Population Dynamics and Insecticide Susceptibility of Anopheles culicifacies in Malaria Endemic Districts of Chhattisgarh, India

A study was undertaken in the villages of Korea and Bastar district (Chhattisgarh) during the years 2012–2015 to investigate the bionomics of malaria vectors and the prevalence of their sibling species complexes. Entomological surveys carried out every month included indoor resting collections, pyrethrum spray catches, light trap catches, and insecticide susceptibility status of Anopheles culicifacies using World Health Organization (WHO) methods. Anopheles culicifacies and Anopheles fluviatilis species were assayed by polymerase chain reaction (PCR) for the detection of malaria parasite, and sibling species were identified using PCR and DNA sequencing. A total of 13,186 samples of Anopheles comprising 15 species from Bastar and 16 from Korea were collected. An. Culicifacies was recorded as the most dominant species and also the only active vector at both sites. This species was found to be resistant to dichlorodiphenyltrichloroethane (DDT) and Malathion, showing signs of emerging resistance against pyrethroids. Among the sibling species of An. culicifacies, the group BCE was found in maximum numbers, while sibling species T of the An. fluviatilis was recorded to be dominant among its complex. The study provides a comprehensive view of the vector bionomics in the highly malarious regions of India that may have importance in developing vector control strategies.

Identification and Characterization of Aminopeptidase N 1 Gene of the Indian Malaria Vector Anopheles culicifacies (Diptera: Culicidae)

Aminopeptidase N1 (APN) is one of the important enzymes involved in blood digestion and is up-regulated along with several other enzymes in response to bloodmeal ingestion. APN is a zinc metalloprotease that cleaves one amino acid residue at a time from the amino terminus of the protein. The APN1 gene of the Indian malaria vector Anopheles culicifacies Giles was cloned and characterized. The An. culicifacies APN1 (AcAPN1) gene has an Open Reading Frame of 3084 basepairs which encodes a putative protein of 1027 amino acids. The coding region of the gene shares 81% and 78% similarity to the APN1 genes found in An. stephensi (Diptera: Culicidae) and An. gambiae (Diptera: Culicidae), respectively. The organization of the APN1 gene was studied in available mosquito genomes and a three-dimensional structure of AcAPN1 modeled using homology structure modeling. The enzymatic active site was predicted to consist of HEYAH and GAMEN amino acid residues, and a comparison of the protein sequences among different genera revealed the conservation of zinc-binding residues. The expression pattern of AcAPN1 showed that the gene was expressed rapidly in response to the ingestion of the bloodmeal and therefore this gene may be used to exploit its promoter region as an antiparasite candidate molecule.

Insecticide susceptibility status of Anopheles culicifacies to bendiocarb and deltamethrin in a sub-district of Chhattisgarh state, India

Objective: This study assessed the susceptibility status of the malaria vector Anopheles culicifacies within the Keshkal sub-district, Chhattisgarh, India. Anopheles culicifacies were collected in 23 sub-centres (~2000-3000 inhabitants/ sub-centre) of Keshkal sub-district in 2013- 2014. Adult female An. culicifacies were exposed to the World Health Organization (WHO) recommended diagnostic doses of deltamethrin (0.05%) and bendiocarb (0.1%) for one hour. Mortality was recorded after 24 hours and interpreted with respect to WHO criteria. Results: Mosquito collections from only three of 23 clusters were scored as susceptible to deltamethrin, and all clusters except one were recorded as resistant to bendiocarb. The observation to a high frequency of resistance to the carbamate, bendiocarb, is unexpected as carbamates are not used in the study area in public health.

A study of malaria vector surveillance as part of the Malaria Elimination Demonstration Project in Mandla, Madhya Pradesh

Background Understanding of malaria vector density, distribution, insecticide resistance, vector incrimination, infection status, and identification of sibling species are some of the essential components of vector control measures for achieving malaria elimination goals. Methods As part of the malaria elimination demonstration project, entomological surveillance was carried out from October 2017 to October 2019 by collecting indoor resting mosquitoes using hand catch method. Susceptibility test was done for determining the insecticide resistance status of vector mosquito Anopheles culicifacies using standard protocols by the World Health Organization. The cone bioassay method was used for determining the efficacy and quality of insecticide sprayed. Mosquitoes collected from different ecotypes were identified and processed for parasite identification, vector incrimination and sibling species determination. Results The two known malaria vector species (Anopheles culicifacies and Anopheles fluviatilis) were found in the study area, which have been previously reported in this and adjoining areas of the State of Madhya Pradesh. The prevalence of An. culicifacies was significantly higher in all study villages with peak in July while lowest number was recorded in May. Proportion of vector density was observed to be low in foothill terrains. The other anopheline species viz, Anopheles subpictus, Anopheles annularis, Anopheles vagus, Anopheles splendidus, Anopheles pallidus, Anopheles nigerrimus and Anopheles barbirostris were also recorded in the study area, although their prevalence was significantly less compared to the An. culicifacies. In 2017, An. culicifacies was found to be resistant to dichloro-diphenyl-trichloroethane (DDT) and malathion, with possible resistance to alphacypermethrin and susceptible to deltamethrin. However, in 2019, the species was found to be resistant to alphacypermethrin, DDT, malathion, with possible resistance to deltamethrin. The bioassays revealed 82 to > 98% corrected % mortality of An. culicifacies on day-one post-spraying and 35 to 62% on follow-up day-30. Anopheles culicifacies sibling species C was most prevalent (38.5%) followed by A/D and E while B was least pre-dominant (11.9%). Anopheles fluviatilis sibling species T was most prevalent (74.6%) followed by U (25.4%) while species S was not recorded. One An.culicifacies (sibling species C) was found positive for Plasmodium falciparum by PCR tests in the mosquitoes sampled from the test areas. Conclusion Based on the nine entomologic investigations conducted between 2017–2019, it was concluded that An. culicifacies was present throughout the year while An. fluviatilis had seasonal presence in the study areas. Anopheles culicifacies was resistant to alphacypermethrin and emerging resistance to deltamethrin was observed in this area. Anopheles culicifacies was confirmed as the malaria vector. This type of information on indigenous malaria vectors and insecticide resistance is important in implementation of vector control through indoor residual spraying (IRS) and use of insecticide-impregnated bed nets for achieving the malaria elimination goals.

First report on the presence of natural Wolbachia population from major malarial vector mosquitoes Anopheles culicifacies s.l., and Anopheles stephensi from Tamil Nadu, India

Wolbachia is an alpha-proteobacteria present in several arthropods. The present study focussed on the identification of Wolbachia in wild malarial vector mosquitoes. This was achieved by molecular identification of Wolbachia from collected mosquitoes. A total of four hundred and eight seven mosquito samples were collected. Morphometric and molecular analysis revealed that they belong to Anopheles culicifacies s.l., (48.25%) and Anopheles stephensi (51.75%). The presence of Wolbachia was identified using 16S rRNA, wsp and FtsZ genes, where nested PCR of 16S rRNA alone was successful and then sequenced. Only seven mosquitoes (1.4%) were positive for Wolbachia. In silico and restriction digestion of 16S rRNA gene product using RsaI enzyme showed that the identified Wolbachia belongs to supergroup B. The prevalence rate of natural Wolbachia was lesser in native malarial vector An. culicifacies s.l. and An. stephensi was about 1.7% and 1.2%, respectively. This is the first report on the presence of Wolbachia in Anopheles culicifacies s.l. and Anopheles stephensi.

Efficacy evaluation of Veeralin LN, a PBO-incorporated alpha-cypermethrin long-lasting insecticidal net against Anopheles culicifacies in experimental huts in Odisha State

Background The success of malaria control using long-lasting insecticidal nets (LLINs) is threatened by pyrethroid resistance developed by the malaria vectors, worldwide. To combat the resistance, synergist piperonyl butoxide (PBO) incorporated LLINs is one of the available options. In the current phase II hut trial, the efficacy of Veeralin®LN (an alpha-cypermethrin and PBO-incorporated net) was evaluated against Anopheles culicifacies, a pyrethroid resistant malaria vector. Methods The performance of Veeralin®LN was compared with MAGNet®LN and untreated net in reducing the entry, induced exit, mortality and blood feeding inhibition of target vector species. Results The performance of Veeralin was equal to MAGNet in terms of reducing hut entry, inhibiting blood feeding and inducing exophily, and with regard to causing mortality Veeralin was better than MAGNet. When compared to untreated net, a significant reduction in hut entry and blood feeding and an increase in exophily and mortality were observed with Veeralin. In cone bioassays, unwashed Veeralin caused > 80% mortality of An. culicifacies. Conclusions Veeralin performed equal to (entry, exit, feeding) or better than (mortality in huts and cone bioassays) MAGNet and could be an effective tool against pyrethroid resistant malaria vectors.