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. different insecticide-based vector control interventions, such as IRS and long-lasting insecticidal nets (LLINs). The present study was a part of a multi-country WHO-coordinated study of the Implications of Insecticide Resistance (IIR study), to assess the impact of insecticide resistance in malaria vectors on the ecacy of LLINs and IRS 11,12 . As part of this study, insecticide susceptibility status to diagnostic dosages of deltamethrin and bendiocarb were determined in tribal and rural sub-district Keshkal, Chhattisgarh State, India. on the ecotypes and availability of mosquitoes. Adult susceptibility was determined using WHO diagnostic dosages of bendiocarb and deltamethrin by exposing eld-collected mixed-age females or three-ve day-old F 1 progeny of eld-collected mosquitoes in a WHO tube test 13 . Insecticide impregnated papers were procured from Vector Control Research Unit, University Sains Malaysia, Penang,


Introduction
Anopheles culicifacies (Diptera: Culicidae) are responsible for transmitting more than two-thirds of the total malaria cases reported in India 1 and is the primary malaria vector in Chhattisgarh state, India 2 . Indoor residual spraying (IRS) of insecticides was the major component of India's malaria control programme since the early fties and is now being supplemented by distributing insecticide-treated bed nets (ITNs). A variety of chemicals have been deployed for IRS, initially Dichloro-diphenyl trichloroethane (DDT) before malathion, hexachlorocyclohexane (HCH) and later synthetic pyrethroids 3 . The continuous use of insecticides in public health and agriculture has resulted in reports of insecticide resistance in An. culicifacies in different states of India 4 . Pyrethroid resistance is widespread in Tamil Nadu 5 ; Gujarat 6 , Madhya Pradesh 7 , Odisha 8 , and Andhra Pradesh9 including the study area alpha-cypermethrin has been sprayed for more than a decade 2 . Chhattisgarh state has the highest proportion of deaths (17%) due to malaria and contributes 12% of malaria cases in the country 10 . Assessment of mosquitoes' resistance status is essential for assessing the e cacy of different insecticide-based vector control interventions, such as IRS and long-lasting insecticidal nets (LLINs). The present study was a part of a multi-country WHO-coordinated study of the Implications of Insecticide Resistance (IIR study), to assess the impact of insecticide resistance in malaria vectors on the e cacy of LLINs and IRS 11,12 . As part of this study, insecticide susceptibility status to diagnostic dosages of deltamethrin and bendiocarb were determined in tribal and rural sub-district Keshkal, Chhattisgarh State, India.

Methods
A one-year baseline study was conducted in 2013-2014 in the Keshkal block, (20.0847° N, 81.5867° E; Total population ≈100,000 and 632 m avg. elevation) in the southern region of Chhattisgarh. The area has a tropical climate with dry (Jan-May), monsoon (June -Sept), and winter (Oct-Dec) seasons. The average temperature ranges between 11 and 35°C with average relative humidity (RH) of around 70 % and 1500 mm of average annual rainfall.
Keshkal has a community health centre (CHC), four primary health centres (PHC), namely Bahigaon, Dhanora, Keshkal and Singhanpur comprising 28 sub-centres. Four Plasmodium parasite species, i.e. P. falciparum, P. vivax, P. malariae and P. ovale, are present. The annual parasite incidence (API) of Keshkal CHC varied from 3 to 5 during the years 2010-2013) and contributed nearly 2% of the total annual malaria cases recorded. Chhattisgarh 13 . More recent data shows this trend remains with the slide positivity rate (SPR) for these areas being 3.1% while SPR of the state was 2.68 % 14 . There are 105 villages (henceforth clusters) within the study area, of which 80 were selected for the IIR project study based on logistics and security 11 . The major ecotypes are hilly, low land, riverine and plain areas, with forty-ve per cent of the state covered with dense forest.
Among the 80 villages, 23 villages were selected for conducting insecticide susceptibility tests depending on the ecotypes and availability of mosquitoes. Adult susceptibility was determined using WHO diagnostic dosages of bendiocarb and deltamethrin by exposing eld-collected mixed-age females or three-ve day-old F 1 progeny of eld-collected mosquitoes in a WHO tube test 13 . Insecticide impregnated papers were procured from Vector Control Research Unit, University Sains Malaysia, Penang, Malaysia.
For each test, four-ve replicates of 20-25 female mosquitoes were exposed to insecticide-impregnated test paper for one hour. Mosquitoes were transferred into holding tubes; provided with 10% glucose-soaked cotton pads and maintained at standard temperature (27±2 o C) and relative humidity (80%). After one hour and 24 hours, mortality was scored. Abbott's correction was made if the mortality in the control replicates was between ve and 20% 16 . The criteria for characterizing the resistance/ susceptibility status are, > 98% mortality-Susceptible; 91-97% mortality-possible resistance: and < 90 % mortality-Resistance 15 . The data were analyzed using a Kruskal-Wallis test for the differences in susceptibility status among the subcentres' of four PHCs.

Results
The susceptibility status of An. culicifacies mosquitoes collected from 23 clusters to deltamethrin (0.05%) and bendiocarb 0.1% is shown in Table one. The results suggest that pyrethroids' resistance is less common than carbamates in the study area despite prolonged use of pyrethroid based IRS. Anopheles. culicifacies populations from only ve clusters were resistant to deltamethrin, with three clusters scored as susceptible. The remaining 15 clusters showed intermediate levels of mortality that require further veri cation. In contrast, An. culicifacies populations from 22/23 clusters were resistant to bendiocarb with veri cation of status required for the remaining cluster. However, contrast results were observed next year, rendering the species completely susceptible and the exact reason for the contrasting result could not be ascertained. We grouped clusters by PHC and applied a Kruskal-Wallis ranking test to investigate whether there were signi cant differences in the susceptibility of populations to either deltamethrin (P>0.05%; df 3; n=23) or bendiocarb (p > 0.5; df 3; n=23) between administrative boundaries

Discussion
The present study showed for the rst time widespread resistance in An. culicifacies to the carbamate bendiocarb (0.1%) in the Keshkal block, Chhattisgarh, India. Carbamates are never used in the public health programme, and resistance is likely to have been driven by their use in agriculture. In the area, paddy is the major crop and the most commonly used insecticide class for plat protection is carbamates, followed by organophosphates, pyrethroids and neonicotinoids. In a recent report by Bhatt et al. (unpublished) resistance to bendiocarb has also been observed in southern districts including the present study district Kondagaon (Kanker, Kondagaon and Narayanpur) and northern districts (Raipur and Rajnandgaon).
Similarly, the development of resistance in An. culicifacies in Andhra Pradesh 17 to Malathion is also thought to be due to agricultural selection and not due to public health sprays. However, next year the species reported complete susceptibility to bendiocarb and indicated instability of bendiocarb resistance. Later, it led to a signi cant conclusion that the resistance was dynamic and varied between the clusters 18 .
Conversely, only ve of 23 clusters reported resistance to the pyrethroid deltamethrin (0.05%). Earlier studies from Chhattisgarh have also reported resistance to deltamethrin (0.05%) 19 . The species reported 88 % survivorship to DDT (n= 1273), 57% to Malathion (n=389), and 24% to alpha-cypermethrin (n=628).The use of alpha-cypermethrin for ten years has resulted in selection for resistance; it does not confer cross-resistance to deltamethrin.
In conclusion, despite long-term IRS with alpha delta susceptibility is high, LLINs based upon delta are likely to be effective. Widespread use of bendiocarb appears to have removed one of the few active ingredients we have at our disposal before it was deployed.