title: Malpighian tubules are important determinants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi.
abstract: 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.
authors.affiliation: Social Determinants of Health, Research Center, Urmia University of Medical Sciences (UMSU), Urmia, Iran. chavshin@gmail.com.|Department of Medical Entomology and Vector Control, School of Public Health, Urmia University of Medical Sciences (UMSU), Urmia, Iran. chavshin@gmail.com.
authors.forename: Ali Reza
authors.initials: AR
authors.lastname: Chavshin
authors.affiliation: Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences (TUMS), Tehran, Iran. moshaghi@sina.tums.ac.ir.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Oshaghi
authors.affiliation: Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences (TUMS), Tehran, Iran. hvatandoost1@yahoo.com.|Institute for Environmental Research (IER), Tehran, Iran. hvatandoost1@yahoo.com.
authors.forename: Hasan
authors.initials: H
authors.lastname: Vatandoost
authors.affiliation: Department of Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. bolboljan1392@gmail.com.
authors.forename: Bagher
authors.initials: B
authors.lastname: Yakhchali
authors.affiliation: Department of Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. zarenejad_2008@yahoo.com.
authors.forename: Fahimeh
authors.initials: F
authors.lastname: Zarenejad
authors.affiliation: Department of Ecology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. Olle.Terenius@slu.se.
authors.forename: Olle
authors.initials: O
authors.lastname: Terenius
mesh_terms: D000818:Animals; D000852:Anopheles / Q000382:microbiology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics; D007814:Larva / Q000382:microbiology; D008317:Malpighian Tubules / Q000382:microbiology*; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins