Bacterial candidates for paratransgenic control of mosquito vectors

Bacterial symbionts could be used as vehicles for expressing foreign genes in mosquitoes Expression of selected genes could prevent the mosquito from transmitting human and animal pathogens Obviously, the fundamental prerequisite for an effective protocol of paratransgenesis to control mosquito-borne diseases is the identification of suitable bacteria The key features that a bacterium should possess for this use are (Beard et al , 2002; Riehle and Jacobs-Lorena, 2005):

It should be a dominant bacterium within the insect-associated microbiota

It should be cultivable in cell-free media

It should be readily applicable to genetic transformation

It should exhibit stable expression and maintenance of the newly acquired antipatho-gen function

It should have wide distribution in the preadult and adult insect body

It should colocalize with the infectious agent in the relevant insect organs (e g , gut and salivary glands)

The use of molecular identification methods, based on 16S rRNA gene amplification, cloning, and sequencing, in combination with microbiological and biochemical techniques, has made the identification of the microbiota associated with different mosquito species much more rapid . This has allowed the identification of "new" bacterial strains, as in the case of Aeromonas culicicola from the midgut of Culex quinquefasciatus (Pidiyar et al ., 2002) .

This multifaceted approach, performed on field caught specimens of An. gambiae s l and An. funestus from West Kenya, led to the identification of 16 midgut-associated bacterial species belonging to different genera, among these Anaplasma, Mycoplasma, Nocardia, and Janibacter (Lindh et al , 2005) The genus Anaplasma is a "Wolbachia sister taxon" and includes several tick-borne species pathogenic to ruminants and humans The vectorial capacity of mosquito for Anaplasma deserves further investigation The nominal species Mycoplasma wenyoini identified in specimens of An. arabiensis is closely related to Myco-plasma suis, a bacterium mechanically transmitted between pigs by Ae. aegypti (Prullage et al ., 1993), whereas Nocardia coryneobacterioides is closely related to Rhodococcus rhodnii, a symbiont of the triatomine bug Rhodnius prolixus, vector of Chagas' disease . R. rhodnii has already been successfully employed in paratransgenic strategies aimed at controlling the spread of Chagas' disease, indicating the enormous potential of this approach (Durvasula et al ., 1997; Beard et al., 2001, 2002). The Janibacter strains detected in Anopheles arabiensis represent a new species; the name chosen for this species, Janibacter anophelis, emphasizes the close relationship with the mosquito host (Kämpfer et al ., 2006b) . A y-proteobacterium has also been shown to be associated with the midgut of An. arabiensis and has been named Thorsellia anophelis (Kämpfer et al ., 2006a) . Briones and collaborators (2008) suggested that T. anophelis could be usefully employed in paratransgenic control of a malaria vector, because this bacterium appears to be dominant in Kenyan populations of An. gambiae. As stated above, dominance is regarded as one of the key features of a symbiont to be employed in paratransgenesis The dominance of T. anophelis in the Kenyan An. gambiae population has been assessed through the sequencing of major bands generated in denaturing gradient gel electrophoresis (DGGE) experiments . Further analyses are perhaps required to confirm that this bacterium is dominant in Kenyan An. gambiae mosquitoes

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