Background

Arthropod-borne viruses (arboviruses) are transmitted to humans primarily through the bites of infected hematophagous arthropods, for example mosquitoes (Lindsey et al., 2014). These viruses are maintained in the zoonotic cycle with humans as incidental dead-end host with limited role in the maintenance of this cycle (Ochieng et al., 2013). In the ecosystem, the viruses are incubated through intrinsic and extrinsic incubation. Aedes is one of the genera of mosquitoes known to potentially get infected and transmit a range of arboviruses including Dengue and Chikungunya viruses (Lindsey et al., 2014). These viruses cause a ranging degree of clinical syndromes with varying severity in humans and domestic/wild animals (Ranjit and Kissoon, 2011). Infections can be self-limiting characterized by fevers to deadly histopathological manifestation like encephalitis and/or hemorrhagic fevers (Ranjit and Kissoon, 2011).

In Kenya, cases of Dengue and Chikungunya viruses have been reported in the coastal region with a surge of infection during the wet season (Chadee, 2012; Lutomiah et al., 2016). Although Aedes species have been reported to be the primary vectors for arboviral transmission (Moore et al., 2013), the vectors responsible for the outbreaks at the coast have not been mapped. Aedes species have evolved to be effective in the maintenance of viruses in the population due to their ability to get infected and transmit the viruses ( Bravo et al., 2014; da Costa et al., 2017). These vectors of arboviruses are diurnal exophagic with feeding taking place in early morning and late afternoon (Chadee, 2012). Following feeding, Aedes species mosquitoes take shelter under bushes of shrubs or trees to digest their blood meal. 

Globally, Aedes species identified to be primary vectors of Dengue and Chikungunya viruses are Aedes aegypti s.l and Aedes albopictus (Moore et al., 2013). Aedes aegypti s.l is mainly transmitting arboviruses in sub Saharan, tropical and sub-tropical regions. It has two sub species, the presumed ancestral form Aedes aegypti formosus (Aaf) which is a sylvan mosquito believed to be limited to sub Saharan Africa and Aedes aegypti aegypti (Aaa) present globally in the tropical and sub-tropical regions mainly associated with humans (Mattingly, 1967). Aedes albopictus (also known as “Asian tiger” mosquito) is mainly found in the Asian continent and is incriminated in the transmission of arboviruses in that region (Minard et al., 2017)with no reports of its presence in Kenya. The various Aedes species identified in Kenya include Aedes aegypti, Aedes mcintoshi, Aedes ochraceous and Aedes tricholabis (Trpis and Hausermann, 1986; Ochieng et al., 2013).

Arboviruses solely depend on their vectors for transmission from one human/animal host to the other (Petersen et al., 2016). Transmission of the viruses occur during a blood meal by the mosquitoes from one vertebrate host to the other (Harrington et al., 2014). The life cycle of these vectors involves aquatic and terrestrial ecology. Female Aedes mosquito oviposit their eggs above stagnant water in containers (Chadee, 2012; Lutomiah et al., 2016), preferably black in color. These eggs float and hatch into larvae which then develop into pupae. The pupae hatch into an adult. This life cycle takes an average of 10 to 14 days. The female mosquito of Aedes species is responsible for transmission of arboviruses in the population. Transmission can occur through transovarial mechanisms to its offspring, mating and contact with vertebrate hosts (Chadee, 2012). Contact with vertebrate host is mainly for blood meals required for eggs development and reproduction (Harrington et al., 2014). Mosquitoes pick and transmit viruses during the blood meal (Chadee, 2012). These viruses include Dengue and Chikungunya viruses.

Dengue virus is a single stranded RNA virus comprising five distinct serotypes (DEN-1, DEN-2, DEN-3, DEN-4 and DENV-5) (Normile, 2013). In Saudi Arabia, DENV-2 was identified in 1994 (Gubler and Clark, 1995). In a population based seven years cohort study carried out in large factories in West Java, Indonesia by Kosasih et al. (2016), it was demonstrated that all four serotypes of Dengue virus was in circulation in the most years of the study. In a report by Gubler and Clark (1995), Dengue serotypes are reported in various countries: in Mozambique, DENV-3 was reported in 1985, with DENV-2 reported in several African countries including Djibouti in 1991-92, Somalia in 1982 and 1993 and Kenya in the year 1982. DENV-5 was reported in Malaysia (Normile, 2013) and in Saudi Arabia in 1994 (Gubler and Clark, 1995).

Despite poor surveillance for Dengue virus infections in Africa, it is known that epidemic Dengue fever caused by the first four serotypes has increased dramatically since 1980 (Murray et al., 2013). Most activity has occurred in East Africa resulting into major epidemics of Dengue fever in Kenya. Major outbreaks were experienced between the years 2013-2014 in the northern parts (in Mandera) and in coastal parts of Mombasa (Lutomiah et al., 2016).

Chikungunya epidemics have been reported in different corners in Africa, the Middle East, Europe, India and Southeast Asia (Lanciotti and Valadere, 2014). These epidemics are caused by ECSA and Asian genotypes, either in isolation of simultaneously depending on the locality (Petersen et al., 2016). Currently, the on-going epidemics of Chikungunya virus since 2004 involve many tropical and sub-tropical areas of Africa, Asia, Europe, the Pacific and the America. In Kenya, the maiden outbreak of Chikungunya virus was reported in 2004 in Lamu island on the coastal Kenya (Sergon et al., 2008). It involved about 13,500 people which was deemed quite substantial compared to other contemporary African Chikungunya outbreaks (Petersen et al., 2016). 

These outbreaks are usually associated with weather conditions (rainfall and elevated temperatures) conducive for breeding of Aedes mosquitoes. In towns, infrequent replenishment of domestic water stores and elevated temperatures encourages breeding of Aedes mosquitoes. Therefore, surveillance activities to establish presence of these vectors of arboviruses and their viral infection status in a given ecosystem are very crucial to estimate the potential of arboviral transmission (Ochieng et al., 2013)and design appropriate control measures. In this study we took a systematic approach and surveyed representative coastal region of Kenya to investigate the distribution of respective Aedes species and their infection status by Dengue and Chikungunya viruses for identification of areas at risk for arbovirus transmission.

1 Materials and Methods

1.1 Sentinel sites selection criteria

Study sites were identified based on historical reports of Dengue and Chikungunya outbreaks and seroprevalence data (Chretien et al., 2007; Sergon et al., 2008; Kariuki Njenga et al., 2008; Mbaika et al., 2016; Lutomiah et al., 2016). In addition, the favorable climate and ecology for breeding of Aedes species and pathogen development within the vectors make these areas good candidates for the study. All the sampling loci within these sentinel sites were mapped out by determining their co-ordinates using Geographical Positioning System (GPS). 

The study was conducted in the counties of Lamu, Kilifi, Mombasa and Kwale along the Kenyan Coast. A total of 17 sentinel sites from the 4 counties were selected as follows: Mpeketoni in Lamu County; Malindi, Watamu, Kilifi, Rabai, Mazeras in Kilifi County; Port Reiz, Tudor, Tononoka, Nyali, Shimo-la-tewa in Mombasa County; Kwale, Tiwi, Diani, Msambweni, Lunga lunga and Vanga in Kwale County.

Mpeketoni is a peri-urban town in Lamu County lies on 2˚16´10"S and longitude 40˚54´8"E with elevation of between zero and 50 meters above sea level (www.meteo.go.ke). The county experiences a mean of 417.15 mm of rainfall, with more rain experienced in the months of March to May and little rains experienced in the months of October to December. Average temperature is at 27.3°C and relative humidity of 80% (www.meteo.go.ke). The main economic activity in this zone is fishing.

Kilifi County lies on latitude 3˚37´49"S and longitude 39˚50´59"E with elevation of 10-24 meters (78 feet) above sea level (www.meteo.go.ke). Urban, peri-urban and rural sites were included. The County experiences mean daily temperature of 30°C with 82% relative humidity. Two rain seasons are experienced in this County with long rains in the months of March to May and short rains in the months of October to December. The average rainfall is approximately 88.25 mm (www.meteo.go.ke). This is a County with various economic activities with fishing, livestock production and crop farming as the main drivers of the community’s economy.

Mombasa County lies on latitude 4˚03´16"S and longitude 39˚39´48"E with elevation above the sea level at 20 meters (65 feet) (www.meteo.go.ke). The county experiences both hot and wet climate with long rains between the months of March and May while short rains between the months of October to December. The average temperature is 26.3°C with average mean rainfall of 1072.7 mm and 77.6% relative humidity (www.meteo.go.ke). The main economic activities in this zone include tourism and fishing. In this county, urban and peri-urban sites were included.

Kwale County lies on latitude 04.17489°S and longitude 039.45586°E with an elevation of 4 meters in Vanga to 387 meters above sea level in Kwale town. The County experiences what is described as tropical climate with relatively more rainfall experienced in summer and little rainfall in winter. The average temperature is 26°C with average mean rainfall of 1118 mm per year and 67% relative humidity (www.meteo.go.ke). The main economic activities in this zone include tourism and fishing with little agriculture. Samples were done in urban, peri-urban and rural areas of the county.

1.2 Entomological surveillance

Aedes mosquito sampling surveys were done during wet season (April-June and October-December) and dry season (January-March and July-September) to compare vector abundance and infection rates of the vectors by arboviruses. Indoor and outdoor sampling of adult Aedes mosquitoes were done using carbon dioxide baited Bio gent sentinel traps (BG trap) and aspiration technique. The BG traps were hanged at least two meters from the ground. For indoor sampling, the traps were set near potential breeding sites like water storage tanks, jurricans and buckets. Outdoor sampling was done in the vectors’ natural habitats including bushes and forests. Sampling was done twice per day: at dawn between 500 hrs to 1,000 hrs and in the afternoon between 1,500 hrs to 1,800 hrs with the BG traps placed randomly between 10-20 meters from each other to ensure good representation of the population in the samples. Aspiration was done from Aedes mosquitos’ resting places in the forest and containers such as car tyres and black tins. Geo-coding of sampling sites was performed using global positioning (GPS). All the caught mosquitoes were transported live in net cages to the field sorting insectary set up at the public health facility in each county with liquid nitrogen canisters for good preservation of the samples before transportation for processing at KEMRI laboratories in Kilifi.

1.3 Identification of Aedes species

Sorting of Aedes mosquito samples was done according to sampling site, sex and Aedes species. Morphological identification was done using keys as described by Gillies and De Meillon (1968)and in the manual “Mosquitoes of the Ethiopian Region” (Edward,1941; Habarch, 1988; Reinert, 2000). Males and females Aedes aegypti s.l mosquito were classified as either Aedes aegypti aegypti or Aedes aegypti formosus using scale pattern system as described earlier by McClelland GAH (1960). Briefly, mosquitoes with any white scales on the first abdominal tergite of the adult were designated Aedes aegypti aegypti. If the first abdominal tergite was completely lacking in white scales then the individual was designated Aedes aegypti formosus.

1.4 RNA processing

The pools of Aedes mosquito samples were homogenized as described by (Mackey and Chomczynski, 1995). Briefly, this was done by a mortar and pestle (cooled to temp in a liquid nitrogen bath). 1 ml of Trizol was added into the pestle with the mosquito pools and ground thoroughly. Vortex was done for one minute. An aliquot of the solution was transferred to eppendorf tubes and left in Trizol at room temperature for five minutes. For phase separation, a 0.2 ml of chloroform was added, samples were capped and vortex for 15 seconds. Incubation of the samples was done at room temperature for 2-3 minutes. Centrifugation was done at 12,000 rpm for 15 minutes at 8°C. RNA precipitation was done by centrifugation. The aqueous phase at the top was transferred into a fresh tube. 0.5 ml of isopropanol was added to the new tube and incubated at room temperature for 10 minutes. This was centrifuged at 12,000 rpm for 10 minutes at 8°C. Following centrifugation, the supernatant was removed and RNA pellet washed with 1 ml of 75% ethanol (vortexing). Centrifugation was done at 7,500 rpm for 5 minutes at 8°C. Supernatant was removed and remaining ethanol was air dried for 2-3 minutes. The pellet was eluted in elution buffer with sodium azide and vortexed for a minute. Tubes were transferred to a digital dry bath at 60°C for 15 minutes before placing them on ice.

Quantification and Quality control: Determination of the quantity and quality of extracted RNA was done using Nanodrop spectrophotometer. 1.5 μl microcuvette with OD at 260 nm and 280 nm was used determine sample concentration and purity.

1.5 Identification of serotypes of Dengue virus

2.5 µl of the RNA samples were used in One Step real time PCR (qRT-PCR) with master mix made as per manufacturer’s instructions (Applied bio systems, USA). DENV specific RNA was amplified in a total of 25 µl reaction mixture (12.5 μL of Amplitaq Gold 360 PCR master mix-Applied bio systems, USA, 50 picomoles each of forward and reverse primer, 2 μL of the cDNA and 9.5 μl of DEPC treated water to top up to 25 μL) (Ochieng et al., 2013; Normile, 2013; Konongoi et al., 2016).

The PCR plates were placed in the real time thermo cycler and were cycled at 50°C for 20 minutes, 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 70°C for 1 minute, and a final extension for 10 minute at 72°C. Analysis of the displayed curves was done to identify the positive samples for various serotypes of Dengue virus. This was a step wise procedure as follows: Samples were first tested using flavivirus family primers. Samples testing positive with flavivirus family primers were further tested with consensus Dengue primers D1 and D2. Samples testing positive with the Dengue consensus primers that target the E/NS1 junction of the virus genome were further tested for the 4 Dengue serotypes using the appropriate primers (Kuno et al., 1998). The primer sequences above were used to detect exposure to the virus using amplification conditions as described in (Kuno et al., 1998) and (Lanciotti et al., 1992). A positive control cDNA and a negative control were included during the setting up of all PCR reactions.

1.6 Identification of Genotypes of Chikungunya virus

20.2 μL volume containing 1 μL of cDNA, 4 μL of 5X reaction buffer, 1.8 μL of 25 mM MgCl2, 1.8 μL of 10 μM dNTP, 0.5 μL of 20 μM of each primers, 0.3 μL of Go-Tag Flex DNA polymerase, and 10.3 μL of nuclease free water (NorgenBiotek, Canada) were used in One Step real time PCR (Applied bio systems, USA). The PCR program consisted of an initial denaturation at 95°C for 2 min and 40 cycles of 30 seconds denaturation at 95°C, 30 seconds annealing at 55°C, 1 minute elongation at 72°C and final extension at 72°C for 5 minutes. Presence of alphaviruses was screened in all the amplified products using the pan-alphavivirus primer (Ochieng et al., 2013). Samples testing positive with alphavirus family primers were further tested with the conventional primers for Chikungunya virus (Redd, et al., 2012). A positive control cDNA and a negative control were included during the setting up of all PCR reactions. The conventional primer amplifies a 646 bp gene product in the Chikungunya envelope region of E1. The amplified gene products were identified by amplification curves.

2 Results

2.1 Aedes species along the Kenyan Coast

A total of 37,220 Aedes mosquitoes were collected from 17 sentinel sites located within four Counties along the coastline of Kenya for two years. 58% of the Aedes mosquitoes were collected during the wet season, with a highest sample size of Aedes species collected from Kilifi County at 31%. Aedes aegypti formosus was the dominant species in both dry and wet seasons at 65% and 58% respectively (Figure 1). However, the proportions of these Aedes species along the Coastline did not differ significantly with the seasons (M=2,068, SD=3,520), t₈=0.03, p<0.001. Similarly, the sex of Aedes species collected during the two seasons did not differ significantly along the coastline with seasons, (M=2,068, SD=3,587), t₈=0.315, p<0.001.

2.2 Distribution of Aedes species along the Kenyan Coast

The spatial and temporal distribution of Aedes species according to habitat (urban, peri-urban, rural and marine) and seasons is shown in Figure 2 and Figure 3. Aedes aegypti formosus and Aedes aegypti aegypti were distributed along the entire coastline with exception in the mangrove microhabitats of Watamu, Tudor and Vanga. Aedes ochraceus and Aedes mcintoshi inhabited the northern parts of the coastline: Kilifi, Malindi and Lamu. Aedes fulgens and Aedes fryeri were only in Mombasa regions of Nyali and Portreiz respectively. Aedes pembaensis and Aedes tricholabis were found to inhabit marine mangrove ecological zones of Watamu, Tudor, Shimo la tewa, Msambweni and Vanga with some spreading to interiors of Lunga lunga. Aedes albicosta was only found in north coast regions of Malindi. However, the spatial and temporal distribution of Aedes species did not vary significantly with the seasons (M=190.125, SD=158.94), t₈=0.14, p<0.001.

2.3 Serotypes of Dengue virus in Aedes mosquitoes

An assay for the serotypes of Dengue virus was done in 1861 pools (n=37,220) of Aedes species. 7.9% of these were positive for Dengue virus. The proportions of respective serotypes of Dengue virus are shown in Figure 4. DENV-2 had the highest frequency among serotypes of Dengue virus at 54% with DENV-4 with the least frequency at 6%. DENV-4 was identified for the first time in Aedes species along the coastline of Kenya. The infections of serotypes in Aedes species mosquito did not differ significantly with seasons (M=190, SD=159), t₃=0.1406, p<0.001.

2.4 Distribution of serotypes of Dengue virus

The seasonal abundance and distribution of serotypes of Dengue virus along the coastline of Kenya is shown in Figure 5 and Figure 6. DENV-1 and DENV-2 were distributed along the entire coastline in all sentinel sites from Vanga to Lamu. DENV-3 was conspicuously absent in Aedes species in marine ecological zones. DENV-4 was not identified in the interior site of the coastline (Rabai and Mazeras).

2.5 Distribution of genotypes of Chikungunya virus

Molecular assay for the genotypes of Chikungunya virus was done in 1861 pools (n=37,220) of Aedes species. 2.1% of these were positive for East Central and Southern Africa (ECSA) genotype of Chikungunya virus. The abundance of this genotype in the mosquito vectors did not vary significantly with seasons (M=7.25, SD=15), t₃=0.325, p<0.001 although more Aedes mosquitoes were positive for the virus during the wet season as shown in Figure 7. The virus is highly concentrated on the southern and northern parts of the coastline in Kwale and Kilifi, Lamu Counties as shown in Figure 8 and Figure 9.

3 Discussion

The composition of Aedes species remained largely the same during dry and wet seasons. However, more Aedes mosquitoes were collected during the wet season. This implies wet season has no impact on the types of Aedes species present in the region but provide more breeding grounds for the increase in their population size. In this study, two Aedes aegypti s.l subspecies (Aedes aegypti formosus and Aedes aegypti aegypti) were identified. A study by Moore et al. (2013)reported only Aedes aegypti formosus in Rabai, which is a coastal inland township in Kilifi County. The maiden identification of Aedes aegypti aegypti can be attributed to the sampling and analysis of Aedes mosquitoes from the entire coastline for a continuous period of time and a possibility of gene dynamics. This Aedes mosquito was identified long ago in Shauri moyo village in the interior of the coastal zone by Trpis and Hausermann (1986). A variety of Aedes mosquito vectors for arboviruses were identified along the coast. These include: Aedes pembaensis, Aedes ochraceus, Aedes mcnitosh, Aedes tricholabis, Aedes fryeri, Aedes fulgens and Aedes albicosta. All these Aedes species were earlier reported in the coastal region of Kenya by  Moore et al. (2013), Sang et al. (2010) and Edwards (1941).

The highest proportion of mosquitoes was sampled from Kilifi County. This may be attributed to the presence of forests and bushes around human settlements with readily available outdoor breeding sites (tyres, tree holes and containers). The increase in number of mosquitoes during wet season could be attributed to increase and sustenance of indoor and outdoor breeding sites for Aedes species. Indoor breeding sites for Aedes mosquitoes include water storage tanks, preferably black in color, jurricanes, and buckets and are common in towns as reported by Lutomiah et al. (2016). Outdoor breeding sites include non-flow house water gutters, tins on open grounds, water tanks, scrap metal dealers sites, tyres, drums, tree holes and leaves. These are common in the rural and peri urban areas.

A higher proportion of Aedes mosquitoes were collected from outdoor sampling. This confirms findings by Lutomiah et al. (2016)that more numbers of Aedes mosquitoes are usually trapped from outdoor sampling in entomological surveys. For Aedes aegypti s.l, the proportion of Aedes aegypti formosus was higher than of Aedes aegypti aegypti. This is attributed to more outdoor sampling as Aedes aegypti formosus is an outdoor subspecies primarily feeding on wild animals (exophagic and zoophilic), with Aedes aegypti aegypti as an indoor subspecies preferring to feed on human blood (endophagic and anthropophilic) (Mattingly, 1967). The numbers of Aedes species did not differ significantly for the two seasons because breeding sites for these vectors are available during both seasons.

The distribution of Aedes species was similar in the two seasons with members of Aedes aegypti s.l distributed on the entire coastline of Kenya. However, these members of Aedes aegypti s.l mosquitoes were absent in marine ecologies of Vanga, Tudor and Watamu. This indicates marine ecologies may not be good breeding grounds for these Aedes species. However, studies should be done to assess any influence of marine ecologies on the habitation behavior of these mosquitoes. Aedes ochraceus and Aedes mcnitosh were concentrated in the northern part of the coastline in Kilifi, Malindi and Lamu. In earlier entomological surveys studies by Ochieng et al. (2013) and by Labeaud et al. (2011), these Aedes species were reported in Garissa County, although in this study Garissa was not included. Aedes fryeri and Aedes fulgens were identified in Mombasa Island and Portreiz while Aedes albicosta were present in Malindi only. Similar observations were reported by Edwards (1941). Aedes pembaensis and Aedes tricholabis were identified in marine ecologies breeding in holes made by ocean crabs as also reported by Sang et al. (2010) and by Edwards (1941). In comparison of this study to the earlier entomological surveys, it is evident that Aedes species have expanded their geographical range. This can be due to frequency of movement by locals and visitors in the coastal towns for either business or tourism. Throughout the sampling period, more males than females were sampled. This is because sampling was mainly done in bushes surrounding homesteads where most males feed on fruit juices and flower nectars (Grech et al., 2010). Females were mainly collected from indoor and areas closely to human dwellings as they need a blood meal for viability of their eggs in terms of fertility (Harrington et al., 2014).

On analysis of arboviral infections in the Aedes mosquitoes, we identified four serotypes of Dengue virus circulating in Aedes species along the coastline of Kenya. DENV-5 was not detected. DENV-5 was identified in Malasyia by Normile (2013), although they used serum for assays. This report confirms findings by Sutherland et al. (2011)but contrast reports by Konongoi et al. (2016)in which three serotypes of Dengue virus were reported (DENV-2, DENV-2 and DENV-3). This study reports higher rate of DENV-2 serotype infection in Aedes species along the entire coastline, similar to reports from serological studies by Sutherland et al. (2011). However, this was not the case in assays by Konongoi et al. (2016) in which DENV-1 was reported to have higher rates of infection. This indicates dynamism in population of circulating serotypes. DENV-4 is the least circulating serotype of Dengue virus in Aedes species mosquitoes along the Kenya’s coastline. This is in support of statistics on Dengue virus by Sutherland et al. (2011). The rates of arboviral infection in the Aedes species did not vary significantly along the coastline. This might mean all serotypes of Dengue virus are in circulation (Ochieng et al., 2013)and human infection can occur in similar proportions upon exposure to Aedes mosquito bites.

The presence of Dengue viruses in the vectors does not depend on the seasons. However, their rates of infection increase during wet seasons which may result to Dengue or Chikungunya outbreaks. This can be attributed to increase in the number of Aedes mosquitoes due to readily available breeding grounds and up scaled human contacts (Chadee, 2012). Human contacts can occur during farming and grazing activities/ pastoralism (Ochieng et al., 2013); (Grech et al., 2010).

The genotype of Chikungunya virus circulating in Aedes species mosquitoes along the Kenya’s coastline was the East Central and Southern Africa (ECSA) genotype. This finding was in line with reports from recent studies by Sergon et al. (2008)and by Kariuki Njenga et al. (2008). This genotype was present in Aedes species along the four coastline counties with its concentration in the southern parts of the coastline. The population and activity of the virus increased during wet season, although the increase was not significant. This is similar to findings by Harrington et al. (2014) where there was no difference in number of mosquito biting frequencies during low and high viral transmission seasons. 

In terms of distribution, Dengue and Chikungunya viruses occurred in similar geographical regions during dry and wet seasons. This scenario was also observed by Le Coupanec et al. (2017); Kosasih et al. (2016) where Chikungunya affected areas overlapped with those endemic for Dengue virus. Selective infection of Aedes species by arboviruses in the same ecological zone may suggest preferential association among viruses, vectors and hosts. Absence of DENV-4 in Aedes macnitosh in Lamu, Malindi and Kilifi may suggest this vector is not suitable for transmission of DENV-4. Similar observations were made in Aedes tricholabis for CHIKV despite the virus being detected in the Aedes pembaensis in the same areas of Watamu, Tudor and Vanga; Aedes albicosta for DENV-3 and DENV-4 despite Aedes aegypti s.l populations testing positive for the virus within Malindi and Shimo la tewa; Aedes fryeri for DENV-1, DENV-3 and DENV-4 in Portreiz; Aedes fulgens for DENV-3 and DENV-4 in Nyali and Aedes ochraceus for DENV-4 in Portreiz. This calls for more studies to establish the vector-host-pathogen relationships for effective control strategies.

Aedes aegypti s.l and Aedes pembaensis were infected by all serotypes of Dengue and Chikungunya viruses. This confirms numerous reports that Aedes aegypti s.l forms the primary vector for arboviruses and can be infected by both Dengue and Chikungunya viruses (Rückert et al., 2017). Aedes aegypti aegypti had the highest Dengue and Chikungunya viral infection among all Aedes species and sub-species screened for the viruses. This finding supports report that members of the Aedes aegypti aegypti subspecies have higher vector competence compared to Aedes aegypti formosus subspecies (Tabachnick et al., 1985); (Mattingly, 1967). In this study, infection of male mosquitoes by arboviruses was found in all Aedes species. Similar assays done earlier by Lutomiah et al. (2016)reported infection of Aedes aegypti males by Dengue virus. However, in their study, they did not identify other arboviruses in males and only Aedes aegypti were screened. Infection in males clearly demonstrates ability of male mosquitoes to play a role in transmission of arboviruses in the environment. Males acquire the virus from their parents through transovarial mode of transmission and subsequently transmit it to the females during mating as reported by da Costa et al. (2017). Males pass the virus through sperms as they mate with females few days following emergence from their pupae stage. Therefore, control measures for arboviral diseases should target both males and females.

4 Conclusion and Recommendation

There are different Aedes species vectors for Dengue and Chikungunya viruses along the coastline of Kenya. These vectors are distributed on the entire coastline and they include: Aedes aegypti formosus, Aedes aegypti aegypti, Aedes mcintoshi, Aedes ochraceus, Aedes pembaensis, Aedes tricholabis, Aedes albicosta, Aedes fulgens and Aedes fryeri. Aedes aegypti aegypti subspecies are the potential main vectors for arboviruses in the coastal region of Kenya due to their higher infection rates by Dengue and Chikungunya arboviruses. Serotypes of Dengue virus circulating in Aedes species along the coastline were DENV-1, DENV-2, DENV-3 and DENV-4. DENV-2 had the highest vector infection rates with DENV-4 having the least. Serotype DENV-3 was absent in marine ecosystems as it was not found in marine ecologies.

Only the ECSA genotype of Chikungunya virus is present in Aedes mosquito population along the coastline of Kenya. Co-infections of Dengue and Chikungunya arboviruses occur in Aedes species. We recommend for continuous survey for vectors of arboviruses to give us a more reliable picture on the epidemiology of vectors and their infection status to estimate the risk of arboviral outbreaks in the region.

Declarations

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on request.

Competing interests

All authors declare no competing interests.

Authors’ contributions

Jonathan Chome Ngala conceived the idea of this study, carried out all field and laboratory experiments and is the main author. Margaret Wangui Muturi contributed to the supervision and writing of this manuscript. Charles M. Mbogo contributed to the provision of laboratory space for experiments and writing of this manuscript. Martin K. Rono contributed to supervision, genetic analysis and writing of this manuscript. All authors read and approved the final manuscript.

Acknowledgements

We would like to acknowledge the National Research Fund-Kenya, Bernhard Nocht Institute for Tropical Medicine-Germany and the Kenya Medical Research Institute Centre for Geographic Medicine-Coast for funding this piece of work. Thanks to Mr. Festus Yaa, Mr. David Shida and Mr. Nzai Gabriel for their professional assistance in the field and identification of the Aedes species. Much thanks to all residents of the counties along the coastline of Kenya for their consent to set traps in their homesteads and farms.

Reference

Bravo L., Roque V.G., Brett J., Dizon R., and L’Azou M., 2014, Epidemiology of dengue disease in the Philippines (2000-2011): a systematic literature review, PLoS Neglected Tropical Diseases [Electronic Resource] 8(11): 3027

https://doi.org/10.1371/journal.pntd.0003027

Castro J.S.M., and Oliveira F.L.S., 2015, East / Central / South African Genotype Chikungunya Virus, Emerging infectious diseases 21(5): 2013-2015

Chadee D.D., 2012, Studies on the post-oviposition blood-feeding behaviour of Aedes aegypti (L.) (Diptera: Culicidae) in the laboratory, Pathogens and Global Health 10: 1179-1188

Chomczynski P.M.K., 1995, Short technical report. Modification of the TRIZOL reagent procedure for isolation of RNA from Polysaccharide-and proteoglycan-rich sources. Biotechniques 19(6): 942-945

Chretien J.P., Anyamba A., Bedno S.A., Breiman R.F., Sang R., Sergon K., Powers A.M., Onyango C.O., Small J., Tucker C.J., et al., 2007, Drought-associated chikungunya emergence along coastal East Africa, Am J Trop Med Hyg 76, 405-407

https://doi.org/10.4269/ajtmh.2007.76.405

da Costa C.F., dos Passos R.A., Lima J.B.P., Roque R.A., de Souza Sampaio V., Campolina T. B.,  Nágila F.C.S., and Paulo F.P.P., 2017, Transovarial transmission of DENV in Aedes aegypti in the Amazon basin: a local model of xenomonitoring, Parasites & Vectors 10: 249

https://doi.org/10.1186/s13071-017-2194-5

Dash P.K., Parida M.M., Santhosh S.R., Verma S.K., Tripathi N.K., Ambuj S., and Sekhar K., 2007, East Central South African genotype as the causative agent in reemergence of Chikungunya outbreak in India, Vector Borne and Zoonotic Diseases 7(4): 519-527

https://doi.org/10.1089/vbz.2006.0648

Edwards F.W., 1941, Mosquitoes of the Ethiopian region III. London, United Kingdom: London British Museum of Natural History, 116-223

Gillies M.T., and DeMeillon B., 1968, The Anophelinae of Africa South of the Sahara (Ethiopian Zoogeographical Region), Publications of the South African Institute for Medical Research, 54: 131-208

Göertz G.P., Vogels C.B.F., Geertsema C., Koenraadt C.J.M., and Pijlman G.P., 2017, Mosquito co-infection with Zika and chikungunya virus allows simultaneous transmission without affecting vector competence of Aedes aegypti, Public Library of Science Neglected Tropical Diseases 10: 371-385

https://doi.org/10.1371/journal.pntd.0005654

Grech M.G., Ludueña-Almeida F., and Almirón W.R., 2010, Bionomics of Aedes aegypti subpopulations (Diptera_ Culicidae) from Argentina - Grech - 2010, Journal of Vector Ecology - Wiley Online Library

Gubler D.J., and Clark G.G., 1995, Dengue/dengue hemorrhagic fever: the emergence of a global health problem, Emerging Infectious Diseases: 1(2): 55-57

https://doi.org/10.3201/eid0102.952004

Harbach R.E., 1988, The mosquitoes of the subgenus Culex in Southwestern Asia and Egypt (Diptera: Culicidae), Control American Entomology Institution 24: 240

Harrington L.C., Fleisher A., Ruiz-Moreno D., Vermeylen F., Wa C.V., Poulson R.L., and Scott T.W., 2014, Heterogeneous Feeding Patterns of the Dengue Vector, Aedes aegypti, on Individual Human Hosts in Rural Thailand, Public Library of Science Neglected Tropical Diseases. 10: 1348-1370

https://doi.org/10.1371/journal.pntd.0003048

Kajeguka D.C., Kaaya R.D., Mwakalinga S., Ndossi R., Ndaro A., Chilongola J.O., and Alifrangis M., 2016, Prevalence of dengue and chikungunya virus infections in north-eastern Tanzania: a cross sectional study among participants presenting with malaria-like symptoms, Biomedical central Infectious Diseases 16(1): 183

https://doi.org/10.1186/s12879-016-1511-5

Kariuki Njenga M., Nderitu L., Ledermann J.P., Ndirangu A., Logue C.H., Kelly C.H.L., and Powers A.M., 2008, Tracking epidemic Chikungunya virus into the Indian Ocean from East Africa, The Journal of General Virology 89(11): 2754-2760

https://doi.org/10.1099/vir.0.2008/005413-0

Kosasih H., Alisjahbana B., Nurhayati de Mast Q., Rudiman I.F., Widjaja S., and Porter K.R., 2016, The Epidemiology, Virology and Clinical Findings of Dengue Virus Infections in a Cohort of Indonesian Adults in Western Java, Public Library of Science Neglected Tropical Diseases 10: 1371

https://doi.org/10.1371/journal.pntd.0004390

Konongoi L., Ofula V., Nyunja A., Owaka S., Koka H., Makio A., and Sang R., 2016, Detection of dengue virus serotypes 1, 2 and 3 in selected regions of Kenya: 2011-2014. Journal of Virology 13: 182

https://doi.org/10.1186/s12985-016-0641-0

Kuno G., Chang G.J., Tsuchiya K.R., and Karabatsos N.C.C., 1998, Phylogeny of the genus flavivirus, Journal of Virology 72(1): 73-83

Labeaud A.D., Sutherland L.J., Muiruri S., Muchiri E.M., Gray L.R., Zimmerman P.A., and King C.H., 2011, Arbovirus Prevalence in Mosquitoes, Kenya, Emerging infectious diseases 17(2): 233-241

https://doi.org/10.3201/eid1702.091666

Lanciotti R.S., and Valadere A.M., 2014, Transcontinental movement of Asian Genotype Chikungunya virus, Emerging Infectious Diseases 20(8): 1400-1402

https://doi.org/10.3201/eid2008.140268

Lanciotti R.S., Calisher C.H., Gubler D.J., Chang G.J., and Vorndam A.V., 1992, Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction, Journal of  Clinical Microbiology 30(3): 545-551

Le Coupanec A., Tchankouo-Nguetcheu S., Roux P., Khun H., Huerre M., Morales-Vargas R. and Choumet V., 2017, Co-infection of mosquitoes with chikungunya and dengue viruses reveals modulation of the replication of both viruses in midguts and salivary glands of Aedes aegypti mosquitoes, International Journal of Molecular Sciences 18(8): 1708

https://doi.org/10.3390/ijms18081708

Lindsey N.P., Lehman J.A., Staples J.E., and Fischer M., 2014, West Nile virus and other arboviral diseases - United States, 2013, Morbidity and Mortality Weekly Report, 12997

Lutomiah J., Barrera R., Makio A., Mutisya J., Koka H., Owaka S., and Sang R., 2016, Dengue Outbreak in Mombasa City, Kenya, 2013-2014: Entomologic Investigations, PLoS Neglected Tropical Diseases 10(10): 2013-2014

https://doi.org/10.1371/journal.pntd.0004981

Mattingly P.F., 1967, Taxonomy of Aedes Aegypti and Related Species. Bulletin of the World Health Organization 36: 552

Mbaika S., Lutomiah J., Chepkorir E., Mulwa F., Khayeka-Wandabwa C., Tigoi C., Oyoo-Okoth E., Mutisya J., Ng'ang'a Z., and Sang R., 2016, Vector competence of Aedes aegypti in transmitting Chikungunya virus: effects and implications of extrinsic incubation temperature on dissemination and infection rates, Virol J 13, 114

https://doi.org/10.1186/s12985-016-0566-7

McClelland G.A.H., 1960, A prelimnary study of the genetics of abdominal color variations in Aedes aegypti (L.) (Diptera: Culicidae), Annals of Tropical Medicine and Parasitology 54: 305-320

https://doi.org/10.1080/00034983.1960.11685991

Minard G., Tran Van V., Tran F.H., Melaun C., Klimpel S., Koch L.K., and Valiente Moro C., 2017, Identification of sympatric cryptic species of Aedes albopictus subgroup in Vietnam: new perspectives in phylosymbiosis of insect vector, Parasites & Vectors 10: 1-10

https://doi.org/10.1186/s13071-017-2202-9

Moore M., Sylla M., Goss L., Burugu M.W., Sang R., Kamau L.W., and Sharakova M., 2013, Dual African Origins of Global Aedes aegypti s. l . Populations Revealed by Mitochondrial DNA, 7(4): 2175. 276

Moro M.L., Gagliotti, C., Silvi, G., Angelini, R., Sambri, V., Rezza, G. and Seyler, T., 2010, Chikungunya Virus in North-Eastern Italy : A Seroprevalence Survey, American journal of Tropical medicine and Hygiene 82(3): 508-511

https://doi.org/10.4269/ajtmh.2010.09-0322

Murray N.E.A., Quam M.B., and Wilder-Smith, A., 2013, Epidemiology of dengue: Past, present and future prospects, Clinical Epidemiology 5: 299-309

Normile D., 2013, Tropicalmedicine, Surprising new dengue virus throws a spanner in disease controlefforts 342:415

Ochieng C., Lutomiah J., Makio A., Koka H., Chepkorir E., Yalwala S., and Sang R., 2013, Mosquito-borne arbovirus surveillance at selected sites in diverse ecological zones of Kenya, Journal of virology 10: 140

https://doi.org/10.1186/1743-422X-10-140

Petersen L.R., Powers A.M., Weaver S.C., Forrester N.L., Zanluca C., Melo V.C.A., and Olowokure B., 2016, Chikungunya: epidemiology, Vector Borne and Zoonotic Diseases 5: 7171

Ranjit S., and Kissoon N., 2011, Dengue hemorrhagic fever and shock syndromes, Pediatric Critical Care Medicine : A Journal of the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies 12(1): 90-100

Reddy V., Ravi V., Desai A., Parida M., Powers A., and Johnson B.W., 2012, Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever, Journal of Medical Virology 84: 1771-1778

https://doi.org/10.1002/jmv.23406

Reinert J.F., 2000, New classification for the composite genus aedes ( diptera : culicidae : aedini ), elevation of subgenus ochlerotatus to generic rank , reclassification of the other subgenera. And notes on certain subgenera and species, Journal of American Mosquito Control Association 16(3): 175-188

Renault P., Solet J.L., Sissoko D., Balleydier E., Larrieu S., Filleul L., and Pierre V., 2007, A major epidemic of chikungunya virus infection on Réunion Island, France, 2005-2006, American Journal of Tropical Medicine and Hygiene 77(4): 727-731

https://doi.org/10.4269/ajtmh.2007.77.727

Rückert C., Weger-Lucarelli J., Garcia-Luna S.M., Young M.C., Byas A.D., Murrieta R.A., and Ebel G.D., 2017, Impact of simultaneous exposure to arboviruses on infection and transmission by Aedes aegypti mosquitoes, Nature Communications 8: 15412

https://doi.org/10.1038/ncomms15412

Sang R., Kioko E., Lutomiah J., Warigia M., Ochieng C., O’Guinn M., and Richardson J., 2010, Rift Valley fever virus epidemic in Kenya, 2006/2007: The entomologic investigations, American Journal of Tropical Medicine and Hygiene 83(2): 28-37

https://doi.org/10.4269/ajtmh.2010.09-0319

Sergon K., Njuguna C., Kalani R., Ofula V., Onyango C., Konongoi L.S., and Breiman R.F., 2008, Seroprevalence of Chikungunya Virus (CHIKV) Infection on Lamu Island, Kenya, October 2004, American Journal of Tropical Medicine and Hygiene 78(2): 333-337

https://doi.org/10.4269/ajtmh.2008.78.333

Sutherland L.J., Cash A.A., Huang Y.J.S., Sang R.C., Malhotra I., Moormann A.M., and LaBeaud A.D., 2011, Serologic evidence of arboviral infections among humans in Kenya, The American Journal of Tropical Medicine and Hygiene 85(1): 158-161

https://doi.org/10.4269/ajtmh.2011.10-0203

Trpis M., and Hausermann W., 1986, Dispersal and other population parametersof Aedes aegypti in an African village and their possible significance in epidemiology of vector-borne diseases, American journal of tropical medicine and hygiene 35(6): 1263-1279

https://doi.org/10.4269/ajtmh.1986.35.1263