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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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Rarefaction curve from DOTUR analysis using partial 16S rRNA gene sequences of isolates and clones from field-collected A. stephensi (male/female/larvae) mosquitoes. 16S rRNA gene sequences were grouped in to same OTUs by using 97% similarity as a cut off value.
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Figure 8: Rarefaction curve from DOTUR analysis using partial 16S rRNA gene sequences of isolates and clones from field-collected A. stephensi (male/female/larvae) mosquitoes. 16S rRNA gene sequences were grouped in to same OTUs by using 97% similarity as a cut off value.

Mentions: In lab-reared and field-collected adult and larval midgut flora of A. stephensi investigated in this work, the estimated OTU number was 215 using 97% sequence identity as the criterion in DOTUR, using the pooled sequence data from all isolates and clones. The ACE estimate for the individual libraries varied from 50 to 173 (Table 3). The individual libraries harbored many sequence types unique to that library, such that, even pooled data set provides a better estimate of the total diversity. Rarefaction curve analyses (Figure 8) revealed that field-collected A. stephensi male, female and larvae midgut microbial flora ("cultured and uncultured microbes") consist of a vast diversity. In clone libraries, with increasing numbers of sequences, the number of OTUs increases, until saturation is reached. In order to cover total diversity a large number of sequences need to be sampled. However, the present analysis indicates that it is more or less sufficient to give an overview of dominating microbial communities for these two, lab-reared and field- collected environments.


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)

Rarefaction curve from DOTUR analysis using partial 16S rRNA gene sequences of isolates and clones from field-collected A. stephensi (male/female/larvae) mosquitoes. 16S rRNA gene sequences were grouped in to same OTUs by using 97% similarity as a cut off value.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Rarefaction curve from DOTUR analysis using partial 16S rRNA gene sequences of isolates and clones from field-collected A. stephensi (male/female/larvae) mosquitoes. 16S rRNA gene sequences were grouped in to same OTUs by using 97% similarity as a cut off value.
Mentions: In lab-reared and field-collected adult and larval midgut flora of A. stephensi investigated in this work, the estimated OTU number was 215 using 97% sequence identity as the criterion in DOTUR, using the pooled sequence data from all isolates and clones. The ACE estimate for the individual libraries varied from 50 to 173 (Table 3). The individual libraries harbored many sequence types unique to that library, such that, even pooled data set provides a better estimate of the total diversity. Rarefaction curve analyses (Figure 8) revealed that field-collected A. stephensi male, female and larvae midgut microbial flora ("cultured and uncultured microbes") consist of a vast diversity. In clone libraries, with increasing numbers of sequences, the number of OTUs increases, until saturation is reached. In order to cover total diversity a large number of sequences need to be sampled. However, the present analysis indicates that it is more or less sufficient to give an overview of dominating microbial communities for these two, lab-reared and field- collected environments.

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