Molecular phylogenetic affiliation of Wolbachia and phage WO among Mansonia mosquitoes from Kerala, India.

cidae) are the main vectors of Brugian filariasis in India and other Southeast Asian countries. Cherthala in Alappuzha district, Kerala, India is highly endemic to Brugia malayi infection for several decades1. Application of insecticides as primary strategy for controlling mosquitoes over the years has led to resistance and environmental problems2. As a consequence, several alternative vector control strategies have attracted the attention in recent times. The ability of Wolbachia strains to affect population biology of their insect hosts ranging from their reproductive biology, ecology and evolution has provided an option to explore the use of Wolbachia in biocontrol of insect pests and biomedical applications3. Wolbachia are maternally inherited obligatory intracellular symbionts, which infect wide range of arthropods and filarial nematodes4. About 66% of all insect species, including mosquitoes are estimated to be infected with Wolbachia5. Wolbachia are known to have both parasitic and mutualistic association with their host’s biology. They manipulate the reproductive fitness of insect hosts to enhance their transmission. Cytoplasmic incompatibility (CI) is one of the most widespread reproductive manipulation found in mosquitoes6. Thus, Wolbachia can be used as an important system to drive suitable gene into disease vector populations and disable them from transmitting the disease agents, induce life-shortening or strongly inhibit the development of pathogens within the mosquito7-8. Arthropod infecting Wolbachia commonly harbours a bacteriophage named WO. However, the presence of mobile elements have been detected, sometimes at high frequency, in the intracellular bacteria genome. Phage WO can be either lysogenic and integrated into the Wolbachia chromosome or lytic and free in the cytoplasm; and effect of Wolbachia may depend at least in part on the phage infection status9. Recently, phage WO has been exploited for its role to counter-attack the Wolbachia biology. The lytic phage is being targeted to destroy Wolbachia and aide filarial treatment10. Exploitation of Wolbachia in biological control of mosquitoes in any area requires knowledge of field parameters such as the vector species, the parasites or related micro-organisms, the rate of natural infection and the interaction of vector with the environment. In the present study, field collected Mansonia mosquitoes were examined for Wolbachia and phage WO using wsp and orf7 genes. In addition, the phylogeny analysis was examined in relation to Wolbachia and phage WO infection. Wild female mosquitoes, Ma. uniformis and Ma. annulifera were collected in stagnated water samples by root nodules of Pistia stratiotes planted from Kadakkarappally village, Cherthala taluk, Alappuzha district, Kerala state (9°42' N–76°19' E), India, during JuneJuly 2012. The genomic DNA of mosquitoes were extracted using ZR Insect/Tissue DNA Kit-5TM (Zymo Research, USA). Individual mosquito was grounded in ZR Bashing BeadTM Lysis tube (Zymo Research Corporation, Irvine, USA) and homogenized in 600 μl lysis solution and DNA was extracted according to the manufacturer’s protocol. DNA was stored at –20°C until it was further used for polymerase chain reaction (PCR). PCR amplification was done using 25 μl reaction mixture volumes to check for the presence of Wolbachia and phage WO using wsp primers: A supergroup (136F 5'-TGAAATTTTACCTCTTTTC-3' and 691R 5'AAAAATTAAACGCTACTCCA-3'), B supergroup (81F 5'-TGG TCCAATAAGTGATGAAGAAAC-3' and 522R 5'-ACCAGCTTTTGCTTGATA-3') and WO orf7 primers: (WO orf7F-5'-CCCACATGA GCCAATGACGT CTG-3' and WO orf7R-5'-CGTTCGCTCTGCAAG TAACTC CATTAAAAC-3'). The PCR thermal profile was used as described by Ravikumar et al6. A negative (Aedes aegypti) and positive control (Ae. albopictus) were J Vector Borne Dis 52, September 2015, pp. 257–260