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.