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Additional file 1. SPIRIT 2013 Checklist.

Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility., Author Summary Wolbachia are maternally inherited bacteria that manipulate invertebrate reproduction. Cytoplasmic incompatibility is embryo death that occurs when males carrying Wolbachia mate with females that do not, or that carry a different Wolbachia variant; its mechanism is poorly understood. In Culex mosquitoes, in the presence of Wolbachia a gene related to a Drosophila melanogaster gene, grauzone, which has been shown to act as a regulator of the meiotic cell cycle, showed an elevated level of expression. When lower levels of expression were achieved through RNA interference, embryo hatch rates were affected and the stage of development at which embryo death occurs was altered. To find Wolbachia genes that influence cytoplasmic incompatibility, we compared the genomes of two variants of Wolbachia from Culex that produce cytoplasmic incompatibility with one another. Although most segments of these genomes were very similar, one newly identified gene is predicted to be a regulator of gene transcription. We cloned this gene into a plasmid, expressed it in adult mosquitoes and found higher levels of expression of the Culex grauzone homolog. This suggests that the Wolbachia transcriptional regulator may play an important role in manipulating the host in order to induce cytoplasmic incompatibility.

Anopheles gambiae is the most prominent vector of human malaria in Africa. The causative agents of this disastrous disease are unicellular eukaryotic parasites of the genus Plasmodium which are transmitted to humans by infected female mosquitoes when they take a blood meal. During its development in the mosquito, Plasmodium undergoes massive losses suggesting that mosquitoes are able to mount an immune response that limits parasite infection. However, the molecular mechanisms underlying parasite invasion, immune evasion, its recognition and killing via the vector are not well understood. We have functionally analysed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles gambiae and showed that they are specifically expressed in midgut cells invaded by Plasmodium ookinetes. Phenotypic analysis via RNAi knock down indicates that AsSRPN6 is involved in the parasite killing process, whereas AgSRPN6 acts on parasite clearance, by promoting parasite lysis. Furthermore, SRPN6 is also a parasite induced salivary gland epithelial marker located at the basal side of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 had no effect on oocyst rupture but significantly increased the number of sporozoites present in salivary glands. Midgut invasion is vital for the parasite life cycle progression. We show that Δpplp5 ookinetes cannot invade midgut epithelial cells and are retained attached to the midgut, possibly, due to the fact that, these mutant parasites can not form pores in the plasma membrane. Haemocytes secrete immune factors such as opsonins, proteases and their negative regulators as well as antimicrobial peptides, all crucial for immune responses. We report the first genome wide molecular characterisation of Anopheles gambiae circulating haemocytes, presenting a list of 1587 genes we strongly suggest are expressed by these cells.