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

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.