Tropical Biomedicine 28(1): 181–187 (2011)

Research Note

Updated distribution records for vagus (Diptera: Culicidae) in the Republic of , and considerations regarding its secondary vector roles in

Rueda, L.M.1*, Pecor, J.E.1 and Harrison, B.A.2 1 Division of Entomology, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Room 3A28, Silver Spring, MD 20910. Current mailing address: Walter Reed Biosystematics Unit, Museum Support Center (MRC 534), Smithsonian Institution, 4210 Silver Hill Road, Suitland, MD 20746, U.S.A. 2 Public Health Pest Management, North Carolina Department of Environment and Natural Resources, 585 Waughtown Street, Winston Salem, North Carolina 27107, U.S.A. * Corresponding author email: [email protected] Received 17 September 2010; received in revised form 17 December 2010; accepted 22 December 2010

Abstract. Distribution records for Anopheles (Cellia) vagus in the Republic of the Philippines were updated, including recent collection and museum records from Luzon and Visayas Provinces. Larval habitats (e.g. rice paddies, irrigation and drainage ditches), associated species, and the vector potential of this species were also noted.

Anopheles vagus Doenitz, a member of from Fort de Kock, [West Coast], Sumatra, the Pyretophorus Series, subgenus Cellia, a male from Banjoe-Biroe, Java, and other is widely distributed in Asia, particularly specimens from different Indonesian , (Kampuchea), localities (Ceram, Borneo, Lombok, New (including Hong Kong), , Guinea, Pulu Raja). Knight & Stone (1977) , Laos, , Mariana reported that the type specimens are Islands, (Burma), , deposited at the Zoologisches Museum Philippines, , and (ZM) des Humboldt Universitaet, Berlin, Vietnam (Christophers, 1933; Reid, 1968; Germany. Recently, Dr. Joachim Ziegler Knight & Stone 1977; Ward, 1984; WRBU (personal communication, 30 August 2010 2010). Regarding records from the Mariana with LMR) noted that An. vagus specimens Islands, Darsie & Cagampang-Ramos at the ZM comprised two pinned specimens (1971a) and Ward (1984) recorded An. (one female and one male without head) vagus, Anopheles indefinitus (Ludlow), and eight mounted specimens on three and Anopheles subpictus Grassi, on Guam, slides (six males and two females). All An. indefinitus is recognized from Saipan specimens were labeled as “paratypes (Pratt & Siren, 1971, Savage et al., 1993), design. F. Peus”, with locality written as and An. indefinitus and An. subpictus are “Padang”, “Bunjol”, or “Batavia” not “Fort recognized on Tinian (Valder et al., 1976). de Kock” (= Bukittinggi, 90 km from Anopheles vagus was originally described Padang). Therefore, the holotype of this by Doenitz (1902), from a female collected species is missing or unknown.

181 Anopheles vagus variety limosus King from the collection site. The Whirl-Pak® (1932) was described from Rizal, Luzon was then tightly closed to retain air, placed Island, Philippines, and was elevated to in a cooler, and brought to the building subspecies by Colless (1948). This (temporary laboratory) where most larvae subspecies of An. vagus was the only were individually link-reared to adult stage, representative of this species recognized in as morphological voucher specimens for the Philippines prior to 1971. The type of this work. Eclosed adults were pinned on An. vagus limosus is deposited in the U.S. paper points, each given a unique collection National Museum of Natural History number, and identified using diagnostic (USNMNH), Smithsonian Institution, morphological characters (Reid 1968, Suitland, Maryland. As late as Baisas & Cagampang-Ramos & Darsie, 1970). Dowell (1967) and Cagampang-Ramos & Voucher specimens and collection records Darsie (1970) An. vagus vagus was not were deposited in the USNMNH. recognized in the Philippines, and Baisas (1974) did not record this subspecies from Molecular identification Subic Bay Naval Base, Luzon. However, DNA was isolated from individual adults (1 three years prior to Baisas (1974), Darsie or 2 legs per adult) by phenol-chloroform & Cagampang-Ramos (1971b) provided the extraction, and direct sequencing was first records of An. vagus vagus in the carried out as described by Wilkerson et al. Philippines based on specimens collected (2003). The rDNA ITS2 was amplified, and and reared from Luzon and Mindanao polymerase chain reaction (PCR) products islands. Shortly thereafter Ramalingam were directly sequenced using Big Dye 3.0 (1974) elevated subspecies An. vagus (Applied Biosystems Inc. – ABI, Foster, CA) limosus to species status when he with an ABI 3100 sequencer (ABI). The collected both subspecies at the same sequence was then edited and analyzed locality in Sabah, Malaysia. In the current using Sequencher (v 4.8, AB). The ITS2 classification of Anopheles, An. vagus and sequences of An. vagus (FJ457631.1, An. limosus are treated as separate FJ654648.1, EU919718) listed in the species (Harbach, 2004; WRBU 2010). GenBank (NCBI 2010) were used to In this study we examined specimens compare and confirm the sequences collected by LMR from the Philippines resulting from the analysis of field together with existing specimens that collected specimens in this survey. are deposited in the USNMNH to better understand the distribution of An. vagus. Distribution Also, we have accrued additional Laguna Province: Calauan (14.150ºN, references indicating that, under certain 121.316ºE), 3 females (F), reared from circumstances, An. vagus should be larvae collected from rice field, 22 July considered a secondary vector of two or 2002, coll. no. PH 3-2, 3-4, 3-100 more pathogens that impact human health [associated with larvae of Anopheles in Southeast Asia. (Cel.) tessellatus Theobald and Aedes (Aedimorphus) caecus (Theobald)]; 1 Specimen collection and morphological male (M), reared from a larva collected identification from rice field, 22 July 2002, coll. no. About 21 An. vagus larvae were collected PH 4-27 [associated with larvae of from three habitats (rice fields, irrigation An. tessellatus, Aedes caecus, Culex ditches and drainage ditches) in Laguna (Culex) tritaeniorhynchus Giles]; 6 F, 3 M, Province in July 2002 using a plastic dipper reared from larvae collected from drainage (5 cm ht, 13 cm diam; Bioquip, Rancho ditch, 29 July 2002, coll. no. PH 9-5, 9-7, Dominguez, CA). Collected larvae were 9-9, 9-12, 9-14, 9-101, 9-102, 9-103, 9-104 placed in plastic Whirl-Pak® bags (118 ml, [associated with larvae of Cx. tritaenior- 8 x 18 cm) (BioQuip, Rancho Dominguez, hynchus and Lutzia (Metalutzia) fuscana CA) filled approximately 1/2 full with water (Wiedemann)]; Siniloan (14.417ºN,

182 121.450ºE), 5 F, 3 M, reared from larvae based primarily on thousands of specimens collected from irrigation ditches, 24 July in many blood feeding studies indicating 2002, coll. no. PH 6-1, 6-1A, 6-2, 6-2A, that throughout Southeast Asia An. vagus 6-8, 6-9, 6-10, 6-107 [associated with fed primarily (over 90%) on cows and water Aedes (Neomelaniconion) lineatopennis buffalos and was usually ranked the least (Ludlow) and Cx. tritaeniorhynchus]. attracted to humans of all the Anopheles Additional specimens of An. vagus from tested (Reid, 1961, 1968; Bruce-Chwatt the USNMNH were examined and recorded. et al., 1966; Ramachandra Rao, 1984). These include the following: Cebu Province However, over time evidence has accrued (?): T. Manga, 12 June 1933, 10 F, 2 M, coll. indicating this species may serve as a F. H. S./W. V. King, WRBU Acc. no. 657; secondary vector under unusual Lanao Del Sur Province: Masia, Talagian, circumstances that include dense 3 March 1970, 10 F, coll. A. C. Ramos; concentrations of humans in association Samar Province: Osmena, 1 F, coll. L. E. with low numbers or absence of bovids and/ Rozeboom, WRBU Acc. no. 1354; Quezon or primates (Baker et al., 1987; Maheswary Province: Taiaong, Lalig, 15 June 1970, 10 et al., 1994; Amerasinghe et al., 1999; F, coll. A. C. Ramos. Darsie & Cagampang- Prakash et al., 2004). These areas included Ramos (1971b) also reported the Thailand, Kampuchea, Bangladesh, Sri distribution of this species in the Lanka, and Assam State (India), Philippines, including Bulacan Province respectively. More recently, Verhaeghen et (San Jose del Monte), Laguna Province al. (2010) considered An. vagus a potential (Calauan, Majayjay), Quezon Province malaria vector in the Mekong Region (Tiaong), Lanao del Norte Province (Kampuchea, Laos, and Vietnam) during (Kolambugan, Tankal), Lanao del Sur their assessment of vector resistance Province (Balindong, Madamba, Marawi, against , while Manguin et al. Masiu), and South Cotabato Province (2008a) noted that it is a confirmed or (Maitum). Mogi et al. (1984) identified and secondary vector of malaria in East Timor. studied An. vagus specimens collected in The findings in these publications usually Iguig, Cagayan Province, while Wooster & occurred when well recognized vectors Rivera (1985) reported them from Mindoro were uncommon or absent. Such different Occidental Province. findings led Manguin et al. (2008a) to list An. vagus as a secondary malaria Habitat information vector, but in Manguin et al. (2008b) it is The larval habitats in Laguna Province not listed as a major malaria vector. where An. vagus larvae were found during Experimental infectivity studies have this study included: rice fields, irrigation yielded contradictory results. Tran-Thi- ditches and drainage ditches with slow Minh-Phuong et al. (1972) found that a flowing water, having pH ranging from 6.79 brackish water strain of An. vagus in - 7.62 (average pH 7.10, n = 3); conductivity Vietnam was not able to develop 0.13 - 0.24 uS (average 0.26 uS, n = 3); (Welch), yet temperature, 27.40 - 27.70ºC (average Somboon et al. (1994) in northwestern 27.97ºC, n = 3). Reid (1968) noted that Thailand found a fresh water strain larvae of this species are typically found in susceptible to both P. falciparum and P. open muddy pools and in hoof marks, vivax (confirmed by gland dissections and ditches, often in foul water and sometimes ELISA). All of the variable vector data in brackish water. about An. vagus may reflect different strains or even sibling species with Vector potential different ecological requirements and For many years An. vagus was not geographical distributions. Baimai et al. considered a vector of human malaria (1996) reported two karyotypic forms (A parasites (Christophers, 1933; Reid, 1968; and B) of An. vagus in Thailand. Currently, Ramachandra Rao, 1984). This status was it is not known if these forms represent

183 intraspecies or interspecies entities. In the with the filarial parasite, Wuchereria Republic of Philippines it has not been bancrofti (Cobbold) (Atmosoedjono et al., reported as a vector of malaria, possibly 1977), and was confirmed as a secondary because of conflicting distribution records vector of this parasite on that island (Lee and the misidentification of specimens due et al., 1983). Harinasuta et al. (1970) found to confusion with An. limosus. An. vagus females with larvae of W. Anopheles vagus is also capable of bancrofti (0.51% infection rate, larval serving as a vector of other parasites to stages I and II) and Dirofilaria spp. (0.51% humans. On Flores Island, Indonesia, this infective rate, larval stage III) in Thailand. species was found susceptible to infections Manguin et al. (2010) listed An. vagus as

Figure 1. Map of the Philippines, showing occurrence or collection sites of An. vagus, in triangle symbol. Province identification: (1) Cagayan, (2) Bulacan, (3) Quezon), (4) Laguna, (5) Mindoro Occidental, (6) Samar, (7) Cebu, (8) Lanao del Norte, (9) Lanao del Sur, (10) South Cotabato

184 one of the 19 Anopheles species that co- institutional support provided by both transmit Plasmodium and W. bancrofti in organizations. The opinions and assertions Asia. Although (JE) contained herein are those of the authors virus was found in 1 out of 42 pools of An. and are not to be construed as official or vagus from Lombok Island, Indonesia reflecting the views of the Department of (Olson et al., 1985), no further isolates have the Army or the Department of Defense, been reported. All of these findings or the North Carolina Department of regarding the vector roles of An. vagus Environment and Natural Resources. infer that this species should not be forgotten or disregarded as a potential vector when outbreaks of human disease REFERENCES occur in unusual scenarios where well recognized vectors and usual hosts (cows Amerasinghe, P.H., Amerasinghe, F.P., and water buffalos) are uncommon or Konradsen, F., Fonseca, K.T. & Wirtz, absent. R.A. (1999). Malaria vectors in a Anopheles vagus should not be traditional dry zone village in Sri considered a primary vector of malaria due Lanka. American Journal of Tropical to its skewed preference for feeding on Medicine and Hygiene 60: 421-429. bovids, however, evidence has accrued Atmosoedjono, S., Partono, F., Dennis, D.T. over the last 3-4 decades showing it can & Purnomo. (1977). Anopheles function as a secondary vector in the barbirostris (Diptera: Culicidae) as a absence of these preferred hosts. Because vector of the Timor filaria on Flores of the preferred larval habitats of this Island: preliminary observations. species and its ability to survive in small Journal of Medical Entomology 13: pools of muddy and organically polluted 611-613. water in full sunlight it will continue to be Baisas, F.E. (1974). The fauna of a common species in Southeast Asia. In a Subic Naval Reservation, Republic of future with increasing human populations, the Philippines. Headquarters, First warming temperatures, increasing ocean Medical Service Wing (PACAF), APO levels, loss of forests and subsequent AP San Francisco, Technical Report increases in larval habitats for An. vagus, No. 72-2: 1-170, + 79 figures. less reliance on single family farming that Baisas, F.E. & Dowell, F.H. (1967). Keys to requires bovids and more reliance on the adult female and larval anopheline commercialized processed and preserved mosquitoes of the Philippines. Journal foods, humans will undoubtedly be forced of Medical Entomology 4: 11-23. into more concentrated environs that are Baker, E.Z., Beier, J.C., Meek, S.R. & likely to promote the role of An. vagus as Wirtz, R.A. (1987). Detection and a secondary vector of human pathogens. quantification of Plasmodium falciparum and P. vivax infections in Acknowledgments. The assistance of Thai-Kampuchean Anopheles (Diptera: Cong Li and Fredy Ruiz (Walter Reed Culicidae) by Enzyme-linked Biosystematics Unit) in conducting the Immunosorbent Assay. Journal of PCR and sequencing is gratefully Medical Entomology 24: 536-541. acknowledged. Special thanks go to Dr. Baimai, V., Kijchalao, U. & Rattanarithikul, Joachim Ziegler for checking the type R. (1996). Metaphase karyotypes of specimens of An. vagus at the Anopheles of Thailand and Southeast Zoologisches Museum des Humboldt Asia. VI. The Pyretophorus and the Universitaet, Berlin, Germany. This Neomyzomyia Series, subgenus Cellia research was performed under a (Diptera: Culicidae). Journal of the Memorandum of Understanding between American Mosquito Control the Walter Reed Army Institute of Research Association 12: 669-675. and the Smithsonian Institution, with

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