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Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector.

Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK - BMC Microbiol. (2009)

Bottom Line: The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes.To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

View Article: PubMed Central - HTML - PubMed

Affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), ICGEB Campus, Aruna Asaf Ali Marg, New Delhi, India. asharani_verma@yahoo.co.in

ABSTRACT

Background: Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.

Results: Lab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74-2.14 for lab-reared and in the range of 2.75-3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.

Conclusion: More than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

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Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene clones from field-collected A. stephensi larvae. Bootstrap confidence values obtained with 1000 resamplings are given at the branch point. Entries with black square represents generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: clone number, generic name and accession number (in parentheses).
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Figure 7: Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene clones from field-collected A. stephensi larvae. Bootstrap confidence values obtained with 1000 resamplings are given at the branch point. Entries with black square represents generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: clone number, generic name and accession number (in parentheses).

Mentions: More than 100 clones were found positive for the insert and were partially sequenced, 80 of which were found to contain the amplified 16S rRNA gene. Of these, four sequences were shown to be chimeras, which were therefore not included for further analysis. The percentage distribution of the clones from the 16S rRNA gene library representing the microbiota of the midgut of A. stephensi larvae was determined (Table 2, Figure 7). The phylogenetic tree based on 16S rRNA gene placed the 16S rRNA gene library clones from field-collected A. stephensi larvae sample into 8 major groups, belonging to 19 different genera (Table 2). These groups were: Cyanobacteria, Actinobacteria, CFB group bacteria, Gram-positive Firmicutes, betaproteobacteria, gammaproteobacteria, Deinococcus xinjiangensis, and the unidentified and uncultured bacteria group. Larval midgut microbial flora was the found to be most diverse as compared to adult mosquito midgut diversity. Cloning revealed that almost 50% of the sequences obtained in library were not related to the known bacteria. Since the percent similarity with the reported closest database matches are less than 97%, these may be categorized among the new bacteria/species. A total of 36 phylotypes were observed from 16S rRNA library based on their less than 97% similarity.


Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector.

Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK - BMC Microbiol. (2009)

Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene clones from field-collected A. stephensi larvae. Bootstrap confidence values obtained with 1000 resamplings are given at the branch point. Entries with black square represents generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: clone number, generic name and accession number (in parentheses).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698833&req=5

Figure 7: Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene clones from field-collected A. stephensi larvae. Bootstrap confidence values obtained with 1000 resamplings are given at the branch point. Entries with black square represents generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: clone number, generic name and accession number (in parentheses).
Mentions: More than 100 clones were found positive for the insert and were partially sequenced, 80 of which were found to contain the amplified 16S rRNA gene. Of these, four sequences were shown to be chimeras, which were therefore not included for further analysis. The percentage distribution of the clones from the 16S rRNA gene library representing the microbiota of the midgut of A. stephensi larvae was determined (Table 2, Figure 7). The phylogenetic tree based on 16S rRNA gene placed the 16S rRNA gene library clones from field-collected A. stephensi larvae sample into 8 major groups, belonging to 19 different genera (Table 2). These groups were: Cyanobacteria, Actinobacteria, CFB group bacteria, Gram-positive Firmicutes, betaproteobacteria, gammaproteobacteria, Deinococcus xinjiangensis, and the unidentified and uncultured bacteria group. Larval midgut microbial flora was the found to be most diverse as compared to adult mosquito midgut diversity. Cloning revealed that almost 50% of the sequences obtained in library were not related to the known bacteria. Since the percent similarity with the reported closest database matches are less than 97%, these may be categorized among the new bacteria/species. A total of 36 phylotypes were observed from 16S rRNA library based on their less than 97% similarity.

Bottom Line: The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes.To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

View Article: PubMed Central - HTML - PubMed

Affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), ICGEB Campus, Aruna Asaf Ali Marg, New Delhi, India. asharani_verma@yahoo.co.in

ABSTRACT

Background: Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.

Results: Lab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74-2.14 for lab-reared and in the range of 2.75-3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.

Conclusion: More than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

Show MeSH
Related in: MedlinePlus