Abstract
BackgroundPseudomonas 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.MethodsA 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.ResultsFluorescent 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.ConclusionsColonization 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.
Highlights
Pseudomonas is a genus of bacteria commonly found in investigations of gut microbes in malaria mosquitoes
In An. stephensi we have shown that for Escherichia coli the bacterial persistence could be due to escape from the metamorphosis using the Malpighian tubules as a covert [27]
It is known that the larval Malpighian tubules remain intact during metamorphosis in Drosophila [28,29] and tubular fluid including the bacteria may drain from Malpighian tubules towards the midgut at the junction point of midgut/hindgut and Malpighian tubules
Summary
Pseudomonas is a genus of bacteria commonly found in investigations of gut microbes in malaria mosquitoes. While great achievements have been made using routine malaria control programs, the current tools are at stake because of problems arising such as insecticide resistance in Anopheles mosquitoes, drug resistance in the malaria parasites and lack of an effective vaccine. This has forced researchers to develop other methods including transgenesis and paratransgenesis. The basic and most important step in paratransgenesis is to find suitable bacteria They should dominate the insect microflora, infest a part of vector body where they are in close contact with the pathogen, and be adapted to the immunological and physiological condition of the vector body. Transstadial transmission is an important criterion since it would enable the genetically engineered bacteria to be introduced into the larval breeding places and be naturally transferred to the adults
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