Ecology of breeding habitats of mosquito population and screening for virus of Japanese encephalitis and West Nile in the coastal area of Kerala, India

R Balasubramanian; V Arathy Nadh; S Sahina

doi:10.4103/0972-9062.318307

RESEARCH ARTICLE

Year : 2021 | Volume : 58 | Issue : 3 | Page : 232-239

Ecology of breeding habitats of mosquito population and screening for virus of Japanese encephalitis and West Nile in the coastal area of Kerala, India

R Balasubramanian, V Arathy Nadh, S Sahina
National Institute of Virology - Kerala unit, Alappuzha 688005, Kerala, India

Date of Submission	01-Feb-2019
Date of Acceptance	04-Feb-2021
Date of Web Publication	15-Feb-2022

Correspondence Address:
Dr R Balasubramanian
National Institute of Virology - Kerala unit, Alappuzha 688005, Kerala
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0972-9062.318307

Abstract

Background & objectives: Japanese encephalitis (JE) and West Nile virus (WNV) are two mosquito-borne diseases of medical and veterinary importance. Climate impacts in certain ecosystems are better understood; however, coastal brackish water ecosystems and their role in vector borne viral diseases have not been well studied. Hence, the aim of this study is to evaluate the distribution, spatial pattern as well as the screening of these mosquitoes for JE and WN virus in coastal area of Alappuzha district, Kerala, India.
Methods: Immature mosquitoes were collected using dipper methods along the coastal wetlands areas. Adult mosquitoes were collected in four sampling sites with light trap filled with dry ice, operating overnight. The collected mosquitoes were identified by species, using the taxonomic keys. Mosquitoes were pooled for virus detection. Water samples were collected using standard methods.
Results: The total quantity of rainfall and salinity varied from zero to 365.2 mm and from 1.5 to 28 ppt respectively. A total of 10,695 adult mosquitoes and 38,083 immatures revealed the presence of five genus and 23 species. The most predominant species was Culex tritaeniorhynchus. Immature collections Cx. sitiens was the only one species collected during the summer season and in the monsoon season the pond was replaced by fresh water mosquitoes such as Cx. tritaeniorhynchus and Cx. gelidus. A total of 186 pools were screened for JEV and WNV. However, none of the pools were found as positive for the virus.
Interpretation & conclusion: The major vector of JEV Cx. tritaeniorhynchus and Cx. gelidus have adapted to immature development in coastal brackish water habitats. The public health authorities need to recognize thepotential impact on human health of brackish water-adapted mosquito vectors that weretraditionally considered to be freshwater species, and take appropriate surveillance and control measures.

Keywords: adaptation; biotic and abiotic factors brackish water; rainfall; salinity

How to cite this article:
Balasubramanian R, Nadh V A, Sahina S. Ecology of breeding habitats of mosquito population and screening for virus of Japanese encephalitis and West Nile in the coastal area of Kerala, India. J Vector Borne Dis 2021;58:232-9

How to cite this URL:
Balasubramanian R, Nadh V A, Sahina S. Ecology of breeding habitats of mosquito population and screening for virus of Japanese encephalitis and West Nile in the coastal area of Kerala, India. J Vector Borne Dis [serial online] 2021 [cited 2023 Mar 29];58:232-9. Available from: http://www.jvbd.org//text.asp?2021/58/3/232/318307

Introduction

Mosquito-borne diseases such as Japanese encephalitis and West Nile virus are thriving worldwide, especially in the southeast Asian counties. The tropical regions of India are also among Japanese encephalitis endemic counties as potential vectors and susceptible host animals are present and disease transmission could arise due to infected animals and vectors entering the country. The vectors are Culex vishnui group; predominantly Cx. tritaeniorhynchus. Virus activity is naturally maintained through bird-mosquito cycles and pigs are important amplifying hosts^[1]. WNV is another important human pathogen of the mosquito-borne flaviviruses. The vectors are Cx.pipiens group, mosquitoes are predominantly Cx. quinquefasciatus. Virus activity is naturally maintained through bird-mosquito cycles. The emergence and re-emergence of vector-borne diseases can often be linked to human land/land cover, agriculture and urbanization^[2]. These land-use changes may influence disease prevalence and distribution by increasing breeding habitats, food resources, and changing vector-host relationships. These characteristics may increase the survival and growth rates of mosquito larvae. An outbreak of encephalitis virus in coastal areas was reported in many countries and it was transmitted by saline tolerant mosquitoes which aggressively bite both birds and mammals^[3]. Coastal wetlands act as nurseries for juvenile fish and invertebrates including mosquitoes, providing habitat for migrating and resident birds. The rise of brackish water habitats in coastal area favors the emergence of saline tolerant mosquitoes and it also leads to the adaptation of freshwater vectors to breed in brackish waters^[4]. This often leads to changes in mosquito population dynamics and species composition.

Coastal brackish water wetlands are among the most common and productive habitats that play an important ecological role in the interface between marine and terrestrial environments^[5]. Because of their location, these wetlands are spatially and temporally dynamic and highly productive ecosystems, with a wide variety of critical habitats and species including vector mosquitoes and significant biting pests. An expansion of brackish and saline water bodies in coastal areas due to rising sea levels can increase the density of salinity-tolerant mosquito vectors and cause freshwater mosquito vectors to adapt to brackish water habitats^[6]. Such developments can lead to an increase in the density of vectors relative to humans. The changes in the temporal and spatial pattern of climate variables due to climate change will affect the biology and ecology of vectors and consequently increase the risk of vector-borne disease transmission. In many recent studies investigators have examined the relation between climatic variation and occurrence of vector-borne diseases especially mosquito-borne disease like Japanese encephalitis virus, West Nile virus, Dengue, Chikungunya virus etc^[7]. As vector-borne diseases are emerging in coastal regions, they are causing a significant health risk.

Mosquito-borne diseases have emerged and re- emerged as a result of multiple factors such as increasing urbanization and climate change^[8],[9]. Kerala state has reported many outbreaks of Chikungunya, Dengue, Japanese encephalitis and West Nile virus in its coastal districts. Alappuzha district is a network of environmentally sensitive wetlands, lagoons, vast area of paddy field, lakes, estuary, and are temperature conducive for mosquito immature growth. So, this district is vulnerable to vector- borne diseases. Many outbreaks of mosquito-borne diseases reported in this district. In 2011 there were reports of encephalitis outbreaks in Kerala attributed to the West Nile virus. A follow-up study shows the long-lasting damage inflicted upon the patients who survived the outbreak. Prevalence of antibodies against Japanese encephalitis virus and West Nile virus was found in people of the district of Alappuzha in Kerala^{[10],[11],[12],[13]}. However, the details of the ecology of various vector mosquitoes have not yet been studied in this area. Our study investigated the mosquito community structure and composition in the coastal wet lands of Alappuzha, Kerala. Our main objectives were to evaluate how mosquito abundance and species composition differed between the four habitat types. Our study presents a template to assess how climate disturbances are able to influence mosquito species composition and distribution.

Material & Methods

Study area

Alappuzha is a district with the Arabian Sea on the west and a vast network of backwaters, lagoons and fresh water rivers crisscrossing the land. The district lies between 9° 5’ to 9° 55’ North latitude and 76° 17’ to 76° 46’ East longitude. It is mainly a coastal district and the major part of the district is occupied by wetland, either as rice cultivated fields or as marshy lands. The district has 82 km stretch of sea shore. This is a good feeding and nursery ground for a variety of residential and migratory birds. These waters are also used extensively for inland transportation, agriculture and other industrial purpose.

Site selection

Preliminary mosquito larvae were sampled at the sites containing potential larval habitat as denoted in [Figure 1]. Mosquitoes were collected from sites in different habitats along the coastal wet lands on the basis that they represented (i) the incidence of confirmed cases of JE and West Nile virus (ii) Wetland sites held high numbers of migratory birds, an important roosting or foraging sites of herons, egrets and abundant larval breeding sites, and (iii) sites situated close to cattle shed had higher density of livestock and human habitation that had the potential for JE/WNV to spill over to the human population. Some of the sites had trouble sufficiently surveying the sites for larval habitat such these were removed from the collection site. Among those sites, four - Thottappally (Salt marsh - 9 habitats), Chethy (Salt marsh - 4 habitats), Punnapra (7 habitats), and Vadackal (4 habitats) were selected [Figure 2], with likely all potential areas of standing water accounted for larval collection. The mean distance between localities was 5–10 km. Selected sites were sampled at least twice over the month, but were not sampled consistently because either no larvae were found or the original sampling site dried up later in the season and during monsoon season. All the sampling sites were geo-referenced with a hand-held GPS (Garmin, Germany) receiver.

Figure 1: Mosquito collection sites along the coastal area of Alappuzha district, Kerala, India

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Figure 2: Immature and adult mosquito collection sites in coastal area of Alappuzha district, Kerala, India

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Mosquito immature sampling

Mosquito larval collections were made along the margin of different larval habitats from January 2017 to March 2018. These fixed potential breeding sites were surveyed for mosquito larvae using a standard dipper method. A White enameled dipper about 4 inches in diameter (350 ml capacity) was used for collecting mosquito larvae. The handle of the dipper was lengthened by inserting a suitable piece of wooden stick (1.5 m long). At each sampling occasion, dips were taken along the margin of ponds at least 10 dips per site were taken from different locations. Third and fourth instar larvae and pupae were counted and immature abundance at each site for each sampling interval was calculated as the mean number of larvae per dipper. Live mosquito larvae were transported in water with a plastic jar. During shipment, care was taken so that the mosquito larva does not get squished.

Immature rearing and identification

Field collected immature were maintained in the laboratory as separately under ideal condition temperature 27°C ± 2°C and 75% to 80% relative humidity (RH). Immature collections were reared in the laboratory on a diet of yeast and dog biscuits at 3:1 ratio. The feeding was continued till the larvae reached the pupal stage. The pupae were transferred to standard mosquito rearing cage (30 cm × 30 cm × 35 cm) in plastic containers (7 cm χ 12 cm) containing 350 ml of water with the help of a dipper. Subsequently, adults were kept in cages and provided with a cotton wick soaked with 10% glucose solution for post- emergence and after they were killed with chloroform vapors for adult identification. The adults were counted and identified using the keys^[14].

Adult mosquito sampling

Mosquitoes were collected from sites in four different habitats along the coastal wet lands. Adult mosquitoes were collected at each of the four sites on separate occasions. At each of the 4 study sites two replicate trap sites were established. Mosquitoes were collected with light trap baited with carbon dioxide (CO₂; 1kg/trap), operating overnight from evening 6 pm to 6 am. All traps were suspended at a height of 1–1.5 m from the ground, either in trees or human-made structures bonds and cattle sheds. Once trapped, the mosquitoes were brought back to the lab and killed by placing them in the freezer. They were then identified by species, using the taxonomic keys and sorted into tubes of up to 50 individuals per tube and kept at -80°C until viral screening. Mosquitoes were pooled together from all traps at each site from each trapping date. Adult mosquito densities were calculated as mean mosquitoes per trap night.

Water quality analysis

The water samples were collected in glass bottles from each habitat by lowering a closed bottle to the bottom, opening and closing it there by hand and bringing at the surface and recording the relevant details of each sample. The collected samples were transported to the laboratory in icebox jars. Samples for Dissolved Oxygen(mg/l), Biological Oxygen Demand (mg/l) and Chemical Oxygen Demand (mg/l) analysis were collected separately and analysis was performed within 6 hr of collection^[15]. Physical conditions like temperature, salinity and pH were measured on site using a hand-held digital thermometer, refractometer (AtagoCo. Ltd, Japan) and digital pH meter (Systronics, India) respectively. Total hardness (mg/l), nitrate (mg/l), chloride (mg/l) and fluoride (mg/l) of the collected water sample were evaluated using standard water analysis kit (Himedia, India).

Viral RNA detection

Mosquito pools were homogenized in 1000μl of Trizol reagent (Invitrogen, Carlsbad, CA) using bead beater (Biospec. Products Inc. USA). The homogenate was centrifuged at 12000 rpm for 10 min at 4°C, and 250 μl of the supernatant was subjected to flavivirus RNA detection. The remaining homogenate was maintained at -80°C for future use. Total RNA was extracted from mosquito homogenates using Trizol reagent according to manufacturer’s instruction. Finally, the RNA pellet was suspended in 50μl of RNase-free water.

Extracted RNA were tested for virus detection using primers developed and recommended by the Centre for the Disease Control and Prevention Centre (CDC) for use in JE and WNV surveillance^[17],[18]. The abm one step Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) kit was used with the following reaction mix: 12.5μL buffer, 3μL RNase-free water, 1.25μL of JEV and WNV primer, 0.5μL reverse transcriptase, 1μL DNA polymerase, 0.5μL RNase inhibitor and 5μL of the sample, for a total of 25μL. The RT-PCR (AB Applied biosystems, US) was carried out under the following conditions: 50°C for 30 min, 94°C for 5 min, and 25 cycles of 94°C for 30 sec, 50°C for 1 min, and 72°C for 1 min, with a final incubation of 72°C for 7 min. PCR products were visualized after electrophoresis on a 1.5% agarose gel stained with SYBR safe. Negative and positive controls will be included in each RT-PCR run.

Meteorological data

Meteorological parameters like atmospheric temperature, rainfall, relative humidity and wind speed were obtained from the Rice Research Station, Kerala Agricultural University Alappuzha, Kerala.

Data analysis

Pearson correlation analysis was run to determine the relationship between mosquito density and different climatic factors using PASW statistics Version 18, 2009 SPSS Inc, SPSS (Hong Kong) Ltd. Pearson strength coefficient (r) and Pearson population coefficient (ρ) were computed and then analyzed the significance between mosquito densities with environmental parameters.

Results

Total immature mosquito species composition

A total of 38750 fourth instar and pupal mosquitoes were collected throughout the course of this study. A total of 24 larval habitats in which 10500 dips were performed over the entire study period. A total of two genera and twelve species identified. Site-wise species composition of mosquito immatures from coastal (four sites) area of Alappuzha district was shown in [Table 1]. Site 1: Thottappally: Altogether 11 species of mosquito larvae under five genera were identified. A total of 34425 immature stages were collected in which Cx. sitiens 7.76/dips and Cx. tritaeniorhynchus 0.97/dips was the predominant species and with a maximum larval density was more than 500 larvae per dip and remaining species were found low percentage. Culex sitiens was the only one species collected during summer season and with a maximum larval density of more than 500 larvae per dip, it was restricted almost entirely in estuary areas. The salinity of pond was from 7 to 35 ppt. Cx. sitiens was absent following the onset of monsoon rain. However, in the same pond Cx. sitiens was replaced by fresh water mosquitoes such as Cx. tritaeniorhynchus and Cx. gelidus while the salinity of the pond was decreased to 0.5-7 ppt. Therefore, analyzing ecological succession at the habitat levels may reveal substantial fluctuations in mosquito species evenness and abundance over time. Site 2: (Punnapra): A total of 1887 individuals of mosquito immature stages representing eight species and three were collected. The most prevalent mosquito species were Cx. tritaeniorhynchus 0.23/dips, Cx. gelidus 0.17/dips and remaining species were low level. Site 3: (Chethy): In total, 544 mosquitoes belonging to four species and four genera were captured. The most prevalent species were Cx. tritaeniorhynchus 0.19 and remaining species were less abundant. Site 4: (Vadackal): A total of 1227 mosquito larvae were collected in which seven species and three genera were found. The most prevalent species were Cx. gelidus (0.6) and the remaining species were less abundant.

Table 1: Immature mosquito species composition in coastal area of Alappuzha district, Kerala, India

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Seasonal fluctuation of immature mosquito population

Study area received a total quantity of rainfall varying from zero to 365.2 mm in which most of the rainfall was received during monsoon season (June till August). In this season Cx. gelidus and Cx. tritaeniorhynchus were the predominant species [Figure 3]. The temperature was negatively correlated with the total rainfall quantity (r = -0.456, ρ = 0.011) and with the relative humidity (r= -0.447, ρ=0.0130). The salinity of different larval habitat varied from 1.5 to 28 ppt. The maximum salinity and temperature was observed during the month of May. A peak of larval abundance was observed in March-May (47.7 per dips) wherein, Cx. sitiens contributed maximum in the entire population (43.65 per dips). The salinity was highly significant with the relative humidity (r= 0.381, ρ= 0.038).

Figure 3: Seasonal prevalence of immature mosquito species with salinity and climatic factors in coastal area of Alappuzha district, Kerala, India

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Water quality analysis

Biological and physical parameters of mosquito breeding water varied between habitats, according to the season and shift the survival of different species. Majority of Cx. sitiens larvae associated with brackish water (7.76/ dips) were in acidic water ranged frompH - 3.3-8, high salinity ranged from1.5–28ppt, low DO 1 to 4.5 mg/l and the pond was replaced by Cx. tritaeniorhynchus and Cx. gelidus with pH 7.2 to 9.5 and DO 7 - 9.2 mg/l and salinity range from 1.5–7 ppt after the rain. The overall range of mean BOD ranged from 2.3 to 21.3 mg/l. Regarding time of collection, larvae occurred more often, and at significantly higher densities, during the dry season compared to the wet season [Figure 4].

Figure 4: The mean water physico chemical parameters (DO, BOD & COD) in different mosquito breeding habitats in coastal area of Alappuzha district, Kerala, India

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Adult collection

A total of 10596 adult mosquitoes were caught from a total of 39 trap nights in sites in which a total of twenty species and five genera were identified. From the 2040 mosquitoes trapped in site 1, there are 16 species and five genera were identified. Of these mosquitoes, Cx. tritaeniorhynchus (50/trap night) was found to be the predominant species followed by Cx. quinquefasciatus (24.5), Ma. uniformis (18.7), Cx. sitiens (17.7), Ma. indiana (11.3), Ma. annulifera (7), Cx. gelidus (5.1), Ar. subalbatus (4), Cx. crossipes (3.2) and Cx. pseudovishnui (2.28) whereas other species were found to be <1/ trap night of the total mosquitoes collected [Table 2]. The 4651 mosquitoes trapped from Site 2 most notably Cx. tritaeniorhynchus (267.1) was found to be the predominant species followed by Cx. quinquefasciatus (44.8), Cx. gelidus (43.6), Ma. annulifera (30.30), Ar. subalbatus (22), Cx. sitiens (19.7), Ma. uniformis (16.1), Ma. indiana (12.1), Cx. pseudovishnui (3.2) and Ae. vexans (2.8) whereas other species were found to be <1% of the total mosquitoes collected.

Table 2: Adult mosquito species composition in coastal area of Alappuzha district, Kerala, India

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The Site 3 had the lowest species richness, with 13 species identified in the 1636 mosquitoes trapped. The most common species found were Cx. quinquefasciatus (44.09), Cx. tritaeniorhynchus (23.72),Cx. gelidus (20.2), Ma. indiana (17.5), Ma. annulifera (14.84), was found to be the predominant species followed by Ma. uniformis (1036), Ar. subalbatus (7.72), Cx. sinensis (4.27), Cx. crossipes (2.45) and whereas other species were found to be <1% of the total mosquitoes collected. A total of 10 species was identified from the 2368 mosquitoes trapped in site 4 of which Cx. tritaeniorhynchus (97.4), Cx. gelidus (31.6), Cx. quinquefasciatus (31), Ar. subalbatus (17.83) was found to be the predominant species followed by Ma. uniformis (7.25), Ma. annulifera (5.66), Ma. indiana (3.88) and Cx. sitiens (1.91) were the most dominant whereas other species were found to be <1/dips of the total mosquitoes collected.

The seasonal fluctuations in the general mosquito population showed bimodal pattern of peak occurrence [Figure 5]. A summer peak was observed in May (525.3 per trap night) wherein, Cx. tritaeniorhynchus contributed maximum in the entire population (407.3 per trap night) and later a medium peak was noticed in December (318.6 per trap night) and once again Cx. tritaeniorhynchus played major role in elevating combined population density (176 per trap night).

Figure 5: Seasonal prevalence of Culex tritaeniorhynchus, Culex gelidus and Culex quinquefasciatus with mean monthly temperature, relative humidity, and rainfall in coastal area of Alappuzha district, Kerala, India

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Viral screening

A total of 186 pools (8446 mosquitoes) were screened for Japanese encephalitis and West Nile Virus, which includes Cx. tritaeniorhynchus 56.44% (n= 4767), Cx. gelidus 29.16% (n=2463), Cx. sitiens 9.66% (n=816), Cx. quinquefasciatus 2.79% (n= 236), Ma. uniformis 0.76% (n=65), Ma. annulifera 0.75% (n=64), Cx. crossipes 0.24% (n=21) and Ar. subalbatus 0.16% (n=14). However, there was none of the pool were found as positive for virus nucleic acid sequences [Table 3].

Table 3: Detection of JE and WN virus in vector mosquitoes collected from coastal area of Alappuzha district, Kerala, India

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Discussion

Understanding the succession ecology of mosquitoes across different landscape is paramount for understanding spatiotemporal shifts in pathogen transmission dynamics. Diversity, similarity and relative abundance of mosquito species varied among the surveyed habitats and each mosquito species found in this study brings a unique disease risk with it. The major vectors of Japanese Encephalitis and West Nile virus, Cx. tritaeniorhynchus and Cx. gelidus were observed in high density where migratory birds are residing in nearby ponds, which further increases the risk of disease transmission. Many mosquito species found in this survey are known vectors of several arboviruses and parasites. Many risk factors are involved in the disease transmission which include climatic factors such as rainfall, temperature, relative humidity and virus infection from the reservoirs and susceptible host animals’ presence. Global warming might change the pattern of temperature and rainfall which may directly or indirectly influence the mosquito species distribution and their abundance^[16],[17]. The present study also revealed the impact of weather on the distribution and abundance of Culex mosquitoes in coastal areas.

The pattern of rainfall also affects immature mosquito population size and their habitats. Site 1 and 3 are associated with salt marsh and these habitats exhibited the highest salinity values, thus it is not surprising that these were where Cx. sitiens, a euryhaline species, had higher densities, of immature forms. The low level of precipitation, along with increased evaporation, during the summer months, with the consequent decrease in water level with increased salinity can be a possible explanation for the high breeding of Cx. sitiens immatures. In mosquito species community the species adaptability is quite limited. However, some species are able to adapt to different environmental conditions. This results in the emergence of fresh water mosquitoes as brackish or saline tolerant mosquitoes. This implies the increased density of fresh water mosquito density in brackish water. The present study showed that due to rainfall Cx. sitiens larvae disappeared from their habitat. Alternatively, intrusion of fresh water may turn former salt habitats into fresh/brackish water which could support Cx. tritaeniorhynchus and Cx. gelidus by altering abiotic and biotic factors. These favorable climatic and habitats conditions may help a mosquito population to persist year-round reproduction. Increasing abundance of these mosquitoes could have important human health implications since these mosquitoes are aggressive biters of both birds and humans, and thus can act as bridge vectors for encephalitis viruses in coastal area^[18]. In Sri Lanka the major vector of malaria Anopheles culicifacies, an established freshwater species, is able to undergo immature development in brackish waters up to 4 ppt of salinity^[19].

Site 2 and 4 were temporary wetlands made by sea water intrusion and rainfall. These sites exhibited a different mosquito immature fauna, with Cx. gelidus as the overly dominant species, thus placing it far apart from the others. A combination of Cx. tritaeniorhynchus and Cx. gelidus larvae were collected from puddles and pools approximately 500 m from the sea in suburban and rural area. The collection site contains freshwater after rain but is also subject to tidal influence. At the time of sampling the water was fresh and contained grass. The bioecology of Mansonia spp., whose immature stages live attached to stems and roots of aquatic vegetation, hence their absence is likely possible in routine immature collections, despite their continued presence as adults in these ecosystems. While there are Anopheles and Culex mosquitoes breed in wetlands and pond, however, other mosquitoes like Aedes species commonly breed in our backyard’s containers. Hence, in our present immature collection these mosquitoes were not found.

In adult collection, among many mosquito species collected, the two main vectors of Japanese encephalitis, Cx. tritaeniorhynchus and Cx. gelidus were considered in support of their abundance. The prevalence of these mosquitoes may constitute a potential risk for emergence of Japanese encephalitis in these areas. Culex tritaeniorhynchus has been incriminated as the principal vector of Japanese encephalitis in many parts of the world. Japanese encephalitis virus isolations also have been made from Cx. gelidus in India. Culex quinquefasciatus, a major vector of Lymphatic filariasis and known vector for West Nile virus and secondary vector for JEV in China were collected in urbanized area. Mansonia spp. abundance peaked during the monsoon months and other species together contributed in few percent to combined population density. Previous human cases of JEV and WNV have been putatively acquired in this region. Despite the fact that no mosquito infections were detected in the present study, which may also be due to the small sample size, surveillance should be continued as previous serology study support the presence of JEV and WNV in these coastal areas.

However, our study had certain limitations. The habitat characteristics like the presence of predation, vegetation cover, and microorganism associated with these habitats have not been studied. These surveys may miss overlooked habitats like resting mosquitoes in bushes, indoor and outdoor habitats. Immature indices may not correlate with adult mosquito abundance due to some mosquitoes having purely bird feeding habits. Challenges included the practical need to locate traps away from public view to prevent human disturbance and vandalism and accessibility. At many occasions unexpected rain and heavy wind collapsed the adult collection traps and battery. In 2018 heavy flood and water logging in our study area caused hindrance in subsequent larva and adult collection due to non-accessibility of study area.

Conclusion

Study results showed that Cx. tritaeniorhynchus and Cx. gelidus have successfully adapted to oviposit and undergo immature developmentin brackish water collections. These changes could have serious consequences for the health of people those who are residing in coastal areas through a higher incidence of many mosquito-borne diseases. The public health authorities need to recognize thepotential impact on human health of brackish water- adapted Cx. tritaeniorhynchus and Cx. gelidus and other mosquito vectors that were traditionally considered to be freshwater species, and institute appropriate surveillance and control measures.

Conflict of interest: None

Ethical statement: Not applicable

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