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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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Phylogenetic tree constructed for partial 16S rRNA gene of isolates cultured from field- collected A. stephensi larvae. Bootstrap values are given at nodes. Entries with black square represent generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: strain number, generic name and accession number (in parentheses).
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Figure 6: Phylogenetic tree constructed for partial 16S rRNA gene of isolates cultured from field- collected A. stephensi larvae. Bootstrap values are given at nodes. Entries with black square represent generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: strain number, generic name and accession number (in parentheses).

Mentions: Five major phyla, CFB, Gram-positive firmicutes, gammaproteobacteria, Deinococcus-thermus and unidentified class of bacteria were identified from 30 isolates of field-collected A. stephensi Larvae. A total of 29 phylotypes were observed with 97% similarity values as cut off. The 16S rRNA gene sequences from a variety of phylogenetic groups are shown in Figure 6. The majority of the cultured isolates (63%) from field-collected A. stephensi larvae were found to belonging gammaproteobacteria class. Distinct genera were Acinetobacter venetianus, Aeromonas sobria, A. popoffii, Pseudomonas anquilliseptica, uncultured pseudoxanthomonas, Thorsellia anopheles and Vibrio chlorae. Gram-positive firmicutes represented second abundant phylotypes (20% of the isolates) containing Bacillus sp., B. cereus, B. firmus and Exiguobacterium sp. CFB group (Chryseobacterium indologenes) and uncultured class of bacteria constituted an equal proportion of 7%. The degree of similarity of isolates and the 16S rRNA gene sequence of its closest relative in the database was in the range of 85–99%. Uncultured class of bacterial sequences obtained was related to unknown, possibly novel bacteria, which did not fall within defined groups (new bacteria/species). A single OTU was observed from Deinococcus xinjiangensis (Table 2).


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)

Phylogenetic tree constructed for partial 16S rRNA gene of isolates cultured from field- collected A. stephensi larvae. Bootstrap values are given at nodes. Entries with black square represent generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: strain 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 6: Phylogenetic tree constructed for partial 16S rRNA gene of isolates cultured from field- collected A. stephensi larvae. Bootstrap values are given at nodes. Entries with black square represent generic names and accession numbers (in parentheses) from public databases. Entries from this work are represented as: strain number, generic name and accession number (in parentheses).
Mentions: Five major phyla, CFB, Gram-positive firmicutes, gammaproteobacteria, Deinococcus-thermus and unidentified class of bacteria were identified from 30 isolates of field-collected A. stephensi Larvae. A total of 29 phylotypes were observed with 97% similarity values as cut off. The 16S rRNA gene sequences from a variety of phylogenetic groups are shown in Figure 6. The majority of the cultured isolates (63%) from field-collected A. stephensi larvae were found to belonging gammaproteobacteria class. Distinct genera were Acinetobacter venetianus, Aeromonas sobria, A. popoffii, Pseudomonas anquilliseptica, uncultured pseudoxanthomonas, Thorsellia anopheles and Vibrio chlorae. Gram-positive firmicutes represented second abundant phylotypes (20% of the isolates) containing Bacillus sp., B. cereus, B. firmus and Exiguobacterium sp. CFB group (Chryseobacterium indologenes) and uncultured class of bacteria constituted an equal proportion of 7%. The degree of similarity of isolates and the 16S rRNA gene sequence of its closest relative in the database was in the range of 85–99%. Uncultured class of bacterial sequences obtained was related to unknown, possibly novel bacteria, which did not fall within defined groups (new bacteria/species). A single OTU was observed from Deinococcus xinjiangensis (Table 2).

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