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Background   Pseudomonas  is a genus of bacteria commonly found in investigations of gut microbes in malaria mosquitoes. Among those mosquitoes is the dominating malaria vector in Asia,  Anopheles stephensi , where  Pseudomonas  is a prevailing bacterium and natural inhabitant of its breeding places. In order to explore the reason for finding  Pseudomonas  so frequently, an investigation of its localization and transstadial properties was undertaken.    Methods  A  Pseudomonas  isolate from  An. stephensi  was transformed successfully with an endogenous plasmid modified to express green fluorescent protein (GFP). Subsequently, the  Pseudomonas -GFP was added to the laboratory larval breeding place of  An. stephensi  and taken up by the larvae. After 24 hours, the larvae were cleaned and moved to a bath with double-distilled water. Also, female adults were fed sugar solution containing  Pseudomonas -GFP. The  Pseudomonas -GFP was traced in the alimentary canal of larvae, pupae and adults.    Results  Fluorescent microscopy and PCR assays showed that the  Pseudomonas  bacteria underwent transstadial transmission from larvae to pupae and then to adults. In blood-fed female mosquitoes, the bacteria increased in numbers and remained in the mosquito body for at least three weeks after eclosion. In addition to the midgut, the Malpighian tubules of both larvae and adult mosquitoes were colonized by the bacteria. Also  Pseudomonas -GFP that was distributed through sugar solution was able to colonize the Malpighian tubules of adult females.    Conclusions  Colonization of the Malpighian tubules by  Pseudomonas  bacteria seems to be important for the transstadial passage from larvae to adult and presumably for the longevity of the bacteria in the adult mosquito. The existence of an entry point in the larval stage, and the long duration in the female gut, opens up for a possible use of  Pseudomonas  in mosquito paratransgenesis.

Malpighian tubules are important determinants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi