J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 108 http://jad.tums.ac.ir Published Online: June 30, 2022 Original Article Bionomics of Anopheles culicifacies Sensu Lato in two Malaria Endemic Districts of Central Gujarat, India *Rajendra K Baharia1, Jaspreet Kaur2, Ankita Sindhania2, Gunjan Sharma2, Harish C Srivastava1, Chandra S Pant1, Manju Rahi3, Kamaraju Raghavendra2, Om P Singh2 1Indian Council of Medical Research (ICMR)-National Institute of Malaria Research, Field unit, Civil Hospital Nadiad, Gujarat, India 2Indian Council of Medical Research (ICMR)-National Institute of Malaria Research, Dwarka Secctor-8, New Delhi, India 3Indian Council of Medical Research (ICMR), Division of Epidemiology and Communicable Diseases, Ansari Nagar, New Delhi, India *Corresponding author: Dr Rajendra Kumar Baharia, E-mail: rajendrabaharia@gmail.com (Received 20 Dec 2020; accepted 26 Apr 2022) Abstract Background: Gujarat State has been witnessing large scale urbanization, in last two decades, resulting changes in local environment and microclimate may have also influenced the resting, feeding habits and development of Anopheles cu- licifacies sensu 1ato. Therefore, a systematic longitudinal study was undertaken to know the bionomics of An. culicifa- cies s.l. in present study. Methods: The study was conducted in four sentinel villages in Kheda and Panchmahal Districts. The mosquitoes rest- ing indoors and outdoors were collected in early morning hours, using mouth aspirator, pyrethrum space spray and light traps. Mosquito landing collections on human volunteers was carried out from dusk to dawn. Species composition, abundance, seasonal prevalence, resting behavior (Endophily and Exophily), sibling species composition, vector poten- tial and insecticide susceptibility status of malaria vectors was studied. Results: Six Anopheles species were collected, An. subpictus s.l. was the predominant species followed by An. culicifa- cies s.l., a known malaria vector was resting indoor and zoophagic behaviour. Anopheles culicifacies, sibling species B (89%) was found. The sporozoite rate (%) and entomological inoculation rate in Kheda was 2.33%, 3.09 per bite/ per- son/annum and they were 1.05% and 0.475 bite/person/annum in Panchmahal, respectively. Anopheles culicifacies s.l. was found possible resistance to alpha-cypermethrin. Conclusion: Anopheles culicifacies s.l. showed endophillic, zoophagic behaviour and found possible resistance to al- pha-cypermethrin. Early biting behaviour of An. culicifacies s.l. in this area is a cause of concern. Therefore, there is need for frequent monitoring and evaluation of vector control measures in order to achieve the elimination target of ma- laria in this area. Keywords: Endophilic; Zoophagic; Alpha-cypermethrin; Susceptibility; Sibling species Introduction Vector spatial selection is greatly influenced either due to species competition or changes in environmental factors such as temperature and lead to the local adaptation (1). It has been described that climate change will escalate the transmission of vector borne diseases. Several authors have claimed decline in certain areas or no change in disease prevalence due to over- looked ecological factors (2–3) and develop- ment in community (4–5). Of the 58 Anophe- lines in India, only six taxa are major malaria vectors with significant regional distribution (6). Anopheles culicifacies s.l. has been reported as the major malaria vector in the plains in In- dian sub-continent. It has been incriminated as malaria vector from rural area of various states Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 109 http://jad.tums.ac.ir Published Online: June 30, 2022 of India (7–11) including in Gujarat State (12). Various aspect of bionomics of An. culicifacies s.l. was studied in India (13). The abundance, prevalence, biting and vector potential of An. culicifacies s.l. in Gujarat State was studied in 1980s and 1990s (14–21). Cytotaxonomic ex- amination of An. culicifacies showed five 5 sib- ling species named as A, B, C, D and E in var- ious parts of the India (22–25). The species has shown differential sympatricity, resting and feeding behavior, insecticide suscep- tibility, vectorial capacity (12, 14, 17, 19, 21– 22) and have indicated their differential role in transmission of malaria (10–11, 26–27). Anopheles culicifacies s.l. is primarily zoophag- ic in behavior and its impact on malaria trans- mission was assessed in Gujarat State (12–14). Resistance in An. culicifacies against three in- secticides, used in public health programme for vector control namely DDT, Malathion and Diealdrin has been reported from Gujarat dur- ing 1960s and 1970s (28). Gujarat being a pro- gressive state has witnessed rapid urbanization, industrial development, and construction of de- velopment projects and associated deforesta- tion during last two decades in the state, re- sulting in changes in local climate and envi- ronment. Accordingly, change in microclimate may have influenced some of the bionomic at- tributes such as resting and feeding habits that may need renewed attention. Malaria is unsta- ble in Gujarat State, western part of India. Dur- ing last one-decade malaria has declined to an- nual parasite incidence (API) of 0.31 in 2019, at the verge of elimination (29). Therefore, a systematic longitudinal study was undertaken to know the bionomics of An. culicifacies s.l. in some districts of Gujarat State. The results will help in making evidence-based decision for suggesting effective vector control strate- gy and understand existing lacunae to control and eliminate malaria in similar ecological set- tings in Gujarat. Materials and Methods Study area The study was undertaken in Kheda Dis- trict (22°45' N, 72°45' E) and Panchmahal (Godhra) District (22°45′ N, 73°36′ E) locat- ed in central Gujarat (Fig.1). Based on malar- ia incidence, potential mosquito breeding sites and baseline entomological survey, four (2 riverine and 2 canal irrigated) villages in each district were included in the study in May- June 2017. The selected villages Angadi, Pali (Kheda), Juni Dhari, Vinzol (Godhara) are riv- erine whereas the villages such as Ravaliya, Muliyad (Kheda), Khazoori and Nandisar (Godhara) have perennial irrigation canal. The villages were approachable at ease by road with good condition throughout the year. Most of the houses are made up of brick with concrete roof; however, a few mud houses with tin or tile roof also exist. Major crops in this area are Paddy, Millet, Corn and Tobacco. The maximum and minimum external air temperature, relative humidity and rainfall in this area were obtained from Anand Agriculture University, field unit at Thasra, District of Kheda. Mosquito Collections The indoor and outdoor resting, bait and trap collections were carried out at monthly frequency in sentinel villages following stand- ard WHO methods (30). In each village, 6 hu- man dwellings (HD) were fixed for indoor rest- ing mosquito collection. Outdoor resting mos- quitoes were collected from underneath of cul- verts, abandoned temples and bushes. Indoor Resting Mosquito Collection from Human Dwellings Adult female Anopheles species resting in- doors in fixed six human dwellings and two cattle sheds were collected with the help of flashlight and mouth aspirators in early hours of the day (0600 to 0800hrs) for 15 minutes in each of the fixed dwellings. After 15 minutes of hand catch, pyrethrum spray collection (PSC) http://jad.tums.ac.ir/ https://tools.wmflabs.org/geohack/geohack.php?pagename=Panchmahal_district¶ms=22_45_N_73_36_E_type:city(2390776)_region:IN-GJ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 110 http://jad.tums.ac.ir Published Online: June 30, 2022 was done from the same room. Mosquito col- lected through hand catch were kept in paper cups and PSC kept in Petri dishes lined with wet cotton and filter paper at the bottom and transported to the laboratory for processing. The total number of mosquitoes/species rest- ing per structure was calculated as density (per man per hour density for HC) or numbers of mosquitoes/room (PSC). Outdoor Resting Mosquito Collection Mosquitoes resting outdoors were collect- ed with the help of flashlight and mouth aspi- rators during morning hours (0600 to 0800hrs) from available different outdoor sites in the village. The mosquitoes collected were kept in paper cup with wet cotton and transported to the laboratory for further processing. The num- ber of mosquitoes/species collected was cal- culated as the density (man hour density). Human Landing Collection (HLC) Hourly whole night collection of mosqui- toes landing on human bait (volunteer) was car- ried out from dusk to dawn (1800 to 0600hrs). Care was taken to collect mosquitoes as soon as they land on the host to evade mosquito bites on volunteer. A paper cup fastened with net- ting was used for each hour of collection. The collected mosquitoes were brought to field la- boratory identified to species and vector(s). Mos- quitoes were processed for parity rates and the head and thorax was preserved for enzyme- linked immunosorbent assay (ELISA) test. Light-Trap Catch (by CDC Light Trap) Two light-traps, one each in indoor and outdoor was used for mosquito collection. The indoor trap was placed 60cm above the ground in the sleeping unit of the household. For out- door collections, traps were placed just out- side the houses where people sit and sleep or in open away from the habitations in the vil- lage. Mosquitoes were collected in morning from the trap was used for estimating propor- tion of different species prevalent in the area. These collections expressed as numbers of mos- quitoes per trap-night (number of mosquitoes/ trap). Processing of Mosquito Samples Anopheline mosquitoes identified to species based on morphological characters using key (31–35), the physiological condition of stom- ach was determined such as unfed (UF), fresh- ly fed (FF), half gravid (HG) and gravid (G), blood meal of An. culicifacies s.l. were collected on Whatman no. 1 filter paper for host source using gel diffusion technique to determine the feeding preference (Human blood Index), half gravid ovaries of An. culicifacies s.l. was col- lected and preserved for sibling species iden- tification, the head and thorax of each speci- men was preserved for sporozoite ELISA test and unfed females used for estimating parity. Susceptibility Status of Vectors to Different Insecticides Anopheles culicifacies s.l. susceptibility sta- tus against pyrethroids was estimated in Octo- ber 2017, January 2019 and March 2019, ac- cording to standard WHO method (36). Field collected mixed age population of An. culic- ifacies s.l. of villages Anghadi, Pali, Revaliya, and Vinzol were tested using WHO prescribed discriminatory concentration impregnated pa- pers of malathion, deltamethrin and alpha-cy- permethrin. A minimum of 75 mosquitoes (25 mosquito/replicate) for test and 50 (25/replicate) for control were used. The test was conducted in National Institute of Malaria Research (NIMR) laboratory maintained at 27±2 ºC temperature and 60–70% relative humidity during test pe- riod. Percent mortality was determined post 24 hrs of holding period after one-hour exposure from the total number of alive and dead mos- quitoes in the replicates. If control mortality was greater than or equal to 5% and less than or equal to 20%, the value for exposure mor- tality was corrected by using the Abbott’s for- mula (37). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 111 http://jad.tums.ac.ir Published Online: June 30, 2022 Sibling Species Identification Morphologically identified specimens stored in isopropanol were processed for An. culic- ifacies sibling species (38). Species A/D and species B/C/E were identified by two allele spe- cific primers ACA (forward, sequence 5’-GCC GTC CCC ATA CAC TG-3’) and ACB (re- verse, sequence 5’ CCG TAA TCC CGT GAT AAC TT-3’). PCR conditions were: one cycle of denaturation at 95 °C for 5min followed by 35 cycles of each of denaturation at 95 °C for 30s, annealing at 55 °C for 30s and extension at 72 °C for 60s, and final extension at 72 °C for 7min. Vector Incrimination The head and thorax of An. culicifacies s.l. preserved were assayed for presence of spo- rozoite using ELISA (39–41). The specimens were tested for species specific circumspo- rozoite antigen (CSP) of Pf, Pv210 and Pv247 using the protocol as described by Akhatar et al. (41). Results Mosquito Abundance and Seasonal Preva- lence A total of 14056 mosquitoes were collected by all methods used of which, 73.94% (10389/ 14056) was Anopheles along with 25.46% Cu- lex and 0.62% Aedes. Among anophelines An. subpictus s.l. (69.03%) species followed by An. culicifacies s.l. (17.93%), An. annularis (12.35 %), An. stephensi (1.02%), An. fluviatalis s.l. (0.16%) and An. vagus (0.0096%). Anopheles culicifacies s.l. was encountered from all hab- itats in both districts. From outdoor resting hab- itats An. subpictus s.l., An. culicifacies s.l. spe- cies and Culex spp were found. Indoor Resting Collections Human Dwelling The indoor resting mosquito collections were made for 13 months (July 2017 to July 2018) from fixed Human dwellings in sentinel vil- lages of Kheda and Panchmahal Districts. In both the districts, bimodal population increase of An. culicifacies s.l. densities were observed, a minor peak in March and a major peak in Au- gust. The indoor resting density in canal irri- gated area was higher during most of the study period as compared to in riverine areas in both the districts. In post-monsoon months (December to March), indoor resting density was significantly high in both the areas of Panch- mahal compared to Kheda (Fig. 2). During the transmission season, An. culicifacies s.l. den- sity of Kheda irrigated versus Panchmahal riv- erine and Kheda riverine versus Panchmahal irrigated were significant (p= 0.0092, t= 2.77, p< 0.05 and p= 0.0357, t= 2.0, p< 0.05). Cattle Shed Indoor resting sampling in cattle sheds re- vealed that the density of An. culicifacies s.l. was greater in cattle sheds than human dwell- ings in Kheda and Panchmahal. In riverine ar- ea of Kheda, An. culicifacies s.l. density was higher as compared to irrigated area through- out the study except in post monsoon period, whereas in Panchmahal, it was high in river- ine area in all the seasons. Physiological Condition It was observed that in indoor resting sam- ples from riverine villages of Kheda District, the proportion of half-gravid + gravid An. cu- licifacies s.l. was higher compared to full fed and unfed in pre-monsoon, monsoon, and post- monsoon seasons. In canal-irrigated areas of this district, the proportion of fully fed mos- quito was higher compared to half-gravid + gravid, and no unfed was encountered during pre-monsoon period. While in monsoon and post-monsoon period, canal-irrigated villages had higher proportion of half-gravid + gravid as compared to fully fed and unfed. The riv- erine and canal-irrigated villages of Panchma- hal had higher proportion of half-gravid + gravid compared to fully fed. It indicated that http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 112 http://jad.tums.ac.ir Published Online: June 30, 2022 large proportion of An. culicifacies s.l. bites in the evening and late evening and rests indoor in both the districts indicating endophillic be- havior. Parous Rate Altogether 1223 ovaries (Kheda 534 ova- ries, Panchmahal 689 ovaries) of An. culicifa- cies s.l. were examined for parity. Overall, 30 % (200/689) were found to be parous in Panch- mahal District throughout the study period while in Kheda District it was low in winter 13% (3/22) and summer 7% (1/14). Anopheles cu- licifacies s.l. parity varied in different months, ranging from 7% in May to 60% in July. In the transmission season, parity rate of Kheda and Panchmahal District were significant (p= 0.0305, t= 3.9, p< 0.05). Light Trap In Kheda, the densities of An. culicifacies s.l. (3.08 per trap) and other mosquitoes (25.58 per trap) were higher in outdoor trap com- pared to indoor. In Panchmahal, the density of other mosquitoes (22.41 per trap) was 10-fold higher than An. culicifacies s.l. in both traps. In outdoor traps, the density was higher in Kheda compared to in Panchmahal (Figs. 3 and 4). Anopheles culicifacies s.l. density from the districts of Kheda and Panchmahal were sig- nificant with outdoor other mosquitoes’ densi- ty (p= 0.0030, t= 3.4, p< 0.05 and p= 0.0044, t= 3.18, p< 0.05). Human Landing Collection (HLC) The HLC yielded five anopheline species comprising of An. culicifacies s.l., a few An. stephensi, An. annularis, An. fluviatilis s.l., An. vagus, and a few specimens of Aedes spp and Culex spp. In general, the bite rate of An. cu- licifacies s.l. was greater in Kheda compared to Panchmahal in all seasons and found biting throughout the night in both districts. In Kheda, the mean biting rate of An. culicifacies (9.0 bite/bait/night) was high during post-monsoon period, September to October. Whereas biting activity of other mosquitoes was maximum dur- ing March-May, it was 20 times higher than An. culicifacies s.l. In both the districts, most of the landing mosquitoes were caught either in early night or in early morning. Thus, two peaks of biting activities were observed in each night, first between 20–21hrs and second at 02–04hrs in both districts (Fig.5). Human land- ing catch were significant in between Kheda and Panchmahal Districts (p= 0.0292, t= 2.11, p< 0.05). Host Blood Preference A total of 577 (320, riverine and 257 irri- gated area) blood meal samples of An. culic- ifacies s.l. were collected from sentinel villag- es in Kheda District. From villages in Panch- mahal District altogether 573 (327 riverine and 246 irrigated villages) blood meal samples were assayed for blood meal sources. The anthro- pophilic index (AI) of An. culicifacies s.l. in Kheda District, in canal irrigated area was 2.33 % (6/257) and in riverine area none was posi- tive for human blood (0/320). In Panchmahal District, AI was 1.62% (4/246) in canal ir- rigated area and 1.52% (5/327) in riverine ar- ea. Majority of blood meal of An. culicifacies s.l. were found bovine indicating its established zoophagic behavior in both the districts. Insecticide Susceptibility Status The susceptibility status of An. culicifacies s.l. was assessed against Malathion, Deltame- thrin, Permethrin and Alpha-cypermethrin using WHO discriminatory dose impregnated pa- pers. It was observed that An. culicifacies s.l. has developed resistance against Deltamethrin (24 hrs mortality: 75%) and Malathion (24hrs mortality 70%), whereas it was found possible resistance to Alpha-cypermethrin with 24hrs mortality 95% in Kheda (Table 1). In Panch- mahal District, An. culicifacies s.l. was possi- ble resistance to pyrethroid Alpha-cyperme- thrin with 24hrs mortality 95%, but has devel- oped resistance against Malathion (24hrs mor- tality 75%). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 113 http://jad.tums.ac.ir Published Online: June 30, 2022 Sibling Species Composition Altogether 67 and 223 An. culicifacies s.l. from Kheda and Panchmahal, respectively were examined for sibling species identification. Its sibling species distribution pattern was found similar in both the study areas. The primers were specified for detection of the A/D and B/C/E sibling species complex. In Kheda, An. culicifacies species B (82%; 55/67) was pre- dominant species followed by species A/D (12 %; 8/67), in Panchmahal B and A was 91% (203/ 223) and 8% (17/223), respectively (Table 2). Sporozoite and Entomological Inoculation Rates In Kheda and Panchmahal Districts 916 and 900 An. culicifacies s.l. were analyzed for vector incrimination studies. In Kheda, 12 (Plas- modium falciparum: 7, P. vivax: 5) and in Panch- mahal District, 04 (P. falciparum: 3, P. vivax:1) specimens were found positive for sporozoite. The sporozoite and biting rate in Kheda was 1.33% and 2.33 bite/bait/night and 0.45% and 1.05 bite/bait/night in Panchmahal, respectively. Entomological inoculation rate (EIR) in Kheda and Panchmahal was estimated to be 3.09 and 0.475 per bite/person/annum, the higher EIR in the Kheda may be the result of high biting rate of An. culicifacies s.l. (Table 3). Additional Data Additional observations were made on cat- tle shed location, sleeping behaviour of inhab- itants (indoors/outdoors) and livestock enumer- ation to assess any possible impact on the be- haviour of vector and its transmission poten- tial. A total of 120 households were visited in the both districts to know the location of cat- tle shed in study villages. The maximum cat- tle sheds were adjacent to human dwelling in Kheda (60%) and Panchmahal (55%). The mix-dwellings (cattle and human habitations un- der same roof) were more than those of cattle shed away from human dwellings in both dis- tricts. In all the sentinel villages, livestock cen- sus was undertaken by NIMR staff to estimate the cattle human ratio in 2018. The buffalos were found as major livestock followed by cow and other animals (goat and chicken) in each village. The cattle to human ratio were low in both Panchmahal (0.19: 1) and Kheda (0.20: 1) districts. These indicate the greater risk of human vector contact in both districts and ex- plain the observed epidemiological attributes sporozite rates and EIR. The inhabitants of these dwellings were in- terviewed to know the sleeping behavior (in- door/outdoor) of villagers in different seasons. The behaviour of villagers was similar in all three seasons in both the districts. More than 60% inhabitants sleep indoors during winter and monsoon in both districts and > 50% sleep outdoors in summer. Table 1. Susceptibility status of Anopheles culicifacies s.l. against different insecticides Insecticides (%) Kheda Panchmahal 1h Knockdown (%) 24hrs mortality (%) 1h Knockdown (%) 24hrs mortality (%) Control Group 0.0 0.0 0.0 0.0 Deltamethrin (0.05%) 70 75 75 85 Permethrin (0.75%) 80 85 83 90 Alpha cypermethrin (0.05%) 90 95 90 95 Malathion (5%) 60 70 65 75 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 114 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 2. Sibling species composition of the Anopheles culicifacies complex from July 2017 to July 2018 District n Genotype (%) A/D B/C/E NA (Not amplified) UK (Unknown) Kheda 67 08 (12) 55 (82) 04 00 Panchmahal 223 17 (8) 203 (91) 01 02 Grand Total 290 25 (9) 258 (89) 05 02 Fig. 1. Location of study districts of Kheda and Panchmahal in Gujarat State Fig. 2. Mean indoor densities of Anopheles culicifacies s.l. in Kheda and Panchmahal Districts from July 2017 to July 2018 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 115 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 3. Sporozoite rate, biting rate and entomological inoculation rate (EIR) of Anopheles culicifacies s.l. in study area from July 2017 to July 2018 Districts n Pf +ve Pv 210 +ve Pv 245 +ve Total +ve Sporozoite rate % Biting Rate EIR Kheda 916 7 4 1 12 1.33 2.33 3.09 Panchmahal 900 3 1 - 4 0.45 1.05 0.472 Fig. 3. Anopheles culicifacies s.l. and density of other mosquitoes per light trap indoors and outdoors in Kheda District from August 2017 to July 2018 Fig. 4. The density of Anopheles culicifacies s.l. and other mosquitoes per light trap indoors and outdoors in the district of Panchmahal from August 2017 to July 2018 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 116 http://jad.tums.ac.ir Published Online: June 30, 2022 Fig. 5. Mosquito landing rates of Anopheles culicifacies s.l. on human baits from dusk to dawn from July 2017 to July 2018 Discussion The study was carried out in riverine and canal irrigated villages of Kheda and Panch- mahal in July 2017 to July 2018, to update the information on bionomics of An. culicifacies s.1. in these districts of central Gujarat. In vil- lages, the number of brick houses were more compared to mud houses, 50–60% of inhabit- ants reportedly slept indoors in all seasons. Such sleeping behaviour of inhabitants explains ob- served increased endophagic behaviour of mos- quitoes. Using various collection methods, six Anopheles species were collected namely An. culicifacies s.1., An. stephensi, An. fluviatilis s.1., An. subpictus s.1., An. annularis and An. vagus which include three primary vectors and a secondary vector species (16–22). In earlier studies in Kheda, varied prevalence of An. culicifacies s.l, An. stephensi, An. annu- laris, An. subpictus s.1., An. aconitus, An. bar- birostris, An. nigerrimus and An. tessellatus was reported (16–22). Among anophelines, An. sub- pictus s.1. was predominant in human dwell- ings and cattle sheds. While observations from Jabalpur reported An. culicifacies s.1. as most predominant species followed by An. Subpictus s.1. (8). From outdoor habitats, An. culicifacies s.1., An. subpictus s.1. and Culex spp. could be captured, and similar observations were made in earlier studies conducted at Kheda District (14–22). The previous studies have reported large numbers of An. culicifacies resting in out- door habitats in Gujarat (16, 42). On the con- trary we found low outdoor resting densities of An. culicifacies s.l. throughout the study, probably due to non-availability of suitable rest- ing habitats outdoors that were hot and humid due to changes in the local environment dur- ing last few decades. Hence, these observations indicate the need for suitable micro-climate niche for prevalence of mosquitoes either indoors or outdoors. Anopheles culicifacies s.1. was prevalent throughout the year in both the districts in var- ying proportions. In our observations, the den- sity of An. culicifacies s.1. started to build up in the month of February with the increase of temperature suitable for breeding and survival and reached to first peak in March thereafter it declined gradually by July, and with the on- set of monsoons increase in density was ob- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 117 http://jad.tums.ac.ir Published Online: June 30, 2022 served with second peak in September and was similar to observations made in earlier report- ed studies in 1990s (17–18, 21), indicating no differences in seasonal prevalence in major vec- tor, An. culicifacies s.l. in last few decades. Light traps collections yielded An. culicifa- cies s.l., An. stephensi, An. annularis, An. flu- viatilis s.1., An. vagus, Aedes and Culex in var- ied proportions. During post monsoon period, An. culicifacies s.1. density in trap were high- er in both the districts. The density of other mosquito species in traps was nearly ten times more than the density of An. culicifacies s.l. which was like densities in studies in Madhya Pardesh (8). In study from Jabalpur, Madhya Pradesh, in light trap collection, An. culicifa- cies s.1., An. fluviatilis, An. subpictus s.1., An. annularis, An. vagus, An. pallidus, An. splen- didus, An. barbirostris and An. theobaldi have been reported (8). In Bastar, Chattishgarh, in light trap catches, An. culicifacies, An. subpict us s.l., An. vagus and An. annularis were rec- orded (43). In the Odisha State, An. fluviatilis s.l. and An. culicifacies s.l., the recognized primary vectors, and An. aconitus, An. annularis, An. jeyporiensis, An. maculatus and An. varuna the reputed sec- ondary vectors of malaria in India (44). However, significantly high number of mos- quitoes were trapped in tribal villages of tribal Panchmahal District due to availability of large number of suitable mosquito breeding sites. Another possible reason could be low infra- structure development in these villages com- pared to villages in plain areas. High proportion of fed and half-gravid + gravid An. culicifacies s.1. in indoor resting catches in human dwellings in riverine and irrigated villages in both the areas, exhibited en- dophilic and endophagic behaviour of An. cu- licifacies s.1. which was similar to observations in earlier reported studies in Kheda District in early 1990s (17) indicating no change in resting and feeding behaviour of mosquitoes inspite of regular use of insecticidal interventions and changes in housing. The parous rate of An. culicifacies s.1. was nearly 30% in Panchmahal district in all the seasons. While, in Kheda District it was low in winter and summer and varied in different months ranging from the lowest in May to the highest in July. The prolonged ovarian cycle or multiple feeding during same cycle may be responsible for low parous rate in winter and summer. Another study from Kheda District reported overall parous rate of 38.2% during 1991–92 and 36.4% during 2000–2001 (14). An earlier study on seasonal prevalence of An. culicifacies s.1., estimated > 35% parous rate in Kheda District (22). The study clearly shows higher survivorship of An. culicifacies s.l. pop- ulation in all the seasons in Panchmahal, ow- ing to suitable climatic condition. In both the districts, An. culicifacies sib- ling species ‘B’ was predominant but it was not involved in the malaria transmission in this area. Our study confirms the results of earlier study in central Gujarat (NIMR, unpublished report, 2009). The study conducted in 1990s in Kheda district also supports our results which reported > 60% sibling species B (14). These observations contrasted with the distribution in other districts. In Orissa an eastern part of In- dia predominant species was An. culicifacies C (77.9%) followed by B (21.1%) and A (48%) (45–46). Earlier studies in Madhya Pradesh in central part of India also reported highest prev- alence of An. culicifacies C (8, 47). In Chhattis- garh state, central India a study reported equal prevalence of sibling species B and C (48). In Uttar Pradesh, only An. culicifacies sibling spe- cies A and B were found with predominance of A (49). The results of the above reported stud- ies are contradictory to our present study as in the eastern and central states of India, malaria is stable and most of the parts of state are hilly forested. Anopheles culicifacies A and C are known malaria vector in most part of the In- dia (13, 50). In Kheda District, the human biting activi- ty of An. culicifacies s.1. was intense during post-monsoon (September to November) and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 118 http://jad.tums.ac.ir Published Online: June 30, 2022 in Panchmahal (September and November) and low biting in summer in both districts. Similar seasonal shift in biting period was also report- ed in previous studies in early 1990s (17–18, 21). The biting occurred throughout the night with maximum biting in the first quarter and third quarter of night in villages of both districts, our observations agreed with previous reports from Gujarat (14, 18). The early night biting of An. culicifacies s.1. is concern in area where Long lasting insecticide nets (LLINs) is main component of vector control strategy. The low anthropophagic index of An. cu- licifacies s.1. indicated high feeding prefer- ence to bovine blood which is like previously reported studies from Gujarat (12, 48, 51–53). anthropophagic index may be due to low cat- tle–human ration in Gujarat State (14). During summer, villagers were found sleeping out- doors close to cattle shed without protection against mosquito bites in both the districts. It has been observed by previous authors that indoor resting anopheline comes out at dusk, bite outdoor and enters indoor at dawn for resting (20). The present study shows that vil- lagers having low cattle population are at greater risk of malaria transmission by An. culicifacies s.1 in these districts, despite zo- ophagic nature. This could be one of the rea- sons for the increased EIR in Kheda District. Anopheles culicifacies s.1. was found pos- sible resistance to alpha-cypermethrin in the both the districts which is also presently used for indoor residual spraying in this area for malaria vector control under the National Vec- tor Borne Disease Control Programme. Anoph- eles culicifacies s.1. showed resistance to Mal- athion in both the districts. In Kheda district, it has developed resistance to Deltamethrin too. Another study from Kheda District reported resistance in An. culicifacies s.l. against DDT, Dieldrin and Malathion (14). Similarly, An. culicifacies s.1., was reported resistant to DDT, Malathion and Deltamethrin in Surat District of Gujarat in 2005–2006 (28). Extensive use of insecticides increased the resistance. Some of the point mutation in the voltage-gated so- dium channel (NaV) was responsible for the resistance for the Pyrethroids and DDT insecti- cides (54). In the Indian subcontinent, the point mutation leading to Leu-to-Phe substitution in the voltage gated Na+ channel (VGSC) at res- idue 1014, a most common Knockdown re- sistance (kdr) mutation was reported in An. cu- licifacies s.l.-a major malaria vector which is responsible for resistance against DDT and Py- rethroids (55–57). The susceptibility of An. cu- licifacies s.1. status to insecticides reported mul- tiple resistance including to Pyrethroids and could be a concern for vector control as the programme is still reliant on chemical insecti- cides mainly Pyrethroids. Anopheles culicifa- cies s.1. vector is resistant to multiple insecti- cides, and this underlines the need for devel- opment/use of new insecticides for manage- ment of insecticide resistance (58). In present study, a low Anthropophilic in- dex (AI) of An. culicifacies s.1. was recorded in both the districts revealing it’s predominant zoophagic behaviour. Earlier studies carried out on host preference of An. culicifacies s.1. in India have recorded a wide range of variation in the anthropophily (13). However, there are evidence that feeding preference to human changes in different situations, a high AI in ar- eas with high cattle population (14) or during epidemic period (59). Owing to zoophagic na- ture of An. culicifacies s.1., low sporozoite rate was recorded in both the districts. Earlier stud- ies have observed varied degree of sporozoite rates in naturally infected An. culicifacies s.1. in India (12–14). Mean annual entomological inoculation rate was estimated to be low in both the districts. Similar observations were made from studies in central Gujarat have also es- timated low EIR ranging 0.022 to 0.110 infec- tive bite/person/annum in different periods (12, 14). In plain areas of Sundargarh District in Odisha where An. culicifacies s.1. plays main role in malaria transmission, EIR was estimat- ed to 0.014 infective bites/ person/ night (44). Therefore, EIR estimates in our study indicate http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 108–123 RK Baharia et al.: Bionomics of … 119 http://jad.tums.ac.ir Published Online: June 30, 2022 active malaria transmission in Kheda and Panch- mahal Districts of Gujarat. Conclusion For preparation of effective vector control strategy updated knowledge on vector biology and behavior is an essential requirement. Our present study generated important information on prevalence and behaviour of An. culicifa- cies s.l. and other attributes related to malaria transmission. The abundance, physiological con- ditions, and blood meal analysis of indoor rest- ing An. culicifacies s.l. showed endophilic and endophagic behavior in both districts. In both the districts, its highest biting during monsoon and post monsoon coincides with malaria trans- mission season. We found An. culicifacies s.l. possible resistance to alpha-cypermethrin, cur- rently used insecticide. Hence, indoor residual spray (IRS) with alpha-cypermethrin may be evaluated in other villages of the districts to control An. culicifacies s.l. in this area. Effec- tiveness of LLINs depends on site of use (in- door/outdoor) and on feeding time of biting hence early biting behaviour of An. culicifa- cies s.l. in this area is a cause of concern. Alt- hough, there have been changes in human hous- ing and local ecology due to developmental activities in villages of both districts during last two decades in Gujarat, but our study clearly demonstrated no major change in behaviour of An. culicifacies s.l. Therefore, there is need for frequent monitoring and evaluation of vector control measures to achieve the elimination tar- get of malaria in this area. Acknowledgements We are thankful to the Director General, Indian Council of Medical Research for kind- ly permitting to undertake the study and also for providing fund. We also thank to Director, NIMR for their kind support in all aspects. We also thank State Programme Officers of NVBDCP, Gujarat, for providing support dur- ing the study period. We appreciate sincere ef- forts of the staff of the National Institute of Ma- laria Research and health staff of Government of Gujarat in successful conduct of the study. Ethical considerations Institutional Ethical Committee approval was obtained vide letter no. ECR/NIMR/EC/2017/ 142 dated 21 June 2017. A meeting with villagers was convened with the help of Sarpanch and other opinion leaders to apprise them about the purpose of the study. NIMR staff coordinated with the Primary Health Centre staffs, Medical Officer, Multipurpose health worker (MPHW) (male and female) and Accredited Social Health Activist (ASHA) to solicit the cooperation of the villagers during study. Informed consent of householders of se- lected sentinel sites for collection and of vol- unteer to be baits for landing mosquito collec- tions were obtained prior to initiating studies. Conflict of interest statement Authors declare that there is no conflict of interest. References 1. Eleanore D Sternberg, Matthew B Thomas (2014) Local adaptation to Temperature and implications for vector-borne diseases. Trends Parasitol. 30(3): 115–122. 2. Thomas CJ, Davie G, Dunn CE (2004) Mixed picture for change in stable malaria dis- tribution with future climate in Africa. Trends Parasitol. 20: 216–220. 3. Hay SI, Cox J, Rogers DJ, Randolph SE, Stern DI, Shanks GD, Myers MF, Snow RW (2002) Climate change and the resurgence of malaria in East African highland. 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