Species diversity pattern of mosquitoes (Diptera: Culicidae) breeding in different permanent, temporary and natural container habitats of Peshawar, KP Pakistan

To determine the species composition, relative abundance and seasonal variation of different mosquitoes Genera ( Aedes, Anopheles, Armigeres, Culex, and Culiseta) in different habitats the present research work was carried out in Entomology Research Laboratory of The University of Peshawar. Sampling performed from variety of permanent and temporary breeding habitats was carried out on monthly basis from targeted breeding sites for two consecutive years through dipping method. Species diversity in the survey sites was noted. Collection from these seventeen various types of potential larval habitats, yielded a total of 42,430 immature constituting 41,556 larvae and 874 pupae. Among these only 19,651 adult mosquitoes emerged comprising 11,512 female and 8,139 male mosquitoes. 78% (n= 15333) of mosquito larvae were from permanent and 22% (n=4318) were from temporary breeding sites. This study showed that Peshawar valley harbours 15 species from the genera Aedes, Anopheles, Armigeres, Culex and Culiseta. When the density of each species was examined, Culex quinquifasciatus was found to be dominant (79%) and constant in distribution. Among the temporary habitats Aedes albopictus was found as the most prevalent species particularly from tree holes and water cisterns. The highest intensity of mosquitoes was in June (2243 emerged adults) and November (2667 emerged adults) while the lowest was in January (203 emerged adults). A perfect positive correlation (r = +0.8) was found between temperature and population of mosquitoes (df 10 and α 0.05). The species diversity index for mosquitoes remained between 0.12 and 1.76. The Margalef’s richness components was noticeably low for bamboo traps (0.2) and fairly high for rice fields, Percolating water and Animal tracks (1.3) which shows the abundance of mosquito species in these habitats. Similarly Pielou’s Evenness was highest for bamboo traps (E=1) showing species uniform distribution. Animal tracks were presumed not only the diverse habitat rather also possessed high value for species richness and species evenness. Temperature, rainfall, humidity and other related attributes responsible for species variation and abundance need to be analysed further to pave way for controlling vector species in their oviposition targeted sites.

The population expansion and breeding pattern of medically significant mosquitoes are influenced by physicochemical features of their habitats (Amini et al., 2020) as oviposition is regarded as one of the most important component of mosquito-borne infections (Bentley and Day, 1989). These factors not only influence mosquito's oviposition selection sites, but correspondingly effect larval mass and species composition (Hanafi-Bojd et al., 2012;Nikookar et al., 2017).
The current study aimed to update existing status of mosquitoes and deduce the seasonal abundance and habitat preference of mosquitoes. In the course of study the species constitution, reproductive biology and population dynamics determined will help in proposing suitable control measures against mosquito borne diseases.

Study area
The study of mosquito population dynamics was conducted in different areas of Peshawar valley including 14 localities (City, Hayatabad, Islamia College Peshawar, Nasir bagh, Ring road, Reggi, Shoba Bazar, Sherabad, Saddar, Shahi Bala, Shahi Payan, Tehkal, Univ Town, Warsak) representing urban (eight) and rural areas (six) throughout Peshawar district. The distance between the stations varied from 7 km to 15 or 20 Km (Figure 1, Map).

Survey of mosquito larvae
Seasonal larval collection was made from January to December for two consecutive years (2016-17) at twenty

Introduction
Mosquitoes, the deadliest insects on earth are vector agents to bring forth mosquito-borne diseases like Malaria, Dengue, Elephantiasis, Yellow and West Nile fever and many more, critically disturbing human being and animal's health. Many countries round the world are affected yearly by the mosquito borne diseases bringing about excessive economic deficits globally; certainly half of the earth's population is affected by it (WHO, 2004). These bloodthirsty creatures cause serious harm to livestock, domestic animals and even pets (Service, 1993).
A comprehensive checklist of mosquitoes of Pakistan by Aslamkhan (1971) reported 134 mosquitoes from both east and West Pakistan, listing 23 Anopheles species and 63 Culicines with Aedes signified by 32 species in 10 subgenera, three species of Culiseta, two Mansonia species and one species each of Tripteroides, Uranotaenia, Coquilletidia, Ficalbia and Armigere.
24 species of the genus Anopheles have been reported in Pakistan (Alemu et al., 2011) of which ten species usually recovered in KP dwell in different habitats display seasonal fluctuation influenced by ecological factors. Eight Aedes species, nine Culex, two Culiseta and one each of Armigeres and Mansonia were recovered from Peshawar valley (Suleman et al., 1993).
Collection of immature mosquito was undergone monthly between 9:00 to 12:00 hrs on each sampling day, using dipping method. Ten dips (200 ml) were reserved per capture station depending on the density of larvae.

Rearing/preservation of mosquitoes
Samples in plastic cans were transferred to the laboratory where larvae (Early 1 st /2 nd and late 3 rd / 4 th ) and pupae were counted and maintained. All the immature were reared on bakers' yeast until adult emergence. Emerging adults were collected with manual aspirator and killed by chloroform fumes in a cotton swab thereafter, preserved in silica tubes.

Laboratory processing of mosquitoes
Collected specimens were segregated gender wise and the key morphological distinctions within species were noticed and documented. Accurate taxonomic identification under stereo zoom microscope was made by means of standard taxonomic keys available in "Fauna of British India" by Christophers (1933) and Barraud (1934); Knight and Stone (1977).

Meteorological data and habitat characteristics
Weather conditions like Environmental temperature, Relative humidity, heat index and altitude were noted at each sampling station using Kestrel pocket weather tracker Model 4500 NV. All the relevant information regarding the physicochemical parameters i.e., colour, odour, temperature, pH, turbidity, presence or absence of plants, predators and sunlight penetration of the targeted water reservoir were determined. Some other characteristics of larval habitat like area, sampling site, type of reservoir, vegetation cover, number of samples for each locality along with the number of larvae and pupae in each sample were also recorded.

Data analysis
Periodic alteration of mosquito species in terms of relative abundance and distribution was calculated by their respective Formulae 1 and 2 (Rydzanicz and Lonc, 2003;Sengil et al., 2011).
The dominance pattern and diversity of mosquito species in different localities were calculated by Shannon-Wiener diversity Index (Hˈ), Simpson dominance Index (D), Pielou's Evenness Index (E), Margalefs Richness Index (Me) and Berger Parker dominance Index (d).

Sampling of immature
Sampling performed from variety of permanent and temporary breeding habitats yielded a total of 42,430 immature constituting 41,556 Larvae and 874 pupae. Among these 19,651 adult mosquitoes emerged comprising 11,512 female and 8,139 male mosquitoes.

Mosquito's seasonal dynamics as per relative abundance and distribution status
Seasonal dynamics of mosquito immature sampled from various temporary and permanent habitats were reared to adults and were then analysed by density and distribution formulae.

Monthly distribution of mosquito species
A total of 19,651 mosquitoes recovered from immature collected during the field portray a tri-modal distribution with considerable reduction in winter and gradual increased in spring season leading to highest densities in April and climax was attained in November (14%) probably due to the limiting effects of high temperature in summers and cold temperature in winter. Some mosquito species preferably appears in dusk and dawn like Aedes mosquitoes that over crowds the abundance of mosquitoes. From March to November species richness and diversity among mosquitoes were also noticeable possessing more than nine species monthly ( Table 3).
Occurrence of mosquitoes belonging to various species in different months is shown in Table 4. Culex quinquifasciatus, the dominant and constant species was collected throughout the study period therefore, regarded as a cold tolerant species. Ae. albopictus, An. stephensi and Cx. tritaeniorhyncus were also gathered in all the studied months except the coldest months of January and February. The next abundant species was An. culicifacies (347 individuals) reported from March to November followed by Cx. vishnui (193), Cx. bitaeniorhyncus (128) and An. fluviatilus (123) that all were reported co-occuring in same months but there was a significant difference in their numbers, the former being more abundant than the later ones.
Among the genus Aedes, a sole male specimen of Aedes unilineatus was found in November whereas, Aedes aegypti comprise 39 adult mosquitoes caught in the month of June till November from artificial containers. Culiseta longiareolata comprising 74 individuals was captured in February, March, August, September and November from waste water bodies only.
Anopheles pulcherrimus reported in June and July and Armigeres subalbatus recovered in April, May and June were found the least dominant species.

Habitat specificity of different mosquito species
Twenty aquatic habitats including both temporary and permanent water bodies were assessed for the distribution of mosquitoes. The present results demonstrated a total of fifteen different mosquito larva species in these water sources in the study area. Mosquito larvae species harvested belonging to five genera were potential vectors of five human disease. Genera Armigeres and Culiseta were reported solely from Permanent Habitats while Genus Aedes from Temporary habitats only. Genus Anopheles and Genus Culex was found to co-occur equally in both temporary and permanent habitats.
In the current survey the highest collection (78%) was discovered in permanent habitats while 22% collection was from temporary habitats. Regarding species richness, the maximum numbers of species (9 species) were encountered from both sorts of habitats so the species richness was comparable in both types of habitats.

Habitat preference of mosquitoes
Among all studied habitats, drainage channel, percolating water body and animal tracks (9 species) were recorded to be the most preferred breeding habitats of mosquito species during this survey. Former two were permanent water bodies while later represent a temporary water habitat. Species number was low in container habitats like flooded swamps, Rain pools, water cisterns, water fountains and Bamboo glasses comprising of three or two mosquito species.
With regard to relative abundance, polluted water reservoirs contributed 40.4% to the total number of mosquitoes emerged followed by road side ditches (13%), Construction ponds (11.2%), drainage channels (6%) and tree holes (5.2%). Irrigation Canal and water cisterns were comparable constituting 3.6% of the total mosquitoes but the former possess high species richness enclosing eight species while the later resides only three mosquito species. Rest of the species were accounting less than 3% to the total mosquitoes but again a Percolating water body that was contributing very little to the totals included 9 species so was the richest water reservoir (Table 5).
Species confine to one sort of habitat included the Aedes species, that were caught absolutely (100%) from temporary habitat. Current finding reported these species in Bamboo sections, tires, tree holes, water cisterns and  water fountains that were all clear water bodies (Table 6). Similarly, Armigeres subalbatus and Culiseta longiareolata were solely reported from permanent breeding grounds.
Rest of the species found breeding in both of the habitats appeared more or less in one or the other sort of habitat. Anopheles pulcherrimus, Anopheles stephensi and Culex vishnui were the only species that were richly found in temporary water bodies; here also a malarial vector has been reported abundantly in temporary habitat. Culex species were found richly occupying permanent habitats because of the wide surface areas and the accumulation of water throughout irrespective of rainy spell. Culex quinquifasciatus was the most abundant species suitably occurring in polluted water though recorded from all surveyed habitats except from rain pools and water fountains which were found to be the ideal sites for Anopheles mosquitoes (Table 6).

Breeding habitats for mosquito species and their cooccurrence
As far as Aedes species are concerned, bamboo traps, tires, tree holes, water fountains and water cisterns were found to be the acceptable breeding habitats and all these reservoirs of Aedes are temporary water bodies. Aedes albopictus inhabited all of these sites whereas; Aedes aegypti and Aedes unilineatus were reported from single habitats. Aedes aegypti and Aedes albopictus were found to be co-breeding in tires, however, Aedes albopictus and Aedes unilineatus were found to be co-breeding in tree holes.
Concerning Anophelines, Seepage tank was found as the preferred habitat bearing all five Anopheline followed by Drainage Channel, irrigation canal, Percolating water, Animal track and Rice fields (enclosing four species each), Boggy pond (three species each) and the rest with two or one species, with flooded swamps and tires devoid of Anophelines. In contrast to Anophelines, Culicines cover wide range of acceptable habitats, reported in all surveyed habitats, the most favourable habitat with all the five species include Drainage Channel, Percolating water and Animal track followed by Ditches and Irrigation Canal (four species each), Construction Pond, Polluted water and flooded swamps (three species each) others habitats bears few species with the only water fountains devoid of any species. The most frequent, dominating and widely distributed species Culex quinquifasciatus was found positive in fifteen out of the total seventeen surveyed habitats.
Armigeres subalbatus of Genus Armigeres was represented by only specimen caught from Ring road Peshawar area ditches which was the richest habitat in terms of enclosing four different Genera, and this species co breeds with two Anopheline and four Culicine species along with a very rare specimen of Culiseta longiareolata that was also captured in trivial number from the same habitat.

Species diversity, richness and evenness of mosquitoes in different habitats.
In terms of species diversity of different habitats the Animal tracks assessed at Regi area possesses diversified mosquito fauna with comparatively larger index value (Hˉ=1.76) followed by rice fields (Hˉ=1.63), Percolating water (Hˉ=1.61) and Drainage channel (Hˉ=1.1) ( Table 7).
Margalef's Richness of mosquito fauna was found maximum for rice fields, Percolating water and Animal tracks (1.3), which showed the abundance of mosquito species in these habitats was fairly high (Table 7).  Similarly, species evenness (Pielou's Evenness) was highest for bamboo traps (1), which indicated that species were uniformly distributed in this habitat followed by Animal tracks, flooded swamps, rain pools and rice fields (0.8) ( Table 7).

Simpson's diversity index
In parallel with species number, Simpson's diversity index values were higher for Animal tracks (1/D=5) regarded as the richest habitat whereas, for polluted water (1/D=1) the Simpson's diversity was least. Percolating water and drainage channel also reside maximum number of species but the former possessed a higher diversity index as compare to later on the account of different number of mosquitoes collected from the two habitats (Table 8).

Jaccard's (CJ) similarity index for habitats
The Jaccard's similarity analysis based on species composition in their respective habitats revealed top highest relationship of 100% in Drainage Channel ≈ Percolating Water reflecting all the reported species in both the habitats followed by chief similarity of 90% between Drainage Channel ≈ Irrigation Canal, Irrigation Canal ≈ Percolating Water, Irrigation Canal ≈ Rice Field (Table 9). Fager and McGowan (1963) test was used to compute the indices of affinity between sets of Culicidae species in inspected breeding habitat of the study area using Formula 3:

Efficiency indicator/affinity index
The highest affinity of 2.0 was found among Anopheles stephensi and Anopheles culicifacies followed by a value of 1.8 which occurs in Culex quinquifasciatus with Anopheles stephensi as well as with Culex tritaeniorhyncus (Table 10).
The italic values * of affinity between the pairs of species indicates the highest values between the studied pairs of species respectively.

Discussion
Mosquitoes as disease vector and nuisance pest are of remarkable significance but their occurrence in strangely huge numbers during some parts of the year also awards them special status. Vector borne diseases together with dengue have shown a magnificent expansion (Jones et al., 2008) and pose tremendous commercial and public health complications. To consider the availability of diverse breeding grounds for ovipositing mosquitoes in different areas is of great essence before implementing anti-mosquito processing.
The only available information on mosquitoes of KP was presented by Suleman et al. (1993). Here, it may be pointed out that the species composition, relative abundance and seasonal prevalence of mosquitoes may change from year to year at the same site, reflecting great complexities of mosquito ecology (Suleman et al., 1993). So, it requires long term studies to figure out seasonal patterns in species composition and relative abundance of mosquitoes in various ecological zones.
The present study was aimed to identify mosquitoes, characterize their larval grounds and to know their seasonal dynamics in different areas of Peshawar city. This survey revealed that human interventions offer various ideal breeding grounds for mosquitoes to breed that thereby aids in increasing mosquito populations.
This survey has the peculiarity of being the extensive entomological study in the area that reported the existence of both dengue vectors i.e., Ae. aegypti and Ae. albopictus, primary Malarial vectors, i.e., An. stephensi and An. culicifacies and filariasis and West Nile virus vector, i.e., Cx. quinquifasciatus in Peshawar. Recently dengue vectors species were recovered from thirteen towns of Sindh, Punjab and KP including Peshawar (Rasheed et al., 2013) using larval catch.
Current finding revealed that Culicine were more abundant than Anophelines, this difference may be attributed to habitat variability in the localities. So, Culex was considered as the common, the largest important and abundant genus of the tribe Culicini (Service, 1993). Larvae of Culex pipiens quinquifasciatus have been reported from variety of natural as well as artificial water reservoirs (Cranston et al., 1987;Harbach 1988;Service 1993) therefore it has been reported as the dominant and abundant species in several areas (Sengil et al., 2011;Ali and Rasheed, 2009;Aditya et al., 2006;Alten and Bosgelmez, 1996;Suleman et al., 1993). This species can with stand huge mass of pollutants and dwells in wide range of habitats like 18/ 20 habitats were found positive for it in the present study. Hamidian (2007) also reported great number of this mosquito species from manmade larval sites in Iran. Suleman et al. (1993) also revealed the same breeding habitats of the species and reported its larvae even in metal drums holding rain water. Similar results regarding breeding pattern of the species were documented by Ali and Rasheed (2009), Ali et al. (2013, Ilahi and Suleman (2013). Cx. pipiens quinquifasciatus showed a bimodal distribution with a climax in late autumn (November) and a second peak in spring (March) with a declining population in cold winter months. Reisen and Aslamkhan (1978) and Reisen and Milby (1986) reported the same seasonal bimodal pattern for An. stephensi and An. culicifacies in accordance with the present study.
Temporary habitats i.e. pit, crevices and hoofs have been observed to enclose rich Anophlelines (Gillies and Meillon, 1968;Minakawa et al., 1999;Gimnig et al., 2001;Minakawa et al., 2004) that is comparable with the current findings to collect more Anopheles particularly Anopheles stephensi and Anopheles pulcherrimus in the container reservoirs. In temporary habitats, six mosquito species belonging to Aedes, Armigeres, Culex and Toxorhynchites were recovered by Aditya et al. (2006) while inspecting ditches, pools, tanks and polluted drains in Darjeeling sharing two mosquito species Culex pipiens quinquifasciatus and Culex bitaeniorhyncus with the current study. Likewise, Mwangangi et al. (2009) also assessed temporary water bodies i.e. pools, puddles, water tanks and tire tracks in Kenya reported six species (Aedes, Anopheles and Culex) of which only Culex pipiens quinquifasciatus was shared with the present study. Mwangangi et al. (2009) further concluded that both Anopheline and Culicine mosquitoes breed in temporary pools as indicated by this study. Among temporary habitats Animal tracks were presumed not only the diverse habitats as revealed by Hˉ=1.76, rather also possessed high value for species richness and species evenness (Table 7). Devi and Jauhari (2007) explore both permanent and temporary habitats in India, reported thirty four mosquito species belonging to Genera (Aedes, Anopheles, Armigeres, Culex and Uranotaenia) and determined that the zones of lower elevations reside rich mosquito fauna in term of species compared to the higher elevations. No such correspondence of species and elevations was observed in the current study. Mosquito (Culicidae) larvae belonging to Genera Aedes, Anopheles, Culex, Culiseta and Ochlerotatus were recovered in these habitats by Sengil et al. (2011) in Istanbul, Turkey. Common species shared with the present survey include, Culex theleri, Culex tritaeniorhynchus and Culiseta longiareolata. They reported Culex pipiens the dominant species in temporary as well as permanent habitats and a second dominant Culex tritaeniorhynchus that is comparable with our result. Same Genera of mosquito were revealed by Rydzanicz and Lonc (2003) in Poland reporting Culex pipiens as most abundant species in accordance with present results.
The vector species, Culex tritaeniorhynchus in current study was reported from all permanent habitats except seepage tanks but among temporary reservoirs it was got positive in hoofs, swamps, rice fields and tires signifying its inclination for small or large clean water habitats as stated by Alten and Bosgelmez (1996). The assessment of immature mosquitoes in permanent habitat reported a hazardous water body, the percolating water reservoir that encloses nine specie in two Genera (Anopheles and Culex) residing top vector species.
Current survey also include many natural habitats like irrigation canals, ditches, drainage channels, percolating water bodies, animal tracks, boggy ponds, flooded swamps, rice fields, tree holes that were wide and spacious possessing usually fresh water with dense vegetation at the shores so inter species competition trims down as mosquitoes thrives in their natural habitats. In all these habitats richest collection was observed. Moosa-Kazemi et al. (2009) while assessing natural habitats reported almost the same Culicines in Chabahar country, Southeastern Iran. Likewise, Hamidian (2007) while surveying same natural micro-habitats described species corresponding to Genus Culex in Guilan Province, Iran.
Artificial habitats are more troublesome as it can be anything that retains water and progress into a breeding area for mosquitoes. Several studies have reported pest species Aedes albopictus from such habitats (Bartlett-Healy et al. 2012;Okogun et al. 2005) that is in accordance with the current study while surveying seepage tanks, bamboo sections, tires, water cisterns and fountains. Bartlett-Healy et al. (2012) assessed about 276 various types of artificial containers in New Jersey during peak mosquito season and reported Aedes albopictus as abundant and Aedes japonicus as slightly abundant species in urban and rural sites respectively. Richards et al. (2008) reaches to the opinion that such habitats contribute significantly to Aedes albopictus in suburban habitats. Okogun et al. (2005) in Nigeria reported seventeen species in the same genera (Aedes, Anopheles, Culex) as collected in the current study.
Anopheline mosquitoes were targeted by Jude et al. (2010) in Sri Lanka reporting the major (An. culicifascies) and secondary malarial vectors (An. subpictus) in brackish waters. Yadav et al. (1989) depicted sixteen species from canal irrigated, non-canal irrigated zones and riverine region of Kheda District and reported Anopheles annularis, Anopheles culicifacies, Anopheles fluviatilus, Anopheles pulcherrimus and Anopheles stephensi in accord with our findings. Abdoon and Alshahrani (2003) reported species that do not coincide with current findings.
All Anopheline mosquitoes reported in the current study were also reported by Yadav et al. (1989) in irrigated and non-irrigated zones of Kheda Kheda District and solely and Anopheles culicifacies of the current survey was reported by Jude et al. (2010) in brackish waters of Sri Lanka.
Anopheles annularis and Anopheles culicifacies were found co-occuring as reported currently in drainage channel, irrigation canal, percolating waters, seepage tank, animal tracks and rice fields (Reisen et al., 1981;Devi and Jauhari, 2007). Likewise, Anopheles culicifacies and Anopheles fluviatilus were found co-occuring in drainage canal, irrigation canal, percolating waters, seepage tanks, boggy ponds and rice fields in the current study and also reported by Yadav et al. (1989); Devi and Jauhari (2007); Ali and Rasheed (2009); Ilahi (2001) and many more local studies. Jaccards coefficient (Cj) and affinity index was also found maximum for Anopheles stephensi and Anopheles culicifacies. Fager and McGowan (1963) test revealed that (Table 10) species occurring together in more than 50% of the targeted sites perhaps have the same environmental requirements or needs.

Conclusion
The current study has investigated different temporary and permanent larval breeding habitats and seasonal dynamics of mosquito species in different rural and urban areas of Peshawar. Information on the population dynamics of mosquitoes and particularly of vectors is necessary in order to develop an environment friendly control strategy. Environmental factors Temperature, rainfall, humidity and other related climatic attributes affecting the breeding of mosquitoes can help in detecting ovipositional site selection and distribution of vector species thereby provide a way for controlling vectors with great accuracy.