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Wolbachia strains are maternally inherited endosymbiotic bacteria that infect many arthropod species and have evolved several different ways of manipulating their hosts, the most frequent way being cytoplasmic incompatibility (CI). CI leads to embryo death in crosses between infected males and uninfected females as well as in crosses between individuals infected by incompatible Wolbachia strains. The mosquito Culex pipiens exhibits the highest crossing type variability reported so far. Our crossing data support the notion that CI might be driven by at least two distinct genetic units that control the CI functions independently in males and females. Although the molecular basis of CI remains unknown, proteins with ankyrin (ANK) domains represent promising candidates since they might interact with a wide range of host proteins. Here we searched for sequence variability in the 58 ANK genes carried in the genomes of Wolbachia variants infecting Culex pipiens. Only five ANK genes were polymorphic in the genomes of incompatible Wolbachia variants, and none correlated with the CI pattern obtained with 15 mosquito strains (representing 14 Wolbachia variants). Further analysis of ANK gene expression evidenced host- and sex-dependent variations, which did not improve the correlation. Taken together, these data do not support the direct implication of ANK genes in CI determinism.

Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI) resulting in abortive embryonic development. Among all the described host species, mosquitoes of the Culex pipiens complex display the highest variability of CI crossing types. Paradoxically, searches for polymorphism in Wolbachia infecting strains and field populations hitherto failed or produced very few markers. Here, we show that an abundant source of the long-sought polymorphism lies in WO prophage sequences present in multiple copies dispersed in the genome of Wolbachia infecting C. pipiens (wPip). We identified up to 66 different Wolbachia variants in C. pipiens strains and field populations and no occurrence of superinfection was observed. At least 49 different Wolbachia occurred in Southern Europe C. pipiens populations, and up to 10 different Wolbachia were even detected in a single population. This is in sharp contrast with North African and Cretan samples, which exhibited only six variants. The WO polymorphism appeared stable over time, and was exclusively transferred maternally. Interestingly, we found that the CI pattern previously described correlates with the variability of Gp15, a prophage protein similar to a bacterial virulence protein. WO prophage sequences thus represent variable markers that now open routes for approaching the molecular basis of CI, the host effects, the structure and dynamics of Wolbachia populations.

In Ciona intestinalis, the elimination of extra-embryonic test cells during early stage of development is delayed by a fertilization signal. Test cells undergo a caspase-dependent apoptosis event repressed by thyroxine (T4)-activated NF-kappaB. When apoptosis was experimentally blocked, the hatching stage was delayed. The incubation of unfertilized eggs with a 1-h-fertilized egg extract or purified T4 restored apoptosis in test cells at a similar timing than found in fertilized eggs. Ciona expresses specific genes forming a functional IkappaB/NF-kappaB pathway. One, Ci-p65, was transiently induced upon fertilization via T4 and found to exert its anti-apoptotic role in test cells nuclei as well as in a reconstituted cell system. Blocking NF-kappaB activity by dexamethasone-induced overexpression of Ci-IkappaB abrogated the repression of apoptosis in test cells. Overall, the data are consistent for defining a central coupling role of both T4 and NF-kappaB during early embryo development.

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.

We have previously shown that expression of active Rac1 and Cdc4Hs inhibits skeletal muscle cell differentiation. We show here, by bromodeoxyuridine incorporation and cyclin D1 expression, that the expression of active Rac1 and Cdc42Hs but not RhoA impairs cell cycle exit of L6 myoblasts cultured in differentiation medium. Furthermore, expression of activated forms of Rac1 and Cdc42Hs elicits the loss of cell contact inhibition and anchorage-dependent growth as measured by focus forming activity and growth in soft agar. RhoA was once again not found to have this effect. We found a constitutive Rac1 and Cdc42Hs activation in three human rhabdomyosarcoma-derived cell lines, one of the most common causes of solid tumours arising from muscle precursors during childhood. Finally, dominant negative forms of Rac1 and Cdc42Hs inhibit cell proliferation of the RD rhabdomyosarcoma cell line. These data suggest an important role for the small GTPases Rac1 and Cdc42Hs in the generation of skeletal muscle tumours.