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Species composition and blood meal analysis of mosquitoes collected from a tourist island, Koh Chang, Thailand

2014; Wiley; Volume: 39; Issue: 2 Linguagem: Inglês

10.1111/jvec.12122

1948-7134

Supaluk Khaklang, Pattamaporn Kittayapong,

Travel-related health issues

Mosquitoes are vectors of many viruses, bacteria, nematodes, protozoa, and other parasites that can be transmitted and cause illness or death to animals and humans. At present, many mosquito vector-borne diseases are spread worldwide due to ecological, environmental, and human factors, as well as international travel including tourism (Chen and Wilson 2008). Increased tourism is one of the driving forces of land use/cover change due to intensive development accommodations and infrastructure, as well as human settlement (Wang and Lui 2013). Expanding tourism contributes to rapid unplanned urbanization that is often associated with inadequate infrastructure, such as water supplies and waste management, and can lead to an increase in the density of mosquito vectors (Gubler 1998). Moreover, human intrusion into forested areas for recreational purposes (ecotourism) and also for residential development, exposes them to natural hosts and vectors that exist in the areas. These anthropogenic activities could facilitate the emergence of mosquito-borne disease because they can affect larval and adult vector abundance, as well as human-mosquito interactions (Vanwambeke et al. 2007). When travelers visit tourist destinations and lack immunity for exotic diseases, they are at risk for infection with vector-borne disease agents (Chen and Wilson 2008). Mosquito-borne diseases that are frequently reported among international travelers include dengue and chikungunya. Thailand is among the most popular tourist destinations in the world and regularly reports dengue infections among travelers (Schwartz et al. 2000, Sung et al. 2003). Therefore, the present study was conducted in a tourist setting that is considered to be a global outreach hotspot because large numbers of visitors come from various parts of the world and can promote the emergence of vector-borne and zoonotic diseases. Our selected study site was the island of Koh Chang, located in Koh Chang District, Trat Province, off the eastern seaboard in the Gulf of Thailand. The island, covering an area of approximately 650 km2 and consisting of 52 islands, was declared part of the National Marine Park archipelago in 1982. Both dengue and malaria cases have been reported on the island. However, no information has been available regarding mosquito species composition or the risk of mosquito-borne diseases. Therefore, the objectives of our study were to identify species that distribute on Koh Chang, to investigate blood meal sources of collected mosquitoes, and to screen for Flavivirus infection in collected mosquitoes. Mosquito collections were conducted during the dry season in January, 2012 and the wet season in June, 2012. Our study is the first that investigated mosquito vector abundance on this tourist island. The collection was carried out in urban, forest and peri-urban settings located in five villages: Khlong Son, Khlong Prao, Bang Bao, Saluk Phet Nua and Saluk Kok (Figure 1). In order to increase the number of mosquito species collected, a variety of tools including BG traps, CDC light traps, mosquito magnets, and portable vacuum aspirators were used. BG traps baited with BG-lure and mosquito magnets baited with CO2 were set up in each sampling site in the morning. CDC light traps were suspended 150 cm above ground level, turned on in the afternoon, and turned off the next morning. Captured mosquitoes were gathered in the evening of the same day at 18:00 and the next morning at 06:00. After mosquito collection, the traps were rotated to another position. During field collection, captured mosquitoes were placed in plastic ice boxes containing ice to avoid degradation of virus. Portable vacuum aspirators were operated for 20 min in each house for sampling resting mosquitoes, both inside and outside. Collection was mainly done in shaded areas that are preferred resting areas of mosquitoes, such as corners, bathrooms, bedrooms, kitchens, and clothes racks. Besides mosquito collection, available animals near the sampling sites were also recorded during mosquito collections because these animals might be sources of mosquito blood meals. Their antibodies were later used for the identification of blood meal sources. Furthermore, all sampling sites were located with a GPS and mapped to show spatial distribution of the sampling sites. Upon arrival to the laboratory at the Center of Excellence for Vectors and Vector-Borne Diseases (CVVD) at Mahidol University, all mosquito samples were identified using illustrated keys for the mosquitoes of Thailand (Rattanarithikul et al. 2005a, 2005b, 2006a, 2006b, 2007, 2011). Mosquito samples were also classified by sex and blood meal status. The samples were stored at −80º C until used. Blood meals of engorged female mosquitoes were analyzed by ELISA technique previously described by Thapar et al. (1998). ELISA was performed in an isolated air-conditioned laboratory with the normal temperature of 25–27º C using anti-host (IgG) conjugates (Kirkegaard and Perry, Gaithersburg, MD) against human, monkey, and dog. Absorbance value was read by ELISA plate reader at a wavelength of 490 nm. Test specimen was considered to be positive when the absorbance value was greater than the mean cut off plus two standard deviations of the negative controls. Engorged female mosquitoes were individually screened for Flavivirus, while the remainder of female and male mosquitoes were pooled into five individuals by separating them according to sex and collection site. RNA extraction was performed according to the manufacturer's instructions in the easy-RED™ Total RNA extraction kit, iNtRON Biotechnology Inc., Kyungki-Do, Korea. RNA extraction was conducted in an isolated air-conditioned laboratory with the normal temperature of 25–27º C and RNA solution was centrifuged at 4º C. RT-PCR was used for detection of Flavivirus. Three Flavivirus universal primers were used, including PF1-S (5’-TGY RTB TAY AAC ATG ATG GG-3’), PF2-R (5’-GTG TCC CAD CCA GCD GTR TC-3’) (Moreau et al. 2007), and PF3-S (5’-ATH TGG TWY ATG TGG YTD GG-3’) (Cook et al. 2009). Virus cDNA was synthesized and amplified by thermocycler and carried out to 40 cycles at 55º C for 30 min, followed by 94º C for 2 min, 94º C for 15 s, 53º C for 45 s, 68º C for 45 s, 68º C for 5 min, and held at 20º C. The nested PCR was performed for 35 cycles at 95º C for 5 min, 94º C for 30 s, 55º C for 45 s, 72º C for 45 s, 72º C for 10 min and held at 20º C. After cycling, 5 μl of PCR product was run in a 2% agarose gel in TAE buffer strained with ethidium bromide, followed by visualization under UV light inside a GelDoc machine. The total number of collected mosquitoes was 1,124, consisting of 575 males and 549 females, representing seven genera, including Aedes aegypti (n=230), Ae. albopictus (n=123), Ae. desmotes (W-albus G) (n=1), Anopheles dirus (n=1), Armigeres spp. (n=71), Culex fuscocephala (n=15), Cx. gelidus (n=2), Cx. nigropunctatus (n=1), Cx. quinquefasciatus (n=662), Cx. vishnui (n=12), Ficalbia minima (n=2), Tripteroides spp. (n=1), and Uranotaenia spp. (n=3). The most abundant species on the island was Cx. quinquefasciatus (58.90% of total catch), followed by Ae. aegypti (20.46%) and Ae. albopictus (10.94%). Culex quinquefasciatus and Ae. aegypti were principally caught in an urban setting, while Ae. albopictus were abundant in peri-urban and forest areas. Less abundant mosquito species on the island were Ae. desmotes (W-albus G), An. dirus, Armigeres spp., Cx. fuscocephala, Cx. vishnui, Cx. gelidus, Cx. nigropunctatus, Fi. minima, Tripteroides spp., and Uranotaenia spp. (Table 1). Of the 549 collected mosquitoes, 144 were found to be blood engorged, consisting of 30 Ae. aegypti (20.8% of engorged mosquitoes), eight Ae. albopictus (5.6%), two Armigeres spp. (1.4%), 102 Cx. quinquefasciatus (70.8%) and two Cx. vishnui (1.4%). Of the 144 blood engorged mosquitoes 106 (73.6%) were identified, and 38 blood engorged mosquitoes (26.4%) were unidentified. Out of the 144 specimens, 107 were indoor collected mosquitoes (74.3%) and 37 were outdoor collected mosquitoes (25.7%). The majority of blood meals was of dog origin (36.1%), followed by unidentified hosts (26.4%), and mixed blood meals from humans and monkeys (23.61%). Feeding patterns among mosquitoes could be classified into three categories: single host, double hosts, and triple hosts. The proportion of blood meals from a single host of human, dog, and monkey origin was 0.69% (n=1), 36.11% (n=52) and 4.86% (n=7), respectively. Regarding mixed blood meals, the proportion of mixed human-monkey, human-dog, and monkey-dog blood meals was 23.61% (n=34), 1.39% (n=2) and 2.08% (n=3) respectively. The proportion of triple hosts (human-dog-monkey) was 4.86% (n=7) (Table 2). 30 (20.83%) 2 (6.67%) 4 (13.33%) 21 (70.0%) 3 (10.0%) 8 (5.56%) 7 (87.50%) 1 (12.50%) 2 (1.39%) 1 (50.0%) 1 (50.0%) 102 (70.83%) 1 (0.98%) 48 (47.06%) 3 (2.94%) 4 (3.92%) 2 (1.96%) 3 (2.94%) 7 (6.86%) 34 (33.33%) 2 (1.39%) 1 (50.0%) 1 (50.0%) 1 (0.69%) 5 (36.11%) 7 (4.86%) 2 (1.39%) 3 (2.08%) 7 (4.86%) Aedes aegypti took their blood meals of mixed blood mainly from humans and monkeys (70%), followed by monkeys (13.33%) and the remaining were unidentified or of dog origin. Aedes albopictus blood meals were from mixed hosts of humans and monkeys (87%). The blood meals of Armigeres spp. and Cx. vishnui were of mixed human-monkey (50%) and dog origin (50%). Culex quinquefasciatus mainly fed on dog (47.06%), followed by unidentified hosts (33.33%). In addition, seven Cx. quinquefasciatus took their blood meals from triple hosts: human, monkey, and dog, accounting for 6.8%. So far there has been no Flavivirus infection found in these wild-caught mosquito samples after confirmation by DNA sequencing. Our study showed that the most abundant species on the island were Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. We observed that Ae. aegypti and Cx. quinquefasciatus were more urban while Ae. albopictus were found more in rural settings. However, Ae. aegypti were less abundant in urban areas, particularly in resorts and hotels because resort or hotel owners use insecticide spray in order to avoid mosquito bites among tourists. From our study, we found that Ae. albopictus, the forest fringe species, were also collected from villages. It seems that Ae. albopictus are capable of adapting and living in residential areas by changing breeding sites from natural to artificial containers. Our result concurs with the study of Rattanarithikul and Panthusiri (1994) which found that female Ae. albopictus may deposit their eggs in a variety of artificial containers as well as natural containers. Similarly, Chareonviriyaphap et al. (2003) found that broken cans and plastic containers were primary breeding sites for Ae. albopictus during the dry season in Thailand. It seems that mosquito biodiversity is decreasing according to the gradient urban-rural, which concurs with the study conducted in Thailand by Thongsripong et al. (2013), concluding that mosquitoes collected from forest habitats were more diverse than those from residential areas. In this study, An. dirus was collected from a rubber plantation, probably because environmental conditions in rubber plantations, such as humidity, shade, and temperature may provide suitable breeding sites for Anopheles mosquitoes. This condition could also increase the density of Anopheles mosquitoes. In terms of health problems, research conducted in Southeast Asia reported that malaria was associated with rubber plantations (Patz et al. 2000). Workers in rubber plantations may be at risk of malaria infection because they work at night, which coincides with the feeding time of malaria vectors. However, this hypothesis needs to be confirmed by further study and mosquito collections should be conducted with the referenced and frequently used methods such as human landing catch, because the collected number of An. dirus in this study was very small. Although our study site, Koh Chang, is home to many kinds of animals, information regarding whether they serve as blood sources for mosquito vectors, and to what degree, is lacking. Therefore, in this study, blood meals of mosquitoes were tested against only human, monkey, and dog anti-sera, which were commonly observed during the survey. In the literature, it was indicated that the source of blood meals of Ae. aegypti was from several species but predominantly humans. For Ae. albopictus, their blood meals can vary among a wide variety of hosts including humans, and domestic and wild animals such as birds, cattle, cats, and dogs. However, its preferred hosts were mammals (Hawley 1988). The results of our study demonstrated for the first time that the blood source of Ae. aegypti and Ae. albopictus could be from mixed human-monkey, as well as a single blood meal from a monkey. Our unique findings are likely related to humans and monkeys living in close proximity to each other on Koh Chang, which, as part of a national park, is a typical eco-tourism site. The majority of Koh Chang is surrounded by evergreen forests where animals, including monkeys, are abundantly present. Deforestation for the construction of accommodations and infrastructure, driven by intensive development of tourism on the island as well as land clearing for agriculture and human settlement, disturbed the habitats of monkeys. These anthropological activities force monkeys to live in close contact with humans (Malaivijitnond and Hamada 2008). As a consequence, monkeys could be the main blood meal source for mosquitoes in the tourist settings surrounded with national parks. In terms of disease transmission, close contact between humans and monkeys can cause potential risk of disease because monkeys are important reservoir for various viruses. In Malaysia, CHIKV was first isolated from long-tailed macaques and was closely related to human CHIKV causing outbreak in 1998 and 2006 (Apandi et al. 2009). In the Philippines, Inoue et al. (2003) has reported that infections of JE, DENV, and CHIKV were prevalent among monkeys. Because the genetics and evolution of wild primates were closely related to humans, humans are probably susceptible to infections from these animals. As such, mixed blood-feeding behavior on humans and monkeys by Ae. aegypti and Ae. albopictus collected from Koh Chang may enhance pathogen transfer between hosts and become public health issues. However, blood meals of mosquitoes need to be further investigated by PCR, due to low specificity of ELISA, in order to specifically identify vertebrate species. In addition, some blood meals were from unidentified hosts that could be taken from other animals not included in this study. Thus, data about all wild life on the island is required for an accurate investigation of feeding patterns of mosquitoes. Furthermore, sampling size of wild-caught mosquitoes should be increased to confirm the results of our study and to better understand the role of monkeys and mosquitoes in zoonotic diseases transmission. In conclusion, our Koh Chang study has provided baseline information involving mosquito species that present on the island, their feeding pattern, and Flavivirus infection status. Our findings regarding species composition and blood meal sources of mosquitoes should be useful for planning prevention and control of vector-borne diseases, such as dengue and malaria, which have been reported regularly on this island. We thank the local administrative authorities and the local residents of Koh Chang for their full co-operation. This research work was supported by the IDRC/CIDA/AusAID/Global Health Research Initiative under the Ecohealth Emerging Infectious Diseases (EcoEID) Program Grant No. 105509 and Mahidol University.