New Record of Wyeomyia mitchellii (Diptera: Culicidae) on Guam, United States

New Record of Wyeomyia mitchellii (Diptera: Culicidae) on Guam, United States Abstract Wyeomyia (Wyeomyia) mitchellii (Theobald) (Diptera: Culicidae) was recovered for the first time on Guam, United States of America, in 2017. Larval specimens were collected from water-filled axils of bromeliads during a larval survey carried out in a residential neighborhood of the Chalan Pago/Ordot area. Native to the New World, Wy. mitchellii has likely made its way to the Pacific Islands through the possibly illegal import of ornamental bromeliads. While this mosquito does not represent a significant threat to public health, this finding highlights the vulnerability of the Pacific Islands to the introduction of exotic species, including mosquito species that may increase public health risks. mosquito, Wyeomyia mitchellii, exotic species, biosecurity, Pacific The international spread of mosquitoes poses a potentially significant threat to public health as competent vectors of pathogens may be introduced to areas where they are currently absent. Understanding pathways of introduction and maintaining effective quarantine surveillance strategies is critical. Much of the Pacific has experienced an unprecedented level of mosquito-borne activity in recent years (Roth et al. 2014). For instance, in 2016 through 2017 alone, the U.S.-Affiliated Pacific Islands (USAPIs), saw outbreaks of mosquito-borne disease, including transmission of dengue (DENV) and Zika (ZIKV), in the Republic of Palau, the Republic of Marshall Islands (RMI), American Samoa, and in the Federated States of Micronesia (FSM) (Whelan et al. 2016). The spread of Aedes (Stegomyia) aegypti (Linnaeus) (Diptera: Culicidae) across the Pacific is likely a driving factor in increasing outbreak risk, as the distribution of this mosquito is influenced primarily by human activity (Dupont-Rouzeyrol et al. 2014). Thus, surveillance of mosquito introductions to new regions is critical to reducing mosquito outbreak risk. Guam is a tropical island in the North Pacific Ocean (13° 16ʹ 48.0ʺ N: 144° 28ʹ 12.0ʺ E). The island is an organized, unincorporated territory of the United States, and holds policy associations with the United States under the jurisdiction of the Office of Insular Affairs (U.S. Department of Interior). With a land area of 544 km2, Guam is the largest and southernmost island of the Mariana Islands archipelago (Rueda et al. 2011). The north of the island is a forested coral limestone plateau and is densely populated, whereas the south of the island is chiseled with volcanic mountain peaks and is less populated. The climate is tropical marine with very little seasonal temperature variation. The mean high temperature is 31°C, and the mean low temperature is 25°C. Daily precipitation averages 7.8 mm (National Weather Service Forecast Unit 2014). These climatic conditions, combined with a prevalence of suitable habitats, provide ideal conditions for a wide range of mosquito species on Guam year-round. Mosquito-borne disease on Guam has been a concern of local authorities since the early 1900s. As stated in the prewar government handbook of Guam, mosquito-borne diseases were not a recorded issue on the island, and Navy personnel claimed that although present, mosquitoes posed no health threat to the inhabitants of Guam (Cox et al. 1926, Haddock 2010). However, mosquito-borne diseases became a reality on Guam in the years following the American reoccupation of Guam, when Spain ceded Guam to the United States through the Treaty of Paris (1899) (Nowell 1987), and massive movements of humans and goods from other regions of the Pacific where mosquito-borne diseases are endemic took place (Nowell 1987, Haddock 2010). Indeed, the first recorded mosquito species on Guam was in 1905, when J.F. Leys disclosed to the Surgeon General, U.S. Navy, that Stegomyia fasciata (Ae. aegypti) was well-established on the island. In 1936, the first extensive pre-World War II mosquito survey on Guam was conducted by Swezey (1942), in which four mosquito species were collected, all of which belonged to Aedes and Culex. By 1945, 10 mosquito species had been recorded on Guam, none of which were anophelines (Nowell 1980). In 1944, Guam saw its first dengue outbreak with transmission most likely driven by Ae. aegypti. Three years later, Guam faced its first Japanese encephalitis (JEV) outbreak, likely transmitted by the inferred vector species Culex (Culex) annulirostris (Skuse) (Diptera: Culicidae) (Haddock 2010). Today, the main mosquito-borne pathogen threats to Guam are DENV, chikungunya (CHIKV), JEV, yellow fever (YFV), and ZIKV viruses, as well as malaria and filariasis parasites (Rozeboom and Bridges 1972, Rueda et al. 2011). Since the elimination of Ae. aegypti from Guam after World War II and the Vietnam War, the sporadic cases of dengue and malaria on Guam have thus far been nonendemic; all known cases being associated with returning travelers from endemic countries. As of 2016, 24 cases of travel-related dengue have been reported on Guam in the last decade (Guam DPHSS 2017). A wide range of mosquito species have been recorded on, or have been suspected of being present, on Guam (Nowell 1987). However, since the end of World War II, mosquito surveys on Guam have been sporadic. Rueda et al (2011) summarize these post-World War II mosquito assessments. Such surveys demonstrate that the mosquito fauna of Guam changes at an alarming rate, likely as a result from high military traffic, incoming tourists, and island hopping (Rueda et al. 2011). Indeed, although the number of mosquito species on Guam varied from 18 to 42 (Reisen et al. 1972), by the end of 1983, it was established that Guam was home to 24 mosquito species, 17 of which were introduced species (Nowell 1987). This list includes Ae. aegypti because although considered eliminated from Guam, the prevalence of Ae. aegypti is widespread across many neighboring countries. Thus, due to the potential for this mosquito to be transported with human activity, there is a considerable risk of importing Ae. aegypti onto Guam. Out of these mosquito species on Guam, nine are Aedes species, five are Anopheles species, and six are Culex species. Therefore, there is an urgent need to establish mosquito surveillance to ensure any importation of this mosquito is met with a strategic control response. No mosquito surveys from Guam have been published since 2011. While both Santa Rita Navy base and Andersen Air Force base conduct irregular mosquito surveillance on the island, these activities are confined to the base enclosures, and thus, caution must be taken when extrapolating mosquito data from the military bases to the entire island of Guam. Recently, the Guam Division of Environmental Health (DEH), Department of Public Health and Social Services (DPHSS) saw its new Guam Environmental Public Health Laboratory (GEPHL) open in 2017. With the support of Pacific Islands Health Officers Association (PIHOA), mosquito collecting activities on Guam have led to the recent recovery of a previously unreported species on Guam, Wyeomyia mitchellii (Theobald) (Diptera: Culicidae). Surveys of various natural and artificial water-holding containers were undertaken at various locations throughout Guam with any immature mosquitoes collected returned to GEPHL for identification. On 29 March 2017, at least seven larvae were collected from a stand of Aechmea blanchetiana (Baker) bromeliads (Poales: Bromeliacae) in a residential area along Santa Cruz Drive, in Chalan Pago/Ordot municipality (13°26ʹ 16.9ʺ N: 144°46ʹ 07.7ʺ E). Larvae were returned to GEPHL and reared to adult stage in biological rearing chambers placed in an environmental chamber (27.5°C) and provided fish food. All larvae that were collected survived to the adult stage. Adults (approximately 24-h old) were then freeze-killed for identification. Initial identification of specimens was undertaken using the keys of Bohart (1956) and Ramalingam (1976). Among specimens of species known to exist on Guam, such as Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae), Aedes (Stegomyia) guamensis Farner and Bohart (Diptera: Culicidae), Culex (Culex) quinquefasciatus Say (Diptera: Culicidae), and Culex sitiens Wiedemann species group (Diptera: Culicidae), a small number of adult mosquito specimens appeared to be distinct from common mosquitoes collected in Guam based on morphological examination. However, their identification could not be formally resolved using taxonomic keys available for the region. Based on concerns that the specimens may be of an introduced species, the specimens underwent further taxonomic and molecular analysis. Voucher specimens were mounted and placed in the GEPHL’s reference collection. Using the online interactive keys of the Walter Reed Biosystematics Unit (WRBU) [http://www.wrbu.org/VecID_MQ.html], the specimens were strongly believed to be of the genus Wyeomyia based on the presence of mesopostnotal setae, the presence of prespiracular setae, and the presence of a long basal spur on the wing Rs vein. Further reference to Darsie and Ward (1981), and Clark-Gil and Darsie (1983), confirmed the identification as Wyeomyia subgenus Wyeomyia with it most likely being Wy. mitchellii or a closely related species. To confirm the identification based on morphological characteristics, molecular analysis of a single specimen was undertaken. DNA was extracted from two legs of the mosquito by means of magnetic particle separation using the EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany). The legs were homogenized by shaking for 1 h using glass beads and the mosquito grind pretreated with proteinase K at 56°C for another hour. Four hundred microlitres of the homogenate was extracted on the BioRobot EZ1 according to the manufacturer’s instructions. A 1,345-bp segment of the mitochondrial coding gene encompassing the COII and tRNA-Leucine region was amplified with the primers C1-J-1718 (5ʹ-GGN GGA TTT GGA AAT TGA TTA GTN CC-3ʹ) and TL2-N-3014 (5ʹ-TCC ATT GCA CTA ATC TGC CAT ATT-3ʹ) (Simon et al. 1994), respectively. The fragment was amplified by real-time PCR on the Rotogene 6000 (Qiagen) in a total volume of 35 µl containing 8 µl of DNA template, 400 nM of each primer, 1× of 20-fold EvaGreen (Biotium, Fremont, California), 1× MyTaq HS Mix (Bioline, London, United Kingdom), and RNase- and DNAse-free water (Sigma–Aldrich, St. Louis, Missouri). The thermal cycling commenced with enzyme activation (95°C for 2 min), followed by 42 cycles of denaturation (95°C for 18 s), annealing (touch-down 63–53°C for 20 s for 10 cycles, followed by 22 cycles at 53°C), extension (72°C for 30 s), and a final extension at 72°C for 2 min. A pre-hold cycle was set at 50°C for 30 s followed by a melt cycle with ramping temperatures between 75°C and 95°C to serve as a check on purity of the amplified product. The amplicons were verified on a 2% agarose gel. The PCR amplicons were pre-treated with Illustra ExoProStar 1-Step (GE Healthcare, Little Chalfont, United Kingdom) and sent to the Australian Genome Research Facility (AGRF), Sydney, for bi-directional Sanger sequencing using the ABI BigDye Terminator chemistry Version 3.1. The sequenced products were aligned using Geneious 6.0.6 and the consensus sequence (1,221 bp) identified by matching against nucleotide sequences on the National Centre for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) database. The sequence with the highest alignment score, percentage identity and the lowest expect value (E value) of all alignments from the sequence database was considered to be the best match to the query sequence. The sequence (GenBank MF950916) matched known records of Wy. mitchellii, GenBank HM136827, with an alignment score of 2,150, identity of 99 % and E-value of 0.0. Wy. mitchellii has been reported from a range of tropical and subtropical regions of the Americas, such as South Florida, the Greater Antilles, and eastern Mexico (Frank and Fish 2008). The species was initially described from specimens collected in Jamaica. Since the 1980s, the mosquito has been detected outside of its native range in a number of locations across the Pacific including the Hawaiian Islands (Shroyer 1981, Yang et al. 2003) as well as the Society Islands, French Polynesia (Marie and Bossin 2013). These discoveries in the Pacific raise concerns regarding pathways of introduction of other container-inhabiting mosquitoes between countries. Wy. mitchellii is closely associated with natural water holding plants, such as bromeliads and other aroid species where water collects in leaf axils (Shroyer 1981, Frank 1986). Adult mosquitoes lay eggs in these water-impounded leaf axils, typically during the late afternoon (Frank et al. 1985). Once hatched, the larvae require at least 2 wk to complete development, but larvae may take many more weeks to develop, especially when there is minimal organic material in habitats to support larvae development (Frank 1986, Frank and Fish 2008). This ability to survive extended periods of starvation may increase their capacity to be transported in bromeliads moved between locations where naturally accumulating organic material may be minimal. An illegal trade in ornamental bromeliads is suspected to have facilitated the movement of Wy. mitchellii from its native range to the Pacific Islands. In fact, it was proposed that movement of bromeliads from Hawai’i was the likely pathway of Wy. mitchellii into French Polynesia (Marie and Bossin 2013) and it is likely that a similar process facilitated the movement of the mosquito into Guam. It is not possible to know when the mosquito was first introduced on the island, but given the distinctiveness of the mosquito compared to those species known from Guam, it is considered highly unlikely that the specimen would have been overlooked in previous investigations. Additionally, it has not been collected at any other location on Guam during 2017 habitat surveys. Although Wy. mitchellii will bite humans, it is not considered a significant public health threat. Arboviruses, including Venezuelan equine encephalitis virus (VEEV), have been detected in Wy. mitchellii, but the mosquito species’ vector potential remains to be elucidated (Sherer et al. 1971). However, the horticultural trade, whether illegal or not, is a potential pathway of mosquito species introduction which may pose a threat for the movement of mosquitoes of greater public health concern, such as Ae. aegypti. Thus, the discovery of Wy. mitchellii on Guam highlights the importance of mosquito surveillance on the island. Mosquito surveillance is needed not only to assist management of mosquito-borne disease risk associated with currently present species but also for quarantine purposes to ensure rapid identification of other nonendemic species. This is particularly the case of Ae. aegypti, for which the reintroduction of this species on Guam would have significant public health implications. The spread of invasive mosquitoes internationally, particularly Ae. aegypti and Ae. albopictus, has had significant public health implications across the world, including in the Pacific Islands (Roth et al. 2014). Given the threat of Ae. aegypti to Guam, the detection of Wy. mitchellii further highlights the role of local entomological expertise and of the necessity of an effective mosquito surveillance program on the island. Acknowledgments The authors wish to acknowledge the assistance of Matthew Shortus (WHO) during the initial identification process. The authors also acknowledge the assistance of Michelle Lastimoza (Guam Department of Public Health and Social Services) in the coordination of mosquito field surveys on the island. The authors also thank Dr. Russell Campbell (Guam Department of Agriculture) for enabling the export of mosquito specimens out of Guam, and for providing the authors information regarding horticultural trade in the region. Finally, the authors thank the Australian Government Department of Agriculture and Water Resources for assisting with importation of mosquito specimens from Guam to Australia. References Cited Bohart, R.M. 1956. Diptera: Culicidae. Insects Micronesia . 12: 1– 85. Clark-Gil, S. and Darsie R.F.. 1983. The mosquitoes of Guatemala, their identification, distribution, and bionomics, with keys to adult females and larvae. Mosquito Systematics . 15: 151– 284. Cox, P. and Sanchez P.C.. 1926. The island of Guam . Government Printing Office, Washington, DC Darsie R.F. Jr. and Ward R.A.. 1981. Identification and geographical distribution of the mosquitoes of North America, north of Mexico . Walter Reed Army Institute of Research, Washington, DC. Dupont-Rouzeyrol, M., M. Aubry, O. O’Connor, C. Roche, A. C. Gourinat, A. Guigon, A. Pyke, J. P. Grangeon, E. Nilles, S. Chanteauet al.   2014. Epidemiological and molecular features of dengue virus type-1 in New Caledonia, South Pacific, 2001-2013. Virol. J . 11: 61. Google Scholar CrossRef Search ADS PubMed  Frank, J.H. 1986. Bromeliads as ovipositional sites for Wyeomyia mosquitoes: form and color influence behaviour. Fla. Entomol . 69: 728– 742. Google Scholar CrossRef Search ADS   Frank, J.H. and Fish D.. 2008. Potential biodiversity loss in Florida bromeliad phytotelmata due to Metamasius callizona (Coleoptera: Dyrophthoridae), an invasive species. Fla. Entomol . 91: 1– 8. Google Scholar CrossRef Search ADS   Frank, J.H., Lynn H.C., and Goff J.M.. 1985. Diurnal oviposition by Wyeomyia mitchellii and W. vanduzeei (Diptera: Culicidae). Fla. Entomol . 68: 493– 496. Google Scholar CrossRef Search ADS   Guam Department of Public Health and Social Services. 2017. Division of Environmental Health. http://www.dphss.guam.gov/content/division-environmental-health Haddock, R.L. 2010. A history of health on Guam . Crushers Football (Soccer) Club, Hagatña, GU. Marie, J. and Bossin H. C.. 2013. First record of Wyeomyia (Wyeomyia) mitchellii (Diptera: Culicidae) in French Polynesia. J. Med. Entomol . 50: 37– 42. Google Scholar CrossRef Search ADS PubMed  National Weather Service Forecast Unit. 2014. National Weather Service Forecast Unit, Tiyan, Guam. http://w2.weather.gov/climate/local_data.php?wfo=guam Nowell, W.R. 1980. Comparative mosquito collection data from the southern Mariana islands (Diptera: Culicidae). Proc. Calif. Mosq. Control Assoc . 48: 112– 116. Nowell, W. R. 1987. Vector introduction and malaria infection on Guam. J. Am. Mosq. Control Assoc . 3: 259– 265. Google Scholar PubMed  Ramalingam, S. 1976. An annotated checklist and keys to the mosquitoes of Samoa and Tonga. Mosquito Systematics . 8: 298– 318. Reisen, W. K., J. P. Burns, and Basio R. G.. 1972. A mosquito survey of Guam, Marianas islands with notes on the vector borne disease potential. J. Med. Entomol . 9: 319– 324. Google Scholar CrossRef Search ADS PubMed  Roth, A., Mercier A., Lepers A., Hoy D., Duituturaga S., Benyon E., Guillaumot L., and Souares Y.. 2014. Concurrent outbreaks of dengue, chikungunya, and Zika virus infections–an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012–2014. Euro. Surveill . 19: 2– 9. Google Scholar CrossRef Search ADS   Rozeboom, L. E. and Bridges J. R.. 1972. Relative population densities of Aedes albopictus and A. guamensis on Guam. Bull. World Health Organ . 46: 477– 483. Google Scholar PubMed  Rueda, L.M., J.E. Pecor, W.K. Reeves, S.P. Wolf, P.V. Nunn, R.Y. Rabago, T.L. Gutierrez, and Debboun M.. 2011. Mosquitoes of Guam and the Northern Marianas: distribution, checklists, and notes on mosquito-borne pathogens. US Army Med. Dep. J . 17– 28. Sherer, W.F., Dickernman R.W., Campillo-Sainz C., Zarate M.L., and Gonzales E.. 1971. Ecologic studies of Venezuelan encephalitis virus in southeastern Mexico. V. Infection of domestic animals other than equines. Am. J. Trop. Med. Hyg . 20: 989– 993. Google Scholar CrossRef Search ADS PubMed  Shroyer, D.A. 1981. Establishment of Wyeomyia mitchellii on the island of Oahu, Hawaii. Mosq. News . 41: 805– 806. Simon, C.F., Frati A., Beckenbach B., Crespi H., Lui H., and Flook P.. 1994. Evolution, weighting and implication and phylogenetic utility of the mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am . 87: 651– 701. Google Scholar CrossRef Search ADS   Swezey, O.H. 1942. Culicidae of Guam, pp. 199–200. In O.H. Swezey, Insects of Guam . I. B. P. Bishop Mus. Bull . 172. Walter Reed Biosystematics Unit. WRBU mosquito identification resources. http://www.wrbu.org/VecID_MQ.html Whelan, A.C., Becker S.K., Uluiviti V.R., and Maddox N.. 2016. Leveraging Pacific laboratories to boost global health security. Hawaii J. Med. Public Health . 75: 389– 392. Google Scholar PubMed  Yang, P., Furumizo R., Tangalin L., Takekuma C., and Hall K.E.. 2003. Mosquito species breeding in bromeliad azils on the island of Kauai, Hawaii. Proc. Hawaiian Ento. Soc . 36: 95– 101. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Medical Entomology Oxford University Press

New Record of Wyeomyia mitchellii (Diptera: Culicidae) on Guam, United States

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Abstract

Abstract Wyeomyia (Wyeomyia) mitchellii (Theobald) (Diptera: Culicidae) was recovered for the first time on Guam, United States of America, in 2017. Larval specimens were collected from water-filled axils of bromeliads during a larval survey carried out in a residential neighborhood of the Chalan Pago/Ordot area. Native to the New World, Wy. mitchellii has likely made its way to the Pacific Islands through the possibly illegal import of ornamental bromeliads. While this mosquito does not represent a significant threat to public health, this finding highlights the vulnerability of the Pacific Islands to the introduction of exotic species, including mosquito species that may increase public health risks. mosquito, Wyeomyia mitchellii, exotic species, biosecurity, Pacific The international spread of mosquitoes poses a potentially significant threat to public health as competent vectors of pathogens may be introduced to areas where they are currently absent. Understanding pathways of introduction and maintaining effective quarantine surveillance strategies is critical. Much of the Pacific has experienced an unprecedented level of mosquito-borne activity in recent years (Roth et al. 2014). For instance, in 2016 through 2017 alone, the U.S.-Affiliated Pacific Islands (USAPIs), saw outbreaks of mosquito-borne disease, including transmission of dengue (DENV) and Zika (ZIKV), in the Republic of Palau, the Republic of Marshall Islands (RMI), American Samoa, and in the Federated States of Micronesia (FSM) (Whelan et al. 2016). The spread of Aedes (Stegomyia) aegypti (Linnaeus) (Diptera: Culicidae) across the Pacific is likely a driving factor in increasing outbreak risk, as the distribution of this mosquito is influenced primarily by human activity (Dupont-Rouzeyrol et al. 2014). Thus, surveillance of mosquito introductions to new regions is critical to reducing mosquito outbreak risk. Guam is a tropical island in the North Pacific Ocean (13° 16ʹ 48.0ʺ N: 144° 28ʹ 12.0ʺ E). The island is an organized, unincorporated territory of the United States, and holds policy associations with the United States under the jurisdiction of the Office of Insular Affairs (U.S. Department of Interior). With a land area of 544 km2, Guam is the largest and southernmost island of the Mariana Islands archipelago (Rueda et al. 2011). The north of the island is a forested coral limestone plateau and is densely populated, whereas the south of the island is chiseled with volcanic mountain peaks and is less populated. The climate is tropical marine with very little seasonal temperature variation. The mean high temperature is 31°C, and the mean low temperature is 25°C. Daily precipitation averages 7.8 mm (National Weather Service Forecast Unit 2014). These climatic conditions, combined with a prevalence of suitable habitats, provide ideal conditions for a wide range of mosquito species on Guam year-round. Mosquito-borne disease on Guam has been a concern of local authorities since the early 1900s. As stated in the prewar government handbook of Guam, mosquito-borne diseases were not a recorded issue on the island, and Navy personnel claimed that although present, mosquitoes posed no health threat to the inhabitants of Guam (Cox et al. 1926, Haddock 2010). However, mosquito-borne diseases became a reality on Guam in the years following the American reoccupation of Guam, when Spain ceded Guam to the United States through the Treaty of Paris (1899) (Nowell 1987), and massive movements of humans and goods from other regions of the Pacific where mosquito-borne diseases are endemic took place (Nowell 1987, Haddock 2010). Indeed, the first recorded mosquito species on Guam was in 1905, when J.F. Leys disclosed to the Surgeon General, U.S. Navy, that Stegomyia fasciata (Ae. aegypti) was well-established on the island. In 1936, the first extensive pre-World War II mosquito survey on Guam was conducted by Swezey (1942), in which four mosquito species were collected, all of which belonged to Aedes and Culex. By 1945, 10 mosquito species had been recorded on Guam, none of which were anophelines (Nowell 1980). In 1944, Guam saw its first dengue outbreak with transmission most likely driven by Ae. aegypti. Three years later, Guam faced its first Japanese encephalitis (JEV) outbreak, likely transmitted by the inferred vector species Culex (Culex) annulirostris (Skuse) (Diptera: Culicidae) (Haddock 2010). Today, the main mosquito-borne pathogen threats to Guam are DENV, chikungunya (CHIKV), JEV, yellow fever (YFV), and ZIKV viruses, as well as malaria and filariasis parasites (Rozeboom and Bridges 1972, Rueda et al. 2011). Since the elimination of Ae. aegypti from Guam after World War II and the Vietnam War, the sporadic cases of dengue and malaria on Guam have thus far been nonendemic; all known cases being associated with returning travelers from endemic countries. As of 2016, 24 cases of travel-related dengue have been reported on Guam in the last decade (Guam DPHSS 2017). A wide range of mosquito species have been recorded on, or have been suspected of being present, on Guam (Nowell 1987). However, since the end of World War II, mosquito surveys on Guam have been sporadic. Rueda et al (2011) summarize these post-World War II mosquito assessments. Such surveys demonstrate that the mosquito fauna of Guam changes at an alarming rate, likely as a result from high military traffic, incoming tourists, and island hopping (Rueda et al. 2011). Indeed, although the number of mosquito species on Guam varied from 18 to 42 (Reisen et al. 1972), by the end of 1983, it was established that Guam was home to 24 mosquito species, 17 of which were introduced species (Nowell 1987). This list includes Ae. aegypti because although considered eliminated from Guam, the prevalence of Ae. aegypti is widespread across many neighboring countries. Thus, due to the potential for this mosquito to be transported with human activity, there is a considerable risk of importing Ae. aegypti onto Guam. Out of these mosquito species on Guam, nine are Aedes species, five are Anopheles species, and six are Culex species. Therefore, there is an urgent need to establish mosquito surveillance to ensure any importation of this mosquito is met with a strategic control response. No mosquito surveys from Guam have been published since 2011. While both Santa Rita Navy base and Andersen Air Force base conduct irregular mosquito surveillance on the island, these activities are confined to the base enclosures, and thus, caution must be taken when extrapolating mosquito data from the military bases to the entire island of Guam. Recently, the Guam Division of Environmental Health (DEH), Department of Public Health and Social Services (DPHSS) saw its new Guam Environmental Public Health Laboratory (GEPHL) open in 2017. With the support of Pacific Islands Health Officers Association (PIHOA), mosquito collecting activities on Guam have led to the recent recovery of a previously unreported species on Guam, Wyeomyia mitchellii (Theobald) (Diptera: Culicidae). Surveys of various natural and artificial water-holding containers were undertaken at various locations throughout Guam with any immature mosquitoes collected returned to GEPHL for identification. On 29 March 2017, at least seven larvae were collected from a stand of Aechmea blanchetiana (Baker) bromeliads (Poales: Bromeliacae) in a residential area along Santa Cruz Drive, in Chalan Pago/Ordot municipality (13°26ʹ 16.9ʺ N: 144°46ʹ 07.7ʺ E). Larvae were returned to GEPHL and reared to adult stage in biological rearing chambers placed in an environmental chamber (27.5°C) and provided fish food. All larvae that were collected survived to the adult stage. Adults (approximately 24-h old) were then freeze-killed for identification. Initial identification of specimens was undertaken using the keys of Bohart (1956) and Ramalingam (1976). Among specimens of species known to exist on Guam, such as Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae), Aedes (Stegomyia) guamensis Farner and Bohart (Diptera: Culicidae), Culex (Culex) quinquefasciatus Say (Diptera: Culicidae), and Culex sitiens Wiedemann species group (Diptera: Culicidae), a small number of adult mosquito specimens appeared to be distinct from common mosquitoes collected in Guam based on morphological examination. However, their identification could not be formally resolved using taxonomic keys available for the region. Based on concerns that the specimens may be of an introduced species, the specimens underwent further taxonomic and molecular analysis. Voucher specimens were mounted and placed in the GEPHL’s reference collection. Using the online interactive keys of the Walter Reed Biosystematics Unit (WRBU) [http://www.wrbu.org/VecID_MQ.html], the specimens were strongly believed to be of the genus Wyeomyia based on the presence of mesopostnotal setae, the presence of prespiracular setae, and the presence of a long basal spur on the wing Rs vein. Further reference to Darsie and Ward (1981), and Clark-Gil and Darsie (1983), confirmed the identification as Wyeomyia subgenus Wyeomyia with it most likely being Wy. mitchellii or a closely related species. To confirm the identification based on morphological characteristics, molecular analysis of a single specimen was undertaken. DNA was extracted from two legs of the mosquito by means of magnetic particle separation using the EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany). The legs were homogenized by shaking for 1 h using glass beads and the mosquito grind pretreated with proteinase K at 56°C for another hour. Four hundred microlitres of the homogenate was extracted on the BioRobot EZ1 according to the manufacturer’s instructions. A 1,345-bp segment of the mitochondrial coding gene encompassing the COII and tRNA-Leucine region was amplified with the primers C1-J-1718 (5ʹ-GGN GGA TTT GGA AAT TGA TTA GTN CC-3ʹ) and TL2-N-3014 (5ʹ-TCC ATT GCA CTA ATC TGC CAT ATT-3ʹ) (Simon et al. 1994), respectively. The fragment was amplified by real-time PCR on the Rotogene 6000 (Qiagen) in a total volume of 35 µl containing 8 µl of DNA template, 400 nM of each primer, 1× of 20-fold EvaGreen (Biotium, Fremont, California), 1× MyTaq HS Mix (Bioline, London, United Kingdom), and RNase- and DNAse-free water (Sigma–Aldrich, St. Louis, Missouri). The thermal cycling commenced with enzyme activation (95°C for 2 min), followed by 42 cycles of denaturation (95°C for 18 s), annealing (touch-down 63–53°C for 20 s for 10 cycles, followed by 22 cycles at 53°C), extension (72°C for 30 s), and a final extension at 72°C for 2 min. A pre-hold cycle was set at 50°C for 30 s followed by a melt cycle with ramping temperatures between 75°C and 95°C to serve as a check on purity of the amplified product. The amplicons were verified on a 2% agarose gel. The PCR amplicons were pre-treated with Illustra ExoProStar 1-Step (GE Healthcare, Little Chalfont, United Kingdom) and sent to the Australian Genome Research Facility (AGRF), Sydney, for bi-directional Sanger sequencing using the ABI BigDye Terminator chemistry Version 3.1. The sequenced products were aligned using Geneious 6.0.6 and the consensus sequence (1,221 bp) identified by matching against nucleotide sequences on the National Centre for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) database. The sequence with the highest alignment score, percentage identity and the lowest expect value (E value) of all alignments from the sequence database was considered to be the best match to the query sequence. The sequence (GenBank MF950916) matched known records of Wy. mitchellii, GenBank HM136827, with an alignment score of 2,150, identity of 99 % and E-value of 0.0. Wy. mitchellii has been reported from a range of tropical and subtropical regions of the Americas, such as South Florida, the Greater Antilles, and eastern Mexico (Frank and Fish 2008). The species was initially described from specimens collected in Jamaica. Since the 1980s, the mosquito has been detected outside of its native range in a number of locations across the Pacific including the Hawaiian Islands (Shroyer 1981, Yang et al. 2003) as well as the Society Islands, French Polynesia (Marie and Bossin 2013). These discoveries in the Pacific raise concerns regarding pathways of introduction of other container-inhabiting mosquitoes between countries. Wy. mitchellii is closely associated with natural water holding plants, such as bromeliads and other aroid species where water collects in leaf axils (Shroyer 1981, Frank 1986). Adult mosquitoes lay eggs in these water-impounded leaf axils, typically during the late afternoon (Frank et al. 1985). Once hatched, the larvae require at least 2 wk to complete development, but larvae may take many more weeks to develop, especially when there is minimal organic material in habitats to support larvae development (Frank 1986, Frank and Fish 2008). This ability to survive extended periods of starvation may increase their capacity to be transported in bromeliads moved between locations where naturally accumulating organic material may be minimal. An illegal trade in ornamental bromeliads is suspected to have facilitated the movement of Wy. mitchellii from its native range to the Pacific Islands. In fact, it was proposed that movement of bromeliads from Hawai’i was the likely pathway of Wy. mitchellii into French Polynesia (Marie and Bossin 2013) and it is likely that a similar process facilitated the movement of the mosquito into Guam. It is not possible to know when the mosquito was first introduced on the island, but given the distinctiveness of the mosquito compared to those species known from Guam, it is considered highly unlikely that the specimen would have been overlooked in previous investigations. Additionally, it has not been collected at any other location on Guam during 2017 habitat surveys. Although Wy. mitchellii will bite humans, it is not considered a significant public health threat. Arboviruses, including Venezuelan equine encephalitis virus (VEEV), have been detected in Wy. mitchellii, but the mosquito species’ vector potential remains to be elucidated (Sherer et al. 1971). However, the horticultural trade, whether illegal or not, is a potential pathway of mosquito species introduction which may pose a threat for the movement of mosquitoes of greater public health concern, such as Ae. aegypti. Thus, the discovery of Wy. mitchellii on Guam highlights the importance of mosquito surveillance on the island. Mosquito surveillance is needed not only to assist management of mosquito-borne disease risk associated with currently present species but also for quarantine purposes to ensure rapid identification of other nonendemic species. This is particularly the case of Ae. aegypti, for which the reintroduction of this species on Guam would have significant public health implications. The spread of invasive mosquitoes internationally, particularly Ae. aegypti and Ae. albopictus, has had significant public health implications across the world, including in the Pacific Islands (Roth et al. 2014). Given the threat of Ae. aegypti to Guam, the detection of Wy. mitchellii further highlights the role of local entomological expertise and of the necessity of an effective mosquito surveillance program on the island. Acknowledgments The authors wish to acknowledge the assistance of Matthew Shortus (WHO) during the initial identification process. The authors also acknowledge the assistance of Michelle Lastimoza (Guam Department of Public Health and Social Services) in the coordination of mosquito field surveys on the island. The authors also thank Dr. Russell Campbell (Guam Department of Agriculture) for enabling the export of mosquito specimens out of Guam, and for providing the authors information regarding horticultural trade in the region. Finally, the authors thank the Australian Government Department of Agriculture and Water Resources for assisting with importation of mosquito specimens from Guam to Australia. References Cited Bohart, R.M. 1956. Diptera: Culicidae. Insects Micronesia . 12: 1– 85. Clark-Gil, S. and Darsie R.F.. 1983. The mosquitoes of Guatemala, their identification, distribution, and bionomics, with keys to adult females and larvae. Mosquito Systematics . 15: 151– 284. Cox, P. and Sanchez P.C.. 1926. The island of Guam . Government Printing Office, Washington, DC Darsie R.F. Jr. and Ward R.A.. 1981. Identification and geographical distribution of the mosquitoes of North America, north of Mexico . Walter Reed Army Institute of Research, Washington, DC. Dupont-Rouzeyrol, M., M. Aubry, O. O’Connor, C. Roche, A. C. Gourinat, A. Guigon, A. Pyke, J. P. Grangeon, E. Nilles, S. Chanteauet al.   2014. Epidemiological and molecular features of dengue virus type-1 in New Caledonia, South Pacific, 2001-2013. Virol. J . 11: 61. Google Scholar CrossRef Search ADS PubMed  Frank, J.H. 1986. Bromeliads as ovipositional sites for Wyeomyia mosquitoes: form and color influence behaviour. Fla. Entomol . 69: 728– 742. Google Scholar CrossRef Search ADS   Frank, J.H. and Fish D.. 2008. Potential biodiversity loss in Florida bromeliad phytotelmata due to Metamasius callizona (Coleoptera: Dyrophthoridae), an invasive species. Fla. Entomol . 91: 1– 8. Google Scholar CrossRef Search ADS   Frank, J.H., Lynn H.C., and Goff J.M.. 1985. Diurnal oviposition by Wyeomyia mitchellii and W. vanduzeei (Diptera: Culicidae). Fla. Entomol . 68: 493– 496. Google Scholar CrossRef Search ADS   Guam Department of Public Health and Social Services. 2017. Division of Environmental Health. http://www.dphss.guam.gov/content/division-environmental-health Haddock, R.L. 2010. A history of health on Guam . Crushers Football (Soccer) Club, Hagatña, GU. Marie, J. and Bossin H. C.. 2013. First record of Wyeomyia (Wyeomyia) mitchellii (Diptera: Culicidae) in French Polynesia. J. Med. Entomol . 50: 37– 42. Google Scholar CrossRef Search ADS PubMed  National Weather Service Forecast Unit. 2014. National Weather Service Forecast Unit, Tiyan, Guam. http://w2.weather.gov/climate/local_data.php?wfo=guam Nowell, W.R. 1980. Comparative mosquito collection data from the southern Mariana islands (Diptera: Culicidae). Proc. Calif. Mosq. Control Assoc . 48: 112– 116. Nowell, W. R. 1987. Vector introduction and malaria infection on Guam. J. Am. Mosq. Control Assoc . 3: 259– 265. Google Scholar PubMed  Ramalingam, S. 1976. An annotated checklist and keys to the mosquitoes of Samoa and Tonga. Mosquito Systematics . 8: 298– 318. Reisen, W. K., J. P. Burns, and Basio R. G.. 1972. A mosquito survey of Guam, Marianas islands with notes on the vector borne disease potential. J. Med. Entomol . 9: 319– 324. Google Scholar CrossRef Search ADS PubMed  Roth, A., Mercier A., Lepers A., Hoy D., Duituturaga S., Benyon E., Guillaumot L., and Souares Y.. 2014. Concurrent outbreaks of dengue, chikungunya, and Zika virus infections–an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012–2014. Euro. Surveill . 19: 2– 9. Google Scholar CrossRef Search ADS   Rozeboom, L. E. and Bridges J. R.. 1972. Relative population densities of Aedes albopictus and A. guamensis on Guam. Bull. World Health Organ . 46: 477– 483. Google Scholar PubMed  Rueda, L.M., J.E. Pecor, W.K. Reeves, S.P. Wolf, P.V. Nunn, R.Y. Rabago, T.L. Gutierrez, and Debboun M.. 2011. Mosquitoes of Guam and the Northern Marianas: distribution, checklists, and notes on mosquito-borne pathogens. US Army Med. Dep. J . 17– 28. 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WRBU mosquito identification resources. http://www.wrbu.org/VecID_MQ.html Whelan, A.C., Becker S.K., Uluiviti V.R., and Maddox N.. 2016. Leveraging Pacific laboratories to boost global health security. Hawaii J. Med. Public Health . 75: 389– 392. Google Scholar PubMed  Yang, P., Furumizo R., Tangalin L., Takekuma C., and Hall K.E.. 2003. Mosquito species breeding in bromeliad azils on the island of Kauai, Hawaii. Proc. Hawaiian Ento. Soc . 36: 95– 101. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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Journal of Medical EntomologyOxford University Press

Published: Mar 1, 2018

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