Mosquitoes belong to the family Culicidae, which is a monophyletic taxon under the order Diptera (1). This family is characterized by a diverse array of groupings, which are distributed over various tropical and temperate regions worldwide. Mosquitoes possess a slim segmented body, a single pair of wings, three sets of long hair-like legs, and specialized, greatly elongated mouthparts used for biting and sucking (1). Currently, a total of 3523 species belonging to 111 distinct genera have been documented throughout various regions (3). This family consists of three subfamilies, namely, Anophelinae, Culicinae and Toxorhynchitinae. Multiple mosquito species inhabit freshwater environments and fulfill a crucial function in the food chain. Certain species possess the ability to bite and spread illnesses that affect human health, such as malaria, West Nile virus, and dengue fever (Eldridge, 2008). Various mosquito species have been documented in various geographical areas worldwide, such as 113 species in Bangladesh (4). 22 species of Culex, Aedes, and Anopheles mosquitoes found in India (5).  (Margalit et al., 1987) documented 25 different species found in the southern region of Israel. (6) investigated a total of 39 kinds of mosquitoes in Egypt. (7) documented 41 species in Lombok Island; 21 different species that originate from Turkey (Sengil et al., 2011); 384 species from Thailand 8; 249 species from Malaysia (9); 20 different species that originated from the Island of Japan (10) whereas Vietnam has 191 species (11). 12) documented a total of 252 species of Culex mosquitoes and 43 species of Anopheles mosquitoes across the Indian subcontinent included 66 species of Culex mosquitoes and 34 species of Anopheles mosquitoes specifically in Pakistan. (13) reported a total 134 mosquito species in Pakistan, which were classified into the Anophelinae and Culicinae subfamilies. A total of twenty-four Anopheline mosquitoes, consisting of seventeen species from the subgenus Cellia and seven species from the subgenus Anopheles, were documented in Pakistan (14) documented a total of 30 mosquito species found in Changa Manga Lahore also identified that Cx. tritaeniorhynchus, Cx. quinquefasciatus, and Aedes lineatopennis as the specific species responsible for transmitting the West Nile virus. A total of 29 mosquito species were documented for the first time in Lahore, Pakistan (15). Many mosquito species found in the Peshawar Valley and nearby regions, along with their respective levels of abundance (16).

Mosquitoes transmit a variety of infections, including bacteria, viruses and nematodes. Mosquito-borne infections include chikungunya, dengue, malaria, yellow fever, West Nile virus, filariasis, fever, and encephalitis. Mosquitoes are believed to spread numerous infections to around 700 million people each year throughout, Central America, South America, Africa, Russia, Mexico and most of Asia, resulting in millions of fatalities (17). At least two million people die yearly from these infections. Dengue fever is quickly becoming a pandemic in Pakistan. The infection expanded faster last year than in prior years because of to an ideal environmental condition for Aedes spp. breeding season.

Recognizing the significance of mosquitoes as medical pests, the aim of current research to investigate mosquito species diversity, relative abundance, and ecology in order to develop strategies for future mosquito control initiatives in this area of Pakistan. There were no published studies on the mosquito fauna and ecology in District Okara, Pakistan

Study Area

The current study was conducted in the 5 areas of the district Okara, Punjab Pakistan. Okara is a city, of Punjab province located southwest of the city  Lahore and Faisalabad which is 100 km bypassing away from the Ravi River. Its geographical coordinates are 30°48'49.1"N,  73°27'25.2"E. The current metro area population of Okara in 2023 is 425,000, and 2.66% increase from 2022 (Shah et al., 2023). Okara covers an area of 4,377/km². The population density of Okara according to the census of 2017 is 694.7/km² and the annual population change recorded was 1.6% from 1998 to 2017. Okara has a subtropical desert climate and it is situated at an altitude of 183.54 meters (602.17 feet) above sea level. The district's average annual temperature is 31.25 °C (88.25 °F) which is 10.36% higher than the national average for Pakistan. The winters are brief, mild, dry, and mainly clear in Okara while the summers are lengthy, hot, humid, and clear. The average annual temperature ranges from 46 to 104 °F, rarely falling below 41 or rising over 111. Okara generally experiences 47.82 wet days (13.1%) per year and averages approximately 22.35 mm (0.88 inches) of precipitation.

Figure 1: Map displaying the selected research locations of district Okara for mosquito species analysis.

Table 1: Description of data collection sites in the district Okara.

Sampling of Larvae and Adult Mosquitoes

The study on Mosquitoes diversity and larva collection was conducted between April 2022 and April 2023. Okara is a natural mosquito breeding site and a hotspot for mosquito-borne diseases. Larvae and adults were collected from 5 sites comprising Agriculture fields, open irrigation channels, canal freshwater bodies, rice fields, animal sheds, indoors, drains, and sewage that provide great habitat for mosquitoes as shown in Table 1. Larvae were collected during the day time while the adults were collected in the evening time. Mosquito larvae and pupae were harvested using dippers, plastic bottles, and buckets of water in a sunny area of the yard, together with a small amount of hay or straw, from fresh water and polluted water sources at specified areas. In the indoor collection, there were houses and barns for animals. For indoor collection, a mouth aspirator utilized, and a vacuum, by spreading colored clothes that attract mosquitoes. Entomological forceps were used to pick mosquitoes. The outdoor collection consisted of water habitats either fresh or contaminated water, plants, marshes, or tall grasses, standing waters such as birdbaths and faulty gutters, and water containing automobiles tires. During outdoor collection aspirator, handmade net. CDC light traps were set up in each sampling site during the evening and left overnight. These traps were attracting mosquitoes with light and a fan sucks them into a collection bag. The technique of freeze drying was used to preserve collected mosquitoes. This method of treating entomological specimens results in little to no structural deformation, removal of scales and setae, and color loss, however, it may result in the build-up of ice at the scales of wings. Morphological characters like palpi, antennae, proboscis, thorax, scales on the thorax, shape of the scutum, scutellum, femora, tibia, pulvilli, clypeus, the coloration of pleural integument, mesepimeral bristles wing pattern, vein of wings pre spiracular setae, post spiracular setae, abdominal band abdominal sterna, abdominal terga, vertex and occiput was used for the identification of mosquitoes.

Data Analysis

The diversity indices (Shannon, Simpson, Margalef, Dominance, and Evenness) calculated from the acquired fieldwork data on mosquitoes were assessed using PAST version 4.03 software. Relative abundance was found by using the following formula.

Where ‘n’ was the number of mosquitoes, and it is divided by the total number of mosquitoes collected and then it is multiplied by 100.

Species Richness: Mosquito species richness was calculated by using the Margalef index of richness (Dmg).

Where S was be total number of species and N was be total number of individuals.

Mosquitoes monitoring frequency

A total of 71 surveys were conducted between April 2022 and April 2023 to monitor the diversity, relative abundance, and ecology of mosquitoes in five different areas within the Okara region. In June and July, the surveys reached their peak with a maximum of 21, while the lowest number of surveys, 13, was recorded in November and December. The selection of the five study sites was based on the objective of evaluating the diversity, population, and their habitat of mosquitoes. The sites GC, CB, and SC had the maximum number of surveys, totaling 52, while CR and JMS had the lowest number of surveys, with just 19. The study locations were inspected according to the weekly schedule. Due to inclement weather conditions, certain surveys outlined in the fieldwork plan were not executed.

Mosquitoes species composition

From April 2022 to April 2023, A total of 2172 adult mosquitoes and 4317 mosquito larvae were collected, in which 13 mosquito species were identified within a single order and a single family. The Diptera order proved to be the most diverse, encompassing 13 species all belonging to a single family are shown in Table 5. Notably, during the study period, the species with the highest populations were Cx. quinquefasciatus, Cx. theileri, Cx. vagans, Cx. pipiens fatigans, An. annularis, An. subpictus, and Cx. duttoni. During the study period, certain species, such as Ad. albopictus and Ad. vexans were only reported once in a single month. During the current study, 13 species of mosquitoes from three genera, Culex, Anopheles, and Aedes were identified. The identified 7 species belonged to the genus Culex, three species to the genus An. and only 3 species belonged to genus Aedes. The most abundant species was Cx. quinquefasciatus with relative abundance of (17.5%), Cx. theileri (15%), Cx. duttoni (12.01%), Cx. pipiens fatigans (10%), An. subpictus (9.2%), and An. annularis (8.13%). In contrast Cx. vagans, Cx. sitiens, An. stephensi, Cx. tritaeniorhynchus, Ad. aegypti, Ad. albopictus, and Ad. vexans was identified to be the least in number with 5.61%, 5.52%, 4.41%, 4.1%, 3.8%, 3.02%, and 1.7% respective relative abundance as shown in Table 2.

Table 2: Relative abundance of adult mosquito species during collection from district Okara.

Mosquitoes diversity

The monthly average of mosquito population shows the maximum values at Sardar Chok, Green City, Company Bagh, and Junior Model School than Canal Road. The Shannon-Weiner diversity index showed that Sardar Chok (H′=1.601) and Company Bagh (H′=1.458) had the greater diversity of mosquitoes, followed by Junior Model School, Green City, and Canal Road, i.e., 1.354, 1.345, and 0.6833. Species evenness varied among Locations based on diversity, with values of 0.9918 (SC), 0.9902 (CR), 0.9679 (JMC), 0.9592 (GC), and 0.8596 (CB). The Simpson index (1-D) for SC, CB, JMC, GC, and CR was 0.7968, 0.7366, 0.7337, 0.7274, and 0.4902 indicating that SC and CB had the maximum mosquito species as shown in Table 3.

Table 3: Location-wise diversity indices of adult mosquito species in district Okara from April 2022 to April 2023

Habitat Preferences of Mosquito Larvae

The locations of the mosquito larvae at the ecological sites in this region are shown in Table 1. A total of 4317 Culicidae larvae were retrieved from different breeding sites. Table 4 shows the specific attributes of larval habitats and the related occurrence percentages of different mosquito larvae, along with their association with their respective habitats. Culex duttoni was obtained from three larval breeding locations. This species was mostly occupied in natural habitat, with vegetation, and clear water and low sunlight. Cx. pipiens fatigans was not found in adequate larval breeding site. Natural habitats, with no vegetation, clear water, and Full sunlight Cx. quinquefasciatus were collected from 4 larval breeding site. This species was mostly occupied in natural habitat without no vegetation, clear water, low sunlight. Cx. theilarie and Cx. Tritaeniorhynchus were mostly occupied in natural habitat with no vegetation and in clear water, low sunlight. These species were collected from 5 and 2 larval breeding site. Ad. Vexans was mostly occupied in natural habitat without no vegetation, clear water and Full sunlight. Ad. Albopictus were mostly occupied in natural habitat, with vegetation, clear water and low sunlight. Cx. vagans and An. stephensi were collected from 4 and 3 larval breeding sites. This species was mostly occupied in natural habitat, with no vegetation, clear water and low sunlight. An. Annularis, An. Subpictus, Ad. aegypti were mostly inhabited in natural habitat, with vegetation, clear water and low sunlight. Cx. sitiens were collected from 5 larval breeding sites. This specie was mostly inhabited in natural habitat, with no vegetation, clear water and low sunlight.

Table 4: The percentages of different mosquito larvae's their breeding sites and habitat preferences in District Okara, Pakistan from April 2022 to April 2023

Table 4: The percentages of different mosquito larvae's their breeding sites and habitat preferences in District Okara, Pakistan from April 2022 to April 2023

Out of 13 species of mosquitoes from three genera Aedes, Anopheles, and Culex, identified 7 species belonged to genus Culex, three species of genus Anopheles, and only 3 species belonged to the genus Aedes. The identified species were Cx. duttoni, Cx. pipiens fatigans, Cx. quinquefasciatus, Cx. theileri, Cx. tritaeniorhynchus, Ad. vexans, Ad. albopictus, Cx. vagans, An. stephensi, An. annularis, An. subpictus, Ad. aegypti, and Cx. sitiens as shown in Table 4. In an early study that was conducted (21) fifteen species of mosquitoes belonging to four genera Anopheles, Aedes, Culex, and Mansonia were collected from the urban city of Lahore from September 2014 to August 2015.

The current study showed the following percentages among the genera, genus Culex (69.74%), Anopheles (21.74%), and Aedes (8.52%). The most abundant species was Cx. quinquefasciatus with relative abundance of (17.5%), Cx. theileri (15%), Cx. duttoni (12.01%), Cx. pipiens fatigans (10%), An. subpictus (9.2%), and An. annularis (8.13%). In contrast Cx. vagans, Cx. sitiens, An. stephensi, Cx. tritaeniorhynchus, Ae. aegypti, Ad. albopictus, and Ad. vexans was identified to be the least in number with 5.61%, 5.52%, 4.41%, 4.1%, 3.8%, 3.02%, and 1.7% relative abundance as shown in Table 2, while a study by Attaullah et al. (2015) reported biodiversity of mosquitoes have been described in rural areas with the dominance of Armigeres subalbatus (48.1%) followed by Ad. aegypti (13.66), Cx. quinquefasciatus (11.62%) and the least collected species was Ad. vittatus (0.19%) as these results showed the overall agreement to the percentage of genera, Culex, (44.38%), Anopheles, (36.03%), and Aedes, (19.58%). Six Anopheline species were found in the Kasur and Sheikhupura districts of province Punjab, Pakistan. According to the findings of (Oneeb et al., 2016), An. stephensi had the highest relative abundance (62.5%), followed by An. subpictus (25.7%), An. culicifacies (5.48%), An. pulcherrimus (3.65%), An. annularis (1.34%), and An. nigerrimus (1.16%). In the current study, the second most dominant species were An. stephensi with 16.28% relative abundance and An. annularis were identified as subdominant specie.

Pakistan is one of the major places in the world for vector borne diseases. (18) stated that Ae. aegypti and Ae. albopictus are the vectors for the four different serotypes of dengue. (19 evaluate in their study that dengue and other vector-borne diseases have been reported during the last ten to fifteen years, but chikungunya, Japanese encephalitis, and West Nile fever arrived fairly recently in Pakistan and are restricted to some parts of the country. The Palosai Creek near Peshawar's contaminated water was researched (20) where Ad. was missing, they identified Cx. quinquefasciatus and An. stephensi. This demonstrates that Aedes species are the inhabitants of temporary habitats only. An. annularis and An. stephensi are more susceptible to malaria. The mosquitoes of this species transmit plasmodium through bites. The present study discovered nearly all the species such as Cx. duttoni, Cx. pipiens fatigans, Cx. quinquefasciatus, Cx. theileri, Cx. tritaeniorhynchus, Ad. vexans, Ad. albopictus, Cx. vagans, An. stephensi, An. annularis, An. subpictus, Aedes aegypti, and Cx. sitiens which were previously collected from waste-water while An. stephensi are freshwater breeders. Culex species preferred a wider range of habitats than the other genera being studied. While, Aedes are the least diversified. The current study shows that agricultural fields, meadows, and temporary pools and ponds are suitable places for mosquitoes to breed and grow.

The results showed that in the area of Okara, Pakistan mosquito fauna is diverse and represented by Culex., Anopheles, and Aedes. Genus Culex. that includes Cx. quinquefasicatus was identified as the most dominant species in the Okara. The genus Anopheles is the recognized vector of Malaria. An. stephensi and An. subpictus is identified as the dominant species. The genus Aedes includes Ad.  albopictus and Ad. aegypti that were identified as the satellite species. Culex. mosquitoes have a variety of habitat preferences and Anopheles mostly prefer freshwater habitats while Aedes species live in temporary and few sites. The Current findings are very crucial in developing control methods for mosquito-borne diseases.

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