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

Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Zarenejad F, Terenius O - Parasit Vectors (2015)

Bottom Line: A Pseudomonas isolate from An. stephensi was transformed successfully with an endogenous plasmid modified to express green fluorescent protein (GFP).Also Pseudomonas-GFP that was distributed through sugar solution was able to colonize the Malpighian tubules of adult females.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.

View Article: PubMed Central - PubMed

Affiliation: Social Determinants of Health, Research Center, Urmia University of Medical Sciences (UMSU), Urmia, Iran. chavshin@gmail.com.

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.

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Related in: MedlinePlus

Colonization ofPseudomonas-GFP in larval midgut and Malpighian tubules. (A) midgut, (B) midgut negative control (C) Malpighian tubules, (D) Malpighian tubules negative control.
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Fig1: Colonization ofPseudomonas-GFP in larval midgut and Malpighian tubules. (A) midgut, (B) midgut negative control (C) Malpighian tubules, (D) Malpighian tubules negative control.

Mentions: We initiated the investigation by successfully transforming an endogenous plasmid into the Pseudomonas isolate BND-YL1, as determined by GFP expression and PCR amplification of the GFP gene sequence (data not shown). Then the genetically transformed Pseudomonas was added to the breeding water of 1st instar An. stephensi larvae. After 24 hours, the larvae were cleaned and moved to a bath with ddH2O. At the 4th instar stage, all larvae showed expression of GFP in the larval midgut (Figure 1A), but the colonization was particularly intense in the Malpighian tubules (Figure 1C). GFP expression was also seen in all pupae and adults (Figure 2). The transstadial transmission of the Pseudomonas isolate BND-YL1 was confirmed both by fluorescent microscopy and PCR amplification of the GFP gene.Figure 1


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

Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Zarenejad F, Terenius O - Parasit Vectors (2015)

Colonization ofPseudomonas-GFP in larval midgut and Malpighian tubules. (A) midgut, (B) midgut negative control (C) Malpighian tubules, (D) Malpighian tubules negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307889&req=5

Fig1: Colonization ofPseudomonas-GFP in larval midgut and Malpighian tubules. (A) midgut, (B) midgut negative control (C) Malpighian tubules, (D) Malpighian tubules negative control.
Mentions: We initiated the investigation by successfully transforming an endogenous plasmid into the Pseudomonas isolate BND-YL1, as determined by GFP expression and PCR amplification of the GFP gene sequence (data not shown). Then the genetically transformed Pseudomonas was added to the breeding water of 1st instar An. stephensi larvae. After 24 hours, the larvae were cleaned and moved to a bath with ddH2O. At the 4th instar stage, all larvae showed expression of GFP in the larval midgut (Figure 1A), but the colonization was particularly intense in the Malpighian tubules (Figure 1C). GFP expression was also seen in all pupae and adults (Figure 2). The transstadial transmission of the Pseudomonas isolate BND-YL1 was confirmed both by fluorescent microscopy and PCR amplification of the GFP gene.Figure 1

Bottom Line: A Pseudomonas isolate from An. stephensi was transformed successfully with an endogenous plasmid modified to express green fluorescent protein (GFP).Also Pseudomonas-GFP that was distributed through sugar solution was able to colonize the Malpighian tubules of adult females.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.

View Article: PubMed Central - PubMed

Affiliation: Social Determinants of Health, Research Center, Urmia University of Medical Sciences (UMSU), Urmia, Iran. chavshin@gmail.com.

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.

Show MeSH
Related in: MedlinePlus