Journal of Vector Ecology
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Scientific Note Dengue viruses in Aedes albopictus Skuse from a pineapple plantation in Costa Rica Olger Calderón-Arguedas1, Adriana Troyo1, Rolando D. Moreira-Soto1,2, Rodrigo Marín3, and Lizeth Taylor1* Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica, [email protected] 2 Centro de Investigación en Estructuras Microscópicas, Universidad de Costa Rica, San José, Costa Rica 3 Programa de Control de Vectores, Ministerio de Salud, San José, Costa Rica * Deceased
1
Aedes albopictus (Diptera: Culcidae) originated in Asia and has been associated with the biological transmission of various arboviruses including dengue (Lambrechts et al. 2010, Bonizzoni et al. 2013). Outbreaks of dengue in Japan, China, Taipei, Seychelles Islands, La Réunion Island, Malaldive Islands, and Hawaii have been attributed to Ae. albopictus as the only species responsible (Lambrechts et al. 2010). Aedes albopictus was introduced into continental America (Texas and Brazil) in the 1980s through imported tires and bamboo plants, and it has spread rapidly (Bonizzoni et al. 2013). In Costa Rica, Ae. albopictus was first reported in 1998; currently, it is widely distributed in the northeast area of the country, the Caribbean slope, and the Panama border (Calderon-Arguedas et al. 2012). Bromeliad plants, which include pineapples (Ananas comosus), offer conditions that are highly favorable for the oviposition and larval development of Ae. albopictus (Eapen et al. 2010). The pineapple industry is an important economic activity in Costa Rica, and there are large plantations in several areas of the country including the northeast and the Caribbean slope. In August 2012, local vector control personnel attended to a complaint of a high abundance of mosquitoes close to an organic pineapple plantation in the Caribbean region, specifically in the district of La Virgen, Sarapiquí County, Province of Heredia. Mosquitoes were identified preliminarily as Aedes albopictus. Considering that this is a dengue endemic area, the presence of dengue virus was evaluated in Ae. albopictus mosquitoes collected in proximity to this organic pineapple plantation. Adult mosquitoes were collected in the periphery of the organic pineapple plantation (10°26’01’’ N, 84°07’14’’ W) by vector control personnel as part of their response to the complaint of neighbors. This plantation has an active area of 400 hectares, approximately 250 field workers, and a small river that runs through it with forested areas surrounding the river. The site of collection was adjacent to the plantation and close to the main road, where approximately three houses are dispersed in an area of three hectares less than 50 m from the pineapple field. Two people from the local Ministry of Health’s vector control team used a backpack aspirator to collect mosquitoes that approached them, for approximately 30 min. Personnel also evaluated the pineapples in the plantation and confirmed that most of them contained water and numerous mosquito larvae, which were all identified as Ae. albopictus (Miguel Ramírez, personal observation). A total of 1,425 adult mosquitoes, preliminarily identified as Ae. albopictus, was collected. Identification of 700 mosquitoes was
confirmed using taxonomic identification keys (Rueda 2004) at the Tropical Diseases Research Center, University of Costa Rica, where mosquitoes were separated according to sex, and bodies and heads were sectioned and grouped into pools of 20 each. Thirty-five pools of bodies and the corresponding pools of heads were obtained, with 32 pools corresponding to females (p1 to p32) and three pools to males (p33 to p35). All pools of mosquito bodies were analyzed by retrotranscription polymerase chain reaction (RT-PCR) to detect dengue-specific RNA, but the pools of heads were analyzed only when the corresponding pools of bodies were RT-PCR positive for dengue. The content of each pool was macerated in sterile phosphate buffer solution, and genomic RNA was extracted from 100 µl of the macerate using the NucleoSpin RNA Virus kit (Macherey-Nagel), following the manufacturer’s instructions. Retrotranscription was carried out with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) employing 10 µl of the extraction product. For the subsequent PCR, primers 5’UTR-S and 5’UTR-C were used, which are highly specific and amplify a cDNA fragment of 120 bp present in the common dengue types (Aquino et al. 2006, Dos Santos et al. 2008). The positive control for each reaction was the DENV-1 strain Angola D1/AO/XX/1988 and the negative control was VERO E6 cells. To confirm the presence of dengue virus RNA and to determine dengue type in positive mosquito pools, sequencing was performed by first repeating the retrotranscription using the specific primers mentioned instead of the random hexamers. PCRs were performed with the same primers as mentioned above, products were purified with BigDye® XTerminator™ Purification Kit, sequenced with BigDyeTM Terminator v3.1, and visualized in the ABI PRISM 3130 DNA Analyzer (Applied Biosystems). Sequences were edited using DNA Baser software (Heracle BioSoft S.R.L.), and sequence homology searches were performed in the GenBank database using the Basic Local Alignment Search Tool (BLAST). Dengue virus RNA was detected in eight of 32 (25%) pools of Ae. albopictus female bodies and also in two pools of the corresponding female heads. One of three (33%) pools of male bodies also contained dengue virus RNA, although it was not detected in the corresponding pool of heads. The overall minimum infection rate was 12.86 (nine positive pools/700 mosquitoes x 1,000). Sequencing suggested the presence of dengue virus RNA in at least five of the PCR positive pools. Despite several sequencing attempts, two of these mosquito pools generated only one small
Vol. 40, no. 1
Journal of Vector Ecology
fragment each (70 bp and 23 bp). Although these fragments were 100% homologous to the DENV-1 sequence with accession number AY277666, their identity cannot be confirmed due to the small size. The amplicons of female pools p24 (heads), p29 (bodies), and p30 (bodies) generated useful sequences with both primers. In these cases, primer 5’UTR-S yielded longer sequence fragments than expected, but they were of very good quality (QV greater than 21, well-spaced peaks, no noise, and no overlapping). When compared against the GenBank database, the sequence of p24 had the highest homology with sequences of DENV-1, specifically accession numbers KJ189347 (99.5%, 611/614) and HQ332179 (98.9%, 622/629) from Mexico and Venezuela, respectively. The dengue sequence in p29 was most similar to DENV-2 sequences from French Guiana and Cuba, with accession numbers EU920828 (99.4, 623/627) and AY702038 (99.2%, 622/627), while the fragment in p30 had higher homology with DENV-4 accession numbers AF326573 (99.5%, 587/590) and AF375822 (99.5%, 587/590) from Dominica. GenBank accession numbers of sequences obtained from Ae. albopictus pools p24, p29, and p30 are: KJ534633 (DENV-1), KJ534634 (DENV-2), and KJ534635 (DENV-4). The presence of dengue RNA in these three pools was verified by performing the RT-PCR according to the method described by Lanciotti et al (1992). PCR products corresponding to DENV-1 (482 bp), DENV-2 (119 bp), and DENV-4 (392 bp) were obtained from pools p24, p29, and p30, respectively. The role of Ae. albopictus as a vector of dengue virus has been known since the 1960s, when the virus was first isolated from this mosquito species (Rudnick and Chan 1965). Historically, Ae. albopictus was probably the first vector responsible for human transmission of dengue viruses, which seems to have occurred in Asia before the introduction of Aedes aegypti (Halstead 2008). In the American continent, its role is obscure due to the wide distribution of the main vector, Ae. aegypti (Martins et al. 2012). However, studies in Brazil, Mexico, and Colombia have demonstrated natural infection of Ae. albopictus with dengue virus and the occurrence of vertical transmission (Serufo et al. 1993, Ibanez-Bernal et al. 1997, Méndez et al. 2006, Cecílio et al. 2009, Martins et al. 2012). Although Ae. albopictus has been considered less competent than Ae. aegypti (Lambrechts et al. 2010), strains of Ae. albopictus from America have shown high infection and transmission rates in experimental conditions, in some cases even higher than Ae. aegypti (Castro et al. 2004, Buckner et al. 2013). The presence of dengue viruses in field-collected Ae. albopictus associated with an organic pineapple plantation in Costa Rica suggests that there may be transmission or permanence of the virus in this mosquito population. The high mosquito abundance may be the consequence of a favorable environment for oviposition and larval development created by a concentration of pineapple plants (Eapen et al. 2010). Although Ae. albopictus may bite other vertebrates in the absence of humans, it is considered anthropophilic (Lambrechts et al. 2010). In this case, there are very few human residents near the plantation, and the main source of human blood within the pineapple field would be from plantation workers. In these scenarios, uninfected females may be in contact with viremic humans and become infected with dengue virus. It is important to mention that pools of mosquito bodies containing dengue, but without dengue in their corresponding heads, may
185
mean that dissemination has not occurred yet, and the virus is restricted to the gut. When positive bodies and heads are present, this indicates that the virus has disseminated into the hemolymph, which is the step preceding viral establishment in salivary glands (Salazar et al. 2007). It is also possible that some mosquitoes may already be infected through vertical transmission, since results suggest that one pool of male bodies contained dengue virus. Studies have demonstrated that vertical transmission of dengue virus occurs naturally in Ae. albopictus (Cecílio et al. 2009), and it is more efficient than in Ae. aegypti (Lambrechts et al. 2010). If the virus is efficiently transmitted to the progeny in this population of Ae. albopictus, the virus may persist during inter-epidemic periods. Moreover, the presence of Ae. aegypti in the adjacent human environments would be enough to maintain transmission in humans and at least provide uninfected Ae. albopictus with viremic bloodmeals. Considering that the dynamics of dengue transmission in proximity to organic pineapple plantations is unknown, further ecological and epidemiological investigations are required in these areas where densities of Ae. albopictus may be high. DENV-1, DENV-2, and DENV-4 were identified in pools of Ae. albopictus, although DENV-4 had not been detected in Costa Rica by the national dengue surveillance system. According to the official information for the year 2012 (when the Ae. albopictus mosquitoes were collected), Costa Rica only reported DENV-1, DENV-2, and DENV-3 transmission (Ministry of Health 2014). Therefore, surveillance based only on human cases may not reflect the presence of other serotypes in the country and the potential for outbreaks. This reinforces the idea that viral detection in mosquitoes should be a fundamental part of routine surveillance activities in endemic countries. Acknowledgments The authors thank José Valle Arguedas and Miguel Ramírez Alpízar for mosquito collections, Carlos Vargas for technical assistance in the processes of RNA extraction and RT-PCR, as well as Silvia Quesada, Camila Boniche, and Katherine Salazar for their collaboration in preparing mosquito pools. Financial support was provided in part by Universidad de Costa Rica projects ED-548 and ED-2755. REFERENCES CITED Aquino, V.H., E. Anatriello, P.F. Goncalves, E.V. Da Silva, P.F. Vasconcelos, D.S. Vierir, W.C. Batista, M.L. Bobadilla, C. Váquez, M. Mora, and L.T. Figueiredo. 2006. Molecular epidemiology of dengue type 3 virus in Brazil and Paragua, 2002-2004. Am. J. Trop. Med. Hyg. 75: 710-715. Bonizzoni, M., G. Gasperi, X. Chen, and A.A. James. 2013. The invasive mosquito species Aedes albopictus: current knowledge and future perspectives. Trends Parasitol. 29: 460468. Buckner, E.A., B.W. Alto, and L.P. Lounibos. 2013. Vertical transmission of Key West dengue-1 virus by Aedes aegypti and Aedes albopictus (Diptera: Culicidae) mosquitoes from Florida. J. Med. Entomol. 50: 1291-1297.
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Calderón-Arguedas, O., A. Troyo, A. Avendaño, and M. Gutiérrez. 2012. Aedes albopictus (Skuse) en la Región Huetar Atlántica de Costa Rica. Rev. Costarric. Salud Pública 21: 76-80. Castro, M.G., R.M. Nogueira, H.G. Schatzmayr, M.P. Miagostovich, and R. Lourenço-de-Oliveira. 2004. Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil. Mem. Inst. Oswaldo Cruz 99: 809-814. Cecílio, A.B., E.S. Campanelli, K.P. Souza, L.B. Figueiredo, and M.C. Resende. 2009. Natural vertical transmission by Stegomyia albopicta as dengue vector in Brazil. Braz. J. Biol. 69: 123-127. Dos Santos, H.W., T.R. Poloni, K.P. Souza, V.D. Muller, F. Tremeschin, L.C. Nali, L.R. Fantinatti, A.A. Amarilla, H.L. Castro, M.R. Nunes, S.M. Casseb, P.F. Vasconcelos, S.J. Badra, L.T. Figueiredo, and V.H. Aquino. 2008. A simple one-step real-time RT-PCR for diagnosis of dengue virus infection. J. Med. Virol. 80:1426-33. Eapen, A., K.J. Ravindran, and A.P. Dash. 2010. Breeding potential of Aedes albopictus (Skuse, 1895) in chikungunya affected areas of Kerala, India. Indian J. Med. Res. 132: 733-735. Halstead, S.B. 2008. Dengue: Overview and history. In: S.B. Halstead (ed.). Dengue. pp. 1-28. Imperial College Press. London. Ibáñez-Bernal, S., B. Briseño, J.P. Mutebi, E. Argot, G. Rodríguez, C. Martínez-Campos, R. Paz, P. de la Fuente-San Román, R. Tapia-Conyer, and A. Flisser. 1997. First record in America of Aedes albopictus naturally infected with dengue virus during the 1995 outbreak at Reynosa, Mexico. Med. Vet. Entomol. 11: 305-309. Lambrechts, L., T.W. Scott, and D.J. Gubler. 2010. Consequences of the expanding global distribution of Aedes albopictus for dengue virus transmission. PLoS Negl. Trop. Dis. 25: e646.
June 2015
Lanciotti, R.S., C.H. Calisher, D.J. Gubler, G.J. Chang, and A.V. Vorndam. 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptasepolymerase chain reaction. J. Clin. Microbiol. 30: 545-551. Martins, V.E., C.H. Alencar, M.T. Kamimura, F.M. de Carvalho Araújo, S.G. De Simone, R.F. Dutra, and M.I. Guedes. 2012. Occurrence of natural vertical transmission of dengue-2 and dengue-3 viruses in Aedes aegypti and Aedes albopictus in Fortaleza, Ceará, Brazil. PLoS One 7: e41386. Méndez, F., M. Barreto, J.F. Arias, G. Rengifo, J. Muñoz, M.E. Burbano, and B. Parra. 2006. Human and mosquito infections by dengue viruses during and after epidemics in a dengueendemic region of Colombia. Am. J. Trop. Med. Hyg. 74: 678683. Ministry of Health (Costa Rica). 2014. Situación del dengue 2013. Ministerio de Salud, Costa Rica. Available from: http://www. ministeriodesalud.go.cr/index.php/vigilancia-de-la-salud/ inicio-vigilancia-analisis-situacion-salud-ms [cited July 30, 2014]. Rudnick, A. and C. Chan. 1965. Dengue Type 2 Virus in naturally infected Aedes albopictus mosquitoes in Singapore. Science 149: 638-639. Rueda, L.M. 2004. Pictorial keys for the identification of mosquitoes (Diptera: Culicidae) associated with dengue virus transmission. Zootaxa 589: 1-60. Salazar, M.I., J.H. Richardson, I. Sánchez-Vargas, K.E. Olson, and B.J. Beaty. 2007. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes. BMC Microbiol. 7: 9. Serufo, J.C., H. Montes de Oca, V.A. Tavares, A.M. Souza, R.V. Rosa, M.C. Jamal, J.R. Lemos, M.A. Oliveira, R.M.R. Nogueira, and H.G. Schatzmayr. 1993. Isolation of dengue virus type 1 from larvae of Aedes albopictus in Campos Altos City, State of Minas Gerais, Brazil. Mem. Inst. Oswaldo Cruz 88: 503-504.
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Scientific Note Dengue viruses in Aedes albopictus Skuse from a pineapple plantation in Costa Rica Olger Calderón-Arguedas1, Adriana Troyo1, Rolando D. Moreira-Soto1,2, Rodrigo Marín3, and Lizeth Taylor1* Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica, [email protected] 2 Centro de Investigación en Estructuras Microscópicas, Universidad de Costa Rica, San José, Costa Rica 3 Programa de Control de Vectores, Ministerio de Salud, San José, Costa Rica * Deceased
1
Aedes albopictus (Diptera: Culcidae) originated in Asia and has been associated with the biological transmission of various arboviruses including dengue (Lambrechts et al. 2010, Bonizzoni et al. 2013). Outbreaks of dengue in Japan, China, Taipei, Seychelles Islands, La Réunion Island, Malaldive Islands, and Hawaii have been attributed to Ae. albopictus as the only species responsible (Lambrechts et al. 2010). Aedes albopictus was introduced into continental America (Texas and Brazil) in the 1980s through imported tires and bamboo plants, and it has spread rapidly (Bonizzoni et al. 2013). In Costa Rica, Ae. albopictus was first reported in 1998; currently, it is widely distributed in the northeast area of the country, the Caribbean slope, and the Panama border (Calderon-Arguedas et al. 2012). Bromeliad plants, which include pineapples (Ananas comosus), offer conditions that are highly favorable for the oviposition and larval development of Ae. albopictus (Eapen et al. 2010). The pineapple industry is an important economic activity in Costa Rica, and there are large plantations in several areas of the country including the northeast and the Caribbean slope. In August 2012, local vector control personnel attended to a complaint of a high abundance of mosquitoes close to an organic pineapple plantation in the Caribbean region, specifically in the district of La Virgen, Sarapiquí County, Province of Heredia. Mosquitoes were identified preliminarily as Aedes albopictus. Considering that this is a dengue endemic area, the presence of dengue virus was evaluated in Ae. albopictus mosquitoes collected in proximity to this organic pineapple plantation. Adult mosquitoes were collected in the periphery of the organic pineapple plantation (10°26’01’’ N, 84°07’14’’ W) by vector control personnel as part of their response to the complaint of neighbors. This plantation has an active area of 400 hectares, approximately 250 field workers, and a small river that runs through it with forested areas surrounding the river. The site of collection was adjacent to the plantation and close to the main road, where approximately three houses are dispersed in an area of three hectares less than 50 m from the pineapple field. Two people from the local Ministry of Health’s vector control team used a backpack aspirator to collect mosquitoes that approached them, for approximately 30 min. Personnel also evaluated the pineapples in the plantation and confirmed that most of them contained water and numerous mosquito larvae, which were all identified as Ae. albopictus (Miguel Ramírez, personal observation). A total of 1,425 adult mosquitoes, preliminarily identified as Ae. albopictus, was collected. Identification of 700 mosquitoes was
confirmed using taxonomic identification keys (Rueda 2004) at the Tropical Diseases Research Center, University of Costa Rica, where mosquitoes were separated according to sex, and bodies and heads were sectioned and grouped into pools of 20 each. Thirty-five pools of bodies and the corresponding pools of heads were obtained, with 32 pools corresponding to females (p1 to p32) and three pools to males (p33 to p35). All pools of mosquito bodies were analyzed by retrotranscription polymerase chain reaction (RT-PCR) to detect dengue-specific RNA, but the pools of heads were analyzed only when the corresponding pools of bodies were RT-PCR positive for dengue. The content of each pool was macerated in sterile phosphate buffer solution, and genomic RNA was extracted from 100 µl of the macerate using the NucleoSpin RNA Virus kit (Macherey-Nagel), following the manufacturer’s instructions. Retrotranscription was carried out with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) employing 10 µl of the extraction product. For the subsequent PCR, primers 5’UTR-S and 5’UTR-C were used, which are highly specific and amplify a cDNA fragment of 120 bp present in the common dengue types (Aquino et al. 2006, Dos Santos et al. 2008). The positive control for each reaction was the DENV-1 strain Angola D1/AO/XX/1988 and the negative control was VERO E6 cells. To confirm the presence of dengue virus RNA and to determine dengue type in positive mosquito pools, sequencing was performed by first repeating the retrotranscription using the specific primers mentioned instead of the random hexamers. PCRs were performed with the same primers as mentioned above, products were purified with BigDye® XTerminator™ Purification Kit, sequenced with BigDyeTM Terminator v3.1, and visualized in the ABI PRISM 3130 DNA Analyzer (Applied Biosystems). Sequences were edited using DNA Baser software (Heracle BioSoft S.R.L.), and sequence homology searches were performed in the GenBank database using the Basic Local Alignment Search Tool (BLAST). Dengue virus RNA was detected in eight of 32 (25%) pools of Ae. albopictus female bodies and also in two pools of the corresponding female heads. One of three (33%) pools of male bodies also contained dengue virus RNA, although it was not detected in the corresponding pool of heads. The overall minimum infection rate was 12.86 (nine positive pools/700 mosquitoes x 1,000). Sequencing suggested the presence of dengue virus RNA in at least five of the PCR positive pools. Despite several sequencing attempts, two of these mosquito pools generated only one small
Vol. 40, no. 1
Journal of Vector Ecology
fragment each (70 bp and 23 bp). Although these fragments were 100% homologous to the DENV-1 sequence with accession number AY277666, their identity cannot be confirmed due to the small size. The amplicons of female pools p24 (heads), p29 (bodies), and p30 (bodies) generated useful sequences with both primers. In these cases, primer 5’UTR-S yielded longer sequence fragments than expected, but they were of very good quality (QV greater than 21, well-spaced peaks, no noise, and no overlapping). When compared against the GenBank database, the sequence of p24 had the highest homology with sequences of DENV-1, specifically accession numbers KJ189347 (99.5%, 611/614) and HQ332179 (98.9%, 622/629) from Mexico and Venezuela, respectively. The dengue sequence in p29 was most similar to DENV-2 sequences from French Guiana and Cuba, with accession numbers EU920828 (99.4, 623/627) and AY702038 (99.2%, 622/627), while the fragment in p30 had higher homology with DENV-4 accession numbers AF326573 (99.5%, 587/590) and AF375822 (99.5%, 587/590) from Dominica. GenBank accession numbers of sequences obtained from Ae. albopictus pools p24, p29, and p30 are: KJ534633 (DENV-1), KJ534634 (DENV-2), and KJ534635 (DENV-4). The presence of dengue RNA in these three pools was verified by performing the RT-PCR according to the method described by Lanciotti et al (1992). PCR products corresponding to DENV-1 (482 bp), DENV-2 (119 bp), and DENV-4 (392 bp) were obtained from pools p24, p29, and p30, respectively. The role of Ae. albopictus as a vector of dengue virus has been known since the 1960s, when the virus was first isolated from this mosquito species (Rudnick and Chan 1965). Historically, Ae. albopictus was probably the first vector responsible for human transmission of dengue viruses, which seems to have occurred in Asia before the introduction of Aedes aegypti (Halstead 2008). In the American continent, its role is obscure due to the wide distribution of the main vector, Ae. aegypti (Martins et al. 2012). However, studies in Brazil, Mexico, and Colombia have demonstrated natural infection of Ae. albopictus with dengue virus and the occurrence of vertical transmission (Serufo et al. 1993, Ibanez-Bernal et al. 1997, Méndez et al. 2006, Cecílio et al. 2009, Martins et al. 2012). Although Ae. albopictus has been considered less competent than Ae. aegypti (Lambrechts et al. 2010), strains of Ae. albopictus from America have shown high infection and transmission rates in experimental conditions, in some cases even higher than Ae. aegypti (Castro et al. 2004, Buckner et al. 2013). The presence of dengue viruses in field-collected Ae. albopictus associated with an organic pineapple plantation in Costa Rica suggests that there may be transmission or permanence of the virus in this mosquito population. The high mosquito abundance may be the consequence of a favorable environment for oviposition and larval development created by a concentration of pineapple plants (Eapen et al. 2010). Although Ae. albopictus may bite other vertebrates in the absence of humans, it is considered anthropophilic (Lambrechts et al. 2010). In this case, there are very few human residents near the plantation, and the main source of human blood within the pineapple field would be from plantation workers. In these scenarios, uninfected females may be in contact with viremic humans and become infected with dengue virus. It is important to mention that pools of mosquito bodies containing dengue, but without dengue in their corresponding heads, may
185
mean that dissemination has not occurred yet, and the virus is restricted to the gut. When positive bodies and heads are present, this indicates that the virus has disseminated into the hemolymph, which is the step preceding viral establishment in salivary glands (Salazar et al. 2007). It is also possible that some mosquitoes may already be infected through vertical transmission, since results suggest that one pool of male bodies contained dengue virus. Studies have demonstrated that vertical transmission of dengue virus occurs naturally in Ae. albopictus (Cecílio et al. 2009), and it is more efficient than in Ae. aegypti (Lambrechts et al. 2010). If the virus is efficiently transmitted to the progeny in this population of Ae. albopictus, the virus may persist during inter-epidemic periods. Moreover, the presence of Ae. aegypti in the adjacent human environments would be enough to maintain transmission in humans and at least provide uninfected Ae. albopictus with viremic bloodmeals. Considering that the dynamics of dengue transmission in proximity to organic pineapple plantations is unknown, further ecological and epidemiological investigations are required in these areas where densities of Ae. albopictus may be high. DENV-1, DENV-2, and DENV-4 were identified in pools of Ae. albopictus, although DENV-4 had not been detected in Costa Rica by the national dengue surveillance system. According to the official information for the year 2012 (when the Ae. albopictus mosquitoes were collected), Costa Rica only reported DENV-1, DENV-2, and DENV-3 transmission (Ministry of Health 2014). Therefore, surveillance based only on human cases may not reflect the presence of other serotypes in the country and the potential for outbreaks. This reinforces the idea that viral detection in mosquitoes should be a fundamental part of routine surveillance activities in endemic countries. Acknowledgments The authors thank José Valle Arguedas and Miguel Ramírez Alpízar for mosquito collections, Carlos Vargas for technical assistance in the processes of RNA extraction and RT-PCR, as well as Silvia Quesada, Camila Boniche, and Katherine Salazar for their collaboration in preparing mosquito pools. Financial support was provided in part by Universidad de Costa Rica projects ED-548 and ED-2755. REFERENCES CITED Aquino, V.H., E. Anatriello, P.F. Goncalves, E.V. Da Silva, P.F. Vasconcelos, D.S. Vierir, W.C. Batista, M.L. Bobadilla, C. Váquez, M. Mora, and L.T. Figueiredo. 2006. Molecular epidemiology of dengue type 3 virus in Brazil and Paragua, 2002-2004. Am. J. Trop. Med. Hyg. 75: 710-715. Bonizzoni, M., G. Gasperi, X. Chen, and A.A. James. 2013. The invasive mosquito species Aedes albopictus: current knowledge and future perspectives. Trends Parasitol. 29: 460468. Buckner, E.A., B.W. Alto, and L.P. Lounibos. 2013. Vertical transmission of Key West dengue-1 virus by Aedes aegypti and Aedes albopictus (Diptera: Culicidae) mosquitoes from Florida. J. Med. Entomol. 50: 1291-1297.
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Calderón-Arguedas, O., A. Troyo, A. Avendaño, and M. Gutiérrez. 2012. Aedes albopictus (Skuse) en la Región Huetar Atlántica de Costa Rica. Rev. Costarric. Salud Pública 21: 76-80. Castro, M.G., R.M. Nogueira, H.G. Schatzmayr, M.P. Miagostovich, and R. Lourenço-de-Oliveira. 2004. Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil. Mem. Inst. Oswaldo Cruz 99: 809-814. Cecílio, A.B., E.S. Campanelli, K.P. Souza, L.B. Figueiredo, and M.C. Resende. 2009. Natural vertical transmission by Stegomyia albopicta as dengue vector in Brazil. Braz. J. Biol. 69: 123-127. Dos Santos, H.W., T.R. Poloni, K.P. Souza, V.D. Muller, F. Tremeschin, L.C. Nali, L.R. Fantinatti, A.A. Amarilla, H.L. Castro, M.R. Nunes, S.M. Casseb, P.F. Vasconcelos, S.J. Badra, L.T. Figueiredo, and V.H. Aquino. 2008. A simple one-step real-time RT-PCR for diagnosis of dengue virus infection. J. Med. Virol. 80:1426-33. Eapen, A., K.J. Ravindran, and A.P. Dash. 2010. Breeding potential of Aedes albopictus (Skuse, 1895) in chikungunya affected areas of Kerala, India. Indian J. Med. Res. 132: 733-735. Halstead, S.B. 2008. Dengue: Overview and history. In: S.B. Halstead (ed.). Dengue. pp. 1-28. Imperial College Press. London. Ibáñez-Bernal, S., B. Briseño, J.P. Mutebi, E. Argot, G. Rodríguez, C. Martínez-Campos, R. Paz, P. de la Fuente-San Román, R. Tapia-Conyer, and A. Flisser. 1997. First record in America of Aedes albopictus naturally infected with dengue virus during the 1995 outbreak at Reynosa, Mexico. Med. Vet. Entomol. 11: 305-309. Lambrechts, L., T.W. Scott, and D.J. Gubler. 2010. Consequences of the expanding global distribution of Aedes albopictus for dengue virus transmission. PLoS Negl. Trop. Dis. 25: e646.
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