RESEARCH NEWS CURRENT SCIENCE, VOL. 103, NO. 3, 10 AUGUST 2012 254 Microbes, mosquitoes and malaria Avinash Sharma, Kamlesh Jangid and Yogesh Shouche Every living organism harbours microbes that are intimately associated with its body surfaces and mosquitoes are no exception to this. Microbes in the mid gut of mosquitoes have special impor- tance because of their proximity to mos- quito-transmitted pathogens in this tract and potential to influence disease trans- mission. Although microbial community structure in the mid gut of different mos- quito species has been analysed using culture based and culture independent methods for almost a decade 1 , recent de- velopments in high throughput sequenc- ing technologies have allowed studies at much greater depths 2,3 . This additional information has given a new dimension to better understand microbial commu- nity dynamics in the mosquito mid gut and its role in disease transmission. Here, we summarize three recent publications that have made some important observa- tions probably raising more questions than answered. Wang et al. 2 used Roche ® 454- pyrosequencing to study the microbial community structure during the life- cycle of wild and lab-reared Kenyan mosquito strain. Specific regions of the eubacterial 16S rRNA gene amplified from mixed-community DNA during dif- ferent developmental stages of the mos- quito were used to study community dynamics in the mosquitoes. Although the mid gut was found to harbour dis- tinctly different bacteria in different developmental stages, the phyla Cyano- bacteria, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes together represented 90.7–99.9% of the sequences in all the stages. In general, the microbial community diversity decreased progres- sively from larvae to adults and further upon blood meal in adults. Whereas the larvae and pupae microbial communities were dominated by Cyanobacteria, those in newly emerged adults showed pre- dominantly Enterobacteriaceae and Pro- pinobacteriaceae. Restructuring of the larval intestine during metamorphosis and change in the gut environment may be responsible for this shift. At the spe- cies level, as many as 69.4% of the sequences were closely affiliated to Thorsellia anopheles, originally isolated from the mid gut of Anopheles arabien- sis and subsequently detected in Anophe- les gambiae. Adult development was associated with an increase in Flavobac- teriaceae from 13.2% to 61.7% and decrease in Enterobacteriaceae from 37.4% to as low as 5.5%. Family SAR11 which contains oligotrophic bacteria was relatively constant around 4–5%. Upon blood meal, the community structure showed a significant change within 2 days with predominance of Proteobacte- ria. However, after 4 and 7 days post- blood meal, Flavobacteria especially the genus Elizabethkingia which was also reported in lab and field mosquitoes, predominated the communities. Regard- less of this dynamically changing com- munity, both wild and lab-reared mosquitoes seemed to harbour compara- ble microbial communities. In contrast, Boissière et al. 3 using a similar strategy reported significant differences between the microbial com- munities in lab-reared and natural popu- lations of Anopheles gambiae from two different locations in Cameroon. Larvae were reared in laboratory and experimen- tally infected with Plasmodium falcipa- rum from a single donor. Blood-fed females were dissected after 8 days and the total DNA from individual gut sam- ples was used for microbial community analysis and Plasmodium infection status. Although sample-specific differ- ences were noted, microbial communities in general were dominated by Proteobac- teria, Bacteroidetes, Actinobacteria, Fir- micutes and Fusobacteria. Between lab-reared and field collected mosqui- toes, microbial communities in lab- reared mosquitoes were dominated by Flavobacteria, specifically the genus Elizabethkingia as reported by Wang et al. 2 . In contrast, mosquitoes from natural habitats showed predominance of Proteo- bacteria. Further, Gram-positive bacteria were detected only in natural populations and were absent in lab-reared mosqui- toes. The relative proportion of alpha-, beta- and gamma-proteobacteia in the microbial communities differed between the mosquito larvae collected from the two sites. Possibly, the differences re- sulted from either the genetic variation between different mosquito strains or due to difference in the experimental design. Whereas Wang et al. 2 used rain water for propagation of mosquitoes in microcosm, Boissière et al. 3 used habitat water for their study. In addition, Wang et al. used larvae from Anopheles gambiae colony maintained in the insectary, whereas Boissière et al. used larvae collected from their natural habitats. Although microbial community dyna- mics is an important component of trans- genic mosquito research, it cannot directly implicate mid gut microbiota in the transmission of diseases, which has been an actively studied area in this research 4 . Cirimotich et al. 5 studied the role of naturally occurring microbes in 454 Pyrosequencing Pyrosequencing technology as developed by 454 Life Sciences is one of the modern massively parallel sequencing procedures that relies upon the release of inorganic pyrophosphate (hence the name) with every nucleotide incorporation during strand synthesis. In the first step, target DNA is frag- mented and clonally amplified on beads in an emulsion PCR (emPCR) to generate multiple copies. The beads are then overlaid onto a PicoTitre- Plate TM with millions of wells in such a way that each well has a single bead. Subsequent sequencing reactions that occur on each bead make use of po- lymerase, luciferase, ATP sulphurylase and microfluidics cycles of the four nucleotides over the PicoTitrePlate TM . The incorporation of a nucleotide dur- ing strand synthesis on each bead releases a pyrophosphate, which acts as the substrate for a luminescence reaction that is measured by the CCD cam- era. The signal intensity is directly proportional to the number of nucleotides incorporated. Thus, several million reactions are carried out simultaneously and with present-day technology ~ 500 bp sequence read can be generated on each bead leading to massive output.