Bioassay and molecular study for detection of insecticide resistance dengue causing mosquito vectors.

Nowadays, dengue infection creates a major problem across the country. The vector species carrying dengue infection has progressively started to developed resistance against most of the currently used insecticides. Hence, a study was carried out in dengue-endemic areas of Assam and Arunachal Pradesh to find the current situation of insecticide susceptibility status of dengue vectors. Based on the previous history of dengue incidence, Aedes mosquitoes were collected from Dibrugarh, Kamrup, Sivasagar, Tezpur and Tinsukia districts in Assam and Pasighat district in Arunachal Pradesh to test the insecticide resistance status through bioassay and molecular methods. The WHO standard bioassay test kits were used to detect insecticide susceptibility status among dengue vectors. In molecular study, allele-specific polymerase chain reaction (PCR) method was done for the detection of mutations in paratype voltage-gated sodium channel gene of Aedes aegypti and Aedes albopictus mosquitoes. In bioassay method, 100% A. aegypti mosquitoes were found to be resistant towards dichlorodiphenyltrichloroethane (DDT), 8% towards pyrethroid and 4% towards malathion. Similarly, 92% A. albopictus mosquitoes have shown resistance competency towards DDT, 12% towards pyrethroid and 8% towards malathion. In allele-specific PCR methods, V1016G heterozygous mutations were detected from the field collected A. aegypti and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. Similarly, F1534C heterozygous mutations were observed from A. aegypti mosquitoes of Tezpur, Tinsukia and Sivasagar district and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. From the study, it was concluded that the Aedes mosquitoes have progressively started to developed resistance towards commonly used insecticides.

Molecular characterisation and phylogenetic analysis of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India.

BACKGROUND AND OBJECTIVES: Dengue is one of the most prevalent arboviral diseases in the world with 390 million dengue infections per year. In this study, we report the molecular characterisation of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India during 2015. SUBJECTS AND METHODS: : A total of 613 dengue-suspected cases were screened for dengue virus by dengue NS1 Ag and anti-dengue IgM antibody depending on the duration of sample collection and onset of symptom. Further, molecular characterisation was done by amplifying the C-PrM region by real-time polymerase chain reaction followed by phylogenetic analysis. RESULTS: Molecular characterisation revealed that the dengue outbreak was predominantly due to dengue virus serotype-1 (DENV-1) (90.9%) while DENV-2 was detected in 7.5% of samples. Co-infection of DENV-1 and DENV-2 was detected in one case. Phylogenetic analysis of the DENV-1 strains with the prototype revealed that the DENV-1 strains were grouped within genotype III. Similarly, DENV-2 strains were clustered within genotype IV. The study revealed a change in the predominant serotype in recent years with DENV-3 in 2012 to DENV-1, 2, 3 and 4 in 2014 to DENV-1 in 2015 in the study region. A unique L24M mutation was observed in the DENV-1 strains of Arunachal Pradesh which was absent in all the circulating strains in India except one strain from the state of Kerala in South India. Marked variation within the DENV-2 strains was observed at A102V and I163V in one strain similar to earlier circulating isolates in India. CONCLUSIONS: The present study reveals a shift in the serotype dominance in the study region. As serotype shifts and secondary infection with a heterologous DENV serotype are frequently associated with disease severity, there is an urgent need for sustained monitoring of the circulating serotypes and enhanced surveillance operations, especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in this region.

Molecular typing and phylogeny of Wolbachia: A study from Assam, North-Eastern part of India.

BACKGROUND: Wolbachia are maternally inherited endosymbiotic alphaproteobacteria, infecting 40-75% of arthropod species. Knowledge on distribution of native strains infecting mosquito vectors from endemic regions is essential for successful implementation of vector control interventions utilizing potential strains of Wolbachia. Study identified various native strains of Wolbachia inhabiting different mosquito species from field and colonised conditions of Assam. The fly Drosophila melanogaster was also included in our study. METHODS: Different mosquito species collected from field viz; Aedes albopictus, Aedes aegypti, Anopheles hyrcanus, Anopheles annularis, Culex vishnui, Toxorhynchites splendens, Armegeries obturbans and fly Drosophila melanogaster were included in the study. Anopheles stephensi and Culex quinquefasciatus were obtained from RMRC, Dibrugarh mosquito colony y for Wolbachia screening. DNA was extracted from these species, amplified using group specific wsp primers followed by sequencing and phylogenetic analysis. RESULTS: Aedes albopictus from Dibrugarh, Tinsukia and Sivasagar district showed superinfection with A and B group of Wolbachia but, Aedes albopictus from Tezpur district presented infection with A group only. Our study reports for the first time natural infection of Wolbachia A and B group from colonised Anopheles stephensi mosquito but reported no infection from field collected Anopheles hyrcanus or Anopheles annularis. Similarly Armigeres obturbans and Culex vishnui presented infection with only B group of Wolbachia. Drosophila melanogaster showed superinfection with A and B group. Toxorhynchites splendens, Aedes aegypti and Culex quinquefasciatus reported no infection with Wolbachia. CONCLUSION: To the best of our knowledge this is the first study on Wolbachia screening from Northeast part of India and also first report of natural Wolbachia infection from colonised Anopheles stephensi species. The current understanding on distribution of Wolbachia strains naturally present within insect species from this geographical region should aid future Wolbachia mediated vector control strategies.