J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 34 http://jad.tums.ac.ir Published Online: March 31, 2022 Original Article Spatial Distribution, Seasonal Abundance and Physio-Chemical Assessment of Mosquito Larval Breeding Sites in Mardan District, Khyber Pakhtunkhwa, Pakistan Sara Gul1, *Khurshaid Khan1, Muhammad Sajjad1, Muhsin Jamal2, Mujeeb Ullah3, Gauhar Rehman1, Abid Ali1 1Department of Zoology, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan 2Department of Microbiology, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan 3Department of Zoology, Islamia College University Peshawar, Khyber Pakhtunkhwa, Pakistan *Corresponding author: Dr Khurshaid Khan, Email: khurshaidkhan@awkum.edu.pk (Received 18 Dec 2020; accepted 16 Dec 2021) Abstract Background: Mosquitoes (Diptera: Culicidae) are haemotophagus insects and are vectors of many arthropod-borne diseases. Present study aimed to explore species composition, seasonal abundance, spatial distribution and physio- chemical properties of larval breeding sites of mosquitoes in District Mardan, Khyber Pakhtunkhwa, Pakistan. Methods: Both adults and larvae of mosquitoes were collected through light traps, insecticide spray, mouth aspirator and larval standard dipping method in District Mardan from May to November 2017. Water samples from larval sites were physio-chemically analysed. Results: 5078 (3704 adults and 1374 larvae) mosquito specimens were collected in Mardan, Katlang and Takhtbhai tehsils. Six species in four genera were reported. Culex pipiens (89.80%) and Armigeres subalbatus (9.20%) were the most abundant species. Diversity was high in Takhtbhai (0.29) followed by Katlang (0.28) and Mardan (0.25). Greater number of specimens were recorded in peridomestic sites (93.97%) as compared to domestic habitats (6.03%). Culex pipiens larval abundance had negative correlation with pH whereas it correlated positively with electric conductivity, salinity, and TDS (total dissolved sulphur). Mosquito abundance peaked in August and July while the lowest was in May. Their monthly abundance had positive correlation with rainfall (r= 0.5069), relative humidity (r= 0.4439) and mean minimum temperature (r= 0.2866). Number of mosquitoes was highest at low elevation < 347m asl (above sea level) in agriculture land and near to water bodies (streams). Conclusion: Culex pipiens being the most abundant species, was susceptible to high pH. Mosquitoes preferred habitats were at low elevation in agriculture land. Keywords: Diptera; Culicidae; Takhtbhai; Temperature; Salinity Introduction Mosquitoes (Diptera: Culicidae) are hema- tophagous insects distributed throughout the tropical and temperate regions of the globe. To date, a total of 3,500 species and subspecies of mosquitoes have been recorded and are placed in 4 genera, among which important ones are Culex, Anopheles and Aedes. Some species of mosquitoes are the biological vectors of many human and livestock diseases including malar- ia, dengue, yellow fever and filariasis. Malaria is considered a serious mosquitoes born infec- tion that has greatly impacted human health over a century. In addition to their medical im- portance as vectors of human pathogens, fe- male mosquitoes can attack humans and other animals causing nuisance of a magnitude that negatively affects individuals and society (1). Mosquitoes lay their eggs (approximately 100–200 per adult life) in a variety of water sources ranging from small containers to marsh- 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/ mailto:KHURSHAIDKHAN@AWKUM.EDU.PK https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 35 http://jad.tums.ac.ir Published Online: March 31, 2022 land ecosystems. Among them Anopheles spe- cies prefer clean and unpolluted water while Culex species breed in water with high organ- ic content. Majority of them breed in drainage ditches, ponds, tin cans, old tires, and tree holes. Some floodwater mosquitos eggs with- stand desiccation for year/s and hatch when flooded again (2). In larval stages they feed on micro-fauna in aquatic habitats and after emer- gence their survival depends upon plant nec- tars with addition females take blood from a variety of animals. Understanding mosquito species composi- tion, distribution, abundance and seasonality is one of the essential prerequisites for surveil- lance and epidemiology of the infectious dis- eases (3, 4). Their pattern of community com- position are useful aspects for medical ento- mologists to gain broader insight and projec- tions regarding disease dynamics, spread and maintenance of globally spreading pathogens (5). In mosquitoes ecological studies, geograph- ic information system (GIS) and remote sens- ing (RS) tools are used to identify environ- mental conditions associated with vector-borne diseases (6). These techniques have been used to identify conditions favourable for larval mos- quitos development and factors particularly veg- etation, elevation as well as landscape for mos- quitos distribution (7). Pakistan have a rich fau- na of mosquitoes and so far, 134 species of mosquitoes were identified while two species (Anopheles culicifacies s.l and An. stephensi) have been reported as malaria vectors (2, 8- 11). Inadequate research exists on mosquitoes in Khyber Pakhtunkhwa province of Pakistan (2, 12). The previous published literature of the country mainly addressed aspects of mosquito identification, classification, vector potential and breeding sites of some species. Limited researchers documented the general ecology, composition, seasonal variations, and breeding habitats of mosquitoes which is useful for both medical entomologists as well as epidemiolo- gists including health workers. Therefore, the present study aimed to explore species com- position, habitats preferences, seasonal abun- dance, and spatial distribution of mosquitoes in district Mardan, Khyber Pakhtunkhwa, Pakistan. Materials and Methods Study area The present study was carried out in Mardan District (34.20 N and 72.05 E) with total sur- face area of 1,632Km2, Khyber Pakhtunkhwa, Pakistan (Fig. 1). The district is bordered in east with Buner and Swabi Districts, in north with Malakand District, in south with Nowshera Dis- trict and in west with Charsadda District. The region has distinct summer and winter seasons with average temperature 22.2 °C as well an- nual rainfall 559mm. Topographically the area has plain in the south and west while moun- tainous terrains in the north-east. Sugarcane, wheat, maize, tobacco and vegetables are the yielding crops where Kalpani (entered from north) is the main stream. Various industries, factories and mills are sporadically established in the district. Study design, data collection and analysis The entomological study was conducted in 59 localities of three tehsils (administrative subunit of the district) of Mardan including Mardan, Katlang and Takhtbhai from May to November 2017. Adult mosquito specimens were collected using insecticide flit method, light traps, mouth aspirators and hand net. Lar- vae were captured through standard dipping method (13). Light traps were used at night- times (8pm to 6am) while flit method was used for indoor sites collection in early morning. Larval collection was made from water bodies (runny, stagnant, ponds, water tanks, tyres with 300ml water from the spot to ensure food sup- ply in plastic jars). Net cloth was used for clos- ing jar openings and were later shifted to Ento- mology research laboratory where the larvae in jars were reared at room temperature until they emerge into adults. During sampling, co- ordinates of each collection site was recorded http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 36 http://jad.tums.ac.ir Published Online: March 31, 2022 through GPS (global positioning system) de- vice (Garmin Etrex, USA). During the field study adults and larvae were collected once per month in 59 fixed sites in Mardan from May to November 2017. Mos- quitoes were identified up to species level through taxonomic keys as described by Chris- topher (14) and Barraud (14, 15). Water sam- ples of 500ml of each larval site were col- lected in triplicate and analysed for total dis- solved sulphur, salinity, electric conductivity, and pH following Ma et al. (15). All data re- lated to mosquitoes were arranged in Microsoft Excel for further analysis. Annual rainfall, min- imum, maximum temperature, and relative hu- midity data for the respective year (2017) were obtained from Meteorological Department, Re- gional Office, Peshawar (KP), Pakistan. GPS co-ordinates data were entered into ArcGIS version 10.5.0 for spatial distribution where digital elevation model (DEM) was extracted from Advance Spaceborne Thermal Emission and Reflection Radiometer (RASTER). Land- use map of the district was obtained from Na- tional Centre of Excellence in Geology Uni- versity of Peshawar, KP, Pakistan. Results A total of 5078 mosquito specimens (adults and larvae) representing four genera (Culex, Anopheles, Armigers and Aedes) containing six species were collected in 59 sites in Mardan (35), Katlang (14) and Takhtbhai (10) tehsils. Among the collected species Cx. pipiens (N= 4560, 89.80%) was the most abundant species followed by Ar. subalbatus (467, 9.20%) while Ae. albopictus, Cx. vishnui, Cx. tritaeniorhyn- chus and An. walkeri showed least abundance <1%. Highest mosquito abundance was rec- orded in Mardan (3481, 68.55%) followed by Takhtbhai (801, 15.77%) and Katlang (796, 15.68%). A total of 1684 males and 3394 fe- males were recorded and male to female ratio was 1:2 (Table 1). Variations in diversity of mosquitoes among the studied tehsils was ob- served whereas Shannon diversity index H= 0.29 was highest for Takhtbhai while species richness was in peak in Katlang 0.141 and Takhtbhai 0.106 (Table 1). Greater number of Culex pipiens was col- lected in outdoor habitats (N=3009) as compared to indoor sites (N=105) in Mardan Tehsil. Sim- ilarly, number of larvae (N=1089) was highest in Mardan as compared to Takhtbhai (N=204) and Katlang (N=81). Culex pipiens larvae were most abundant in Mardan (N=1013) followed by Takhtbhai (N=186) and Katlang (N=77) (Table 2). During the present study mosquitoes were collected from May to November 2017 and their peak abundance was observed in August and September while least abundance in May and November (Fig. 2) Positive correlation was ob- served between climatic variables and mos- quito abundance in all studied tehsils (Table 3). Culex pipiens larval abundance had nega- tive correlation with pH whereas it correlated positively with electric conductivity, salinity, and TDS. Majority of mosquito larvae were collected in pH of greater than 7.0 (Table 4 and 5). Overall mosquito abundance was high at elevation < 347m above sea level (asl) while their abundance was observed at elevation rang- ing from 347 to 1159m asl in the district. In addition, Cx. pipiens abundance was also high at < 347m elevation in the region. Culex. pipiens abundance was clustered on agriculture land of the district (Fig. 3). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 37 http://jad.tums.ac.ir Published Online: March 31, 2022 Table 1. Species composition, diversity and distribution of mosquitoes (adults and larvae) in Mardan District, 2017 Species Tehsils Grand Total Mardan Katlang Takhtbhai Gender ♂/♀ ♂:♀ ♂/♀ ♂:♀ ♂/♀ ♂:♀ Cx. pipiens 1041/2073 0.5 182/523 0.34 236/505 0.46 4560 Ar. subalbatus 152/185 0.82 36/49 0.73 19/26 0.73 467 Ae. albopictus 8/18 0.45 1/1 0 0/0 0 28 Cx. vishnui 0/0 0 2/2 1 5/10 0.5 19 Cx. tritaeniorhynchus 1/1 1 0/0 0 0/0 0 2 An. walkeri 1/1 1 0/0 0 0/0 0 2 Sub total 1203/2278 221/575 260/541 5078 Species per site 5 4 3 Species Richness 0.084 0.141 0.106 Diversity index (H) 0.25 0.28 0.29 Fig. 1. Study area location map http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 38 http://jad.tums.ac.ir Published Online: March 31, 2022 Table 2. Mosquito adult/larvae and indoor/outdoor abundance in Mardan District, 2017 Tehsils Species Cx. pipiens Ar. subalbatus Ae. albopictus Cx. vishnui Cx. tritaeniorhynchus An. walkeri Sub total Mardan (N/%) Indoor 105(53.29) 73(37.05) 15(7.61) 0 2(1.01) 2(1.01) 197 Outdoor 3009(91.62) 264(8.03) 11(0.33) 0 0 0 3284 Katlang (N/%) Indoor 27(24.77) 78(71.55) 2(1.83) 2(1.83) 0 0 109 Outdoor 678(98.68) 7(1.01) 0 2(0.29) 0 0 687 Takhtbhai (N/%) Indoor 0 0 0 0 0 0 0 Outdoor 741(92.5) 45(5.617) 0 15(1.87) 0 0 801 Mardan (N/%) Adult 2101(85.30) 334(13.56) 24(0.97) 0 2(0.08) 2(0.08) 2463 Larvae 1013(93.08) 74(6.73) 2(0.18) 0 0 0 1089 Katlang (N/%) Adult 628(97.52) 12(1.86) 2(0.31) 2(0.31) 0 0 644 Larvae 77(95.06) 2(2.65) 0 2(2.65) 0 0 81 Takhtbhai (N/%) Adult 555(92.96) 42(7.04) 0 0 0 0 597 Larvae 186(91.18) 3(1.47) 0 15(7.35) 0 0 204 Grand Total 4560 467 28 19 2 2 Table 3. Tehsil wise correlation between abundance and climatic parameters in Mardan, 2017 Tehsils Monthly mean Min. Temp Monthly mean Max. Temp Monthly Total Rain Fall (mm) Relative Humidity (Average) Mardan 0.2704 0.0033 0.5666 0.5127 Katlang 0.2591 0.12 0.1723 0.2182 Tkhtbhai 0.3538 0.1266 0.5054 0.2759 Total Abundance 0.2866 0.0396 0.5069 0.4439 http://jad.tums.ac.ir Published Online: March 31, 2022 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 39 http://jad.tums.ac.ir Published Online: March 31, 2022 Fig. 2. Tehsil wise seasonal abundance of mosquitoes in Mardan District, 2017 Fig. 3. Mosquito abundance projected on elevation and land cover maps, 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 40 http://jad.tums.ac.ir Published Online: March 31, 2022 Table 4. Larvae collection and physiochemical properties of water parameters of the studied sites in Mardan, 2017 Tehsil Collection sites Average pH Average EC (μS/cm) Average TDS Average Salinity (g/L) Mosquitoes larvae (number) Mardan Stagnant water near houses 7.33 1488.33 997.2 1.00 Cx. pipiens (234), Ar. subalbatus (15) Stagnant water under shrubs/ herbs 7.51 1684 1128.3 1.13 Cx. pipiens (200), Ar. sublbatus (31) Stagnant field water 7.39 1946.1 1303.9 1.3 Cx. pipiens (224), Ar. subalbatus (10), Ae. albopictus (2) Sewerages 7.3 2213 1482.7 1.48 Cx. pipiens (220), Ar. subalbatus (2) Stagnant water bodies at dung places/ stockyard and garbage’s 7.58 1495.85 1002.23 1 Cx. pipiens (135), Ar. subalbatus (16) Katlang Clean water ditches in crop field 7.48 1569 1051.25 1.05 Cx. pipiens (77), Ar. subalbatus (2), Cx. vishuni (2) Takhtbhai Drainages 7.4 1886 1263.6 1.26 Cx. pipiens (186), Ar. sulbalbatus (3), An. vishnui (15) Mean 7.450 1697.916 1137.614 1.136 Average SDV 0.107 412.523 276.392 0.276 TDS: total dissolved sulphur, EC; Electric conductivity, Sd: standard deviation Table 5. Species wise correlation between larval abundance and water parameters in Mardan, 2017 Total larvae Cx. pipiens Ar. subalbatus Ae. albopictus Average pH -0.42 -0.4064 0.4642 -0.1628 Average EC 0.36 0.3395 -0.4352 0.3113 Average TDS 0.36 0.3396 -0.4351 0.3114 Average Salinity 0.37 0.3474 -0.4296 0.3077 TDS: total dissolved sulphur, EC: electric conductivity http://jad.tums.ac.ir Published Online: March 31, 2022 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 41 http://jad.tums.ac.ir Published Online: March 31, 2022 Discussion Mosquito biodiversity and ecological pref- erences are poorly known despite the fact that there is a need to explore the diverse mosqui- tos fauna in South Asia including Pakistan (16). Recent dengue epidemics revealed the need for more detailed understanding of the diversity, habitat preferences and distribution of these arthropods in the region (17, 18). Studies on species composition and abundance of local mosquito populations have helped to develop better management strategies for mosquito-borne diseases (19-21). Therefore, this study was aimed to provide information related to species com- position, seasonal variation, larval breeding sites and spatial distribution of mosquitoes in Dis- trict Mardan, Khyber Pakhtunkhwa, Pakistan. During the study period total of 5078 mos- quito’s specimens (adults and larvae) belonged to four genera and six species including Cx. pipiens, Ar. subalbatus, Ae. albopictus, Cx. vish- nui, Cx. tritaeniorhynchus as well as An. walk- er were recorded in the Mardan. Among the collected mosquitoes, medical importance of Cx. pipens is not yet investigated in Pakistan. Although this species is source of nuisance due to its biting pattern. The high abundance of this species was reported in different coun- tries globally (22) and locally in Pakistan (23). This species could transmit filarial parasites to humans and in immunocompromised individ- uals. In the present study Ar. subalbatus were anthropophilic mosquitoes and are known to feed on birds blood showing an opportunistic feeding behaviour (24). However, their medi- cal importance is not adequately studied in Pakistan. Although, this mosquito has the vec- torial capability of Brugian filariasis (25), Jap- anese encephalitis virus (26) and dog heart worm (Dirofilaria immitis) (27). In the current study we reported one spe- cies of Aedes genus, Ae. albopictus primarily because the collection sites were rural, and the species has been reported to be preferentially rural. It’s been known to transmit at least 20 arboviruses, filarial worms and a main vector of dengue in Asia (28). In Pakistan previous studies established that, Ae. albopictus is the vector of dengue and is chiefly responsible for the maintaining the disease transmission in rural areas (12). Culex tritaenorhynchus, Cx. vishnui and An. walkeri were captured in least number in the study area. Among them Cx. tritaenorhynchus usually prefer human settle- ment (24). It is responsible for the transmis- sion of WNV (West Nile virus) in Pakistan (29). Other two species, Cx. vishnui and An. walkeri are suspected to be the vectors of JE (30) and WNV to humans respectively (31). Mosquitoes were collected in three tehsils of Mardan District whereas, highest number of specimens were recorded in tehsil Mardan as compared to Katlang and Takhtbai. This is probably because the collection had Cx. pipiens in highest number, which has been known to prefer polluted and urban landscape for exist- ing and breeding (32). Tehsil Mardan has con- gested human settlements, polluted environment and is a main business hub while other two tehsils have freshwater habitats. Other reason for variations in species composition and dis- tribution is the variation in various biotic fac- tors (food) and abiotic factors (habitat diver- sity, water bodies, weather conditions and ele- vation) (33). Investigating seasonal variation has a key importance in the control of vector-borne dis- eases. In our study we found that mosquitoes had a single peak from July to September in the region. In Pakistan, heavy rainfall occurs in these months that provides suitable breed- ing sites and climate for their existence. Dur- ing the collection period maximum number of specimens were recorded from stagnant water bodies which are reported to be the breeding sites for Culex, Aedes and Anopheles in the province (12, 34). All ground water breeding mosquito larvae require optimal water temperature, TDS and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 34–44 S Gul et al.: Spatial Distribution … 42 http://jad.tums.ac.ir Published Online: March 31, 2022 pH for their survival. We observed that mos- quito larvae favoured pH of 7.4± (6.8-7.4± pref- erable for breeding) as recorded by others (35, 36). There was a positive correlation of larval abundance with TDS. Its known that TDS in its higher concentration reduces water trans- parency and increases oxygen deficiency (37). In our spatial analysis we observed mosquito abundance at low elevation < 347m asl, in ag- riculture land and near water bodies (streams). Utilization of vector distribution maps help to predict the risk for transmission of various dis- eases (38, 39). The current study highlights the mosquito diversity of Mardan region which is poorly ex- plored in the past. The importance of these find- ings may help medical entomologist and health departments for implementing effective control and surveillance of mosquitoes borne diseases. Conclusion A total of sex (06) mosquitoes were col- lected whereas, Culex pipiens being the most abundant species, was susceptible to high pH. Mosquitos species preferred habitats at low elevation in agriculture land in the area. This study will help the medical entomologists and health experts for allocation of the budget. Acknowledgements We would like to thank Dr Basit Rasheed, Department of Zoology, University of Pesha- war in and Dr Muhammad Naeem Department of Chemistry, Abdul Wali Khan University Mardan for assisting in mosquito identifica- tion and in water samples analysis. Ethical considerations This study was approved by the Board of Studies, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan. Conflict of interest statement Authors declare that there is no conflict of interest. References 1. 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