in Anopheles JNK1 and JNK3; (S3) effective depletion of JNK pathway components by RNAi including after double injections of dsJNK1 and dspuc; (S4) showing dsJNK1 reduces pJNK levels after mating; (S5) showing no significant effect of JNK pathway depletion on the process of egg development and; (S6) showing the mating-induced up regulation of wounding-related genes could be inhibited by dsJNK.

本研究探讨了应激与免疫信号通路——c-Jun氨基末端激酶（c-Jun N-terminal kinase, JNK）在交配调控的冈比亚按蚊（Anopheles gambiae）雌蚊产卵行为中的作用。本研究源自对交配雌蚊与未交配雌蚊头部的微阵列分析（microarray analysis），仅鉴定出24个存在显著表达差异的基因，其中16个与创伤及凝血反应（如黑化反应）相关通路存在关联，而黑化反应又可与JNK信号通路相联系。为解释该基因表达特征，我们提出假说：交配可能增强雌蚊头部的JNK信号通路活性。本研究的多数数据来自蛋白质免疫印迹（western blots）实验，旨在记录不同组织及不同刺激条件下磷酸化JNK（phospho-JNK, pJNK）的水平变化。在图1中，我们证实了交配后雌蚊头部的pJNK水平升高，并通过RNA干扰（RNAi）敲低JNK通路相关组分，结果显示交配后吸血雌蚊的产卵数量减少。图1d展示了多次独立实验中，交配后第4天产卵的雌蚊数量。图2显示，通过RNAi敲低JNK磷酸酶puckered（dsp5），可人为升高未交配雌蚊头部的pJNK水平，但在同一雌蚊的生殖道中无显著变化；该操作足以使部分吸血未交配雌蚊产卵，且这一表型可通过同时敲低JNK本身被阻断，表明JNK是puckered调控该表型的主要靶标。本部分的蛋白质免疫印迹数据记录了注射dspuc后头部或生殖道中的pJNK水平变化，以及不同dsRNA处理组、多次独立实验中的产卵雌蚊数量。图3中，我们通过注射类固醇激素20-羟基蜕皮酮（20-hydroxyecdysone, 20E）模拟交配过程——20E通常在交配时由雄蚊转移至雌蚊体内，既往研究已证实其可诱导吸血未交配雌蚊产卵；结果发现，20E同样可选择性升高雌蚊头部的pJNK水平（生殖道中未观察到一致变化），并诱导吸血未交配雌蚊产卵，且该效应可被JNK通路敲低所抑制。本部分数据包含20E或对照（溶剂）注射后的pJNK蛋白质免疫印迹结果，以及不同RNAi处理组的产卵雌蚊数量。补充数据从以下方面验证了本文的主要结论：(S1) 证实交配后雌蚊头部pJNK激活的组织与通路特异性；(S2) 比较冈比亚按蚊已知的两个JNK基因——JNK1与JNK3的相对转录水平；(S3) 证实RNAi可有效敲低JNK通路组分，包括两次注射dsJNK1与dspuc后的敲低效果；(S4) 证实敲低dsJNK1可降低交配后的pJNK水平；(S5) 证实JNK通路敲低对卵子发育过程无显著影响；(S6) 证实交配诱导的创伤相关基因上调可被dsJNK抑制。