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Blood Meal Identification and Prevalence of Avian MalariaParasite in Mosquitoes Collected at Kushiro Wetland, A SubarcticZone of JapanAuthor(s): Hiroko Ejiri, Yukita Sato, Kyeong Soon Kim, Yoshio Tsuda, KoichiMurata, Keisuke Saito, Yukiko Watanabe, Yoshiharu Shimura, and MasayoshiYukawaSource: Journal of Medical Entomology, 48(4):904-908. 2011.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/ME11053URL: http://www.bioone.org/doi/full/10.1603/ME11053

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VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION

Blood Meal Identification and Prevalence of Avian Malaria Parasite inMosquitoes Collected at Kushiro Wetland, a Subarctic Zone of Japan

HIROKO EJIRI,1 YUKITA SATO,1,2 KYEONG SOON KIM,3 YOSHIO TSUDA,3 KOICHI MURATA,4

KEISUKE SAITO,5 YUKIKO WATANABE,5 YOSHIHARU SHIMURA,6 AND MASAYOSHI YUKAWA1

J. Med. Entomol. 48(4): 904Ð908 (2011); DOI: 10.1603/ME11053

ABSTRACT In Japan, the prevalence of avianPlasmodium in birds and mosquitoes has been partiallyexamined in the temperate and subtropical zones; however, mosquitoes in the Japanese subarctic zonehave not been adequately investigated. In this study, mosquito collections and avian Plasmodiumdetections from the mosquito samples were carried out to demonstrate the avian Plasmodium trans-mission between vector mosquitoes and birds inhabiting in Kushiro Wetland, subarctic zone of Japan.A total of 5,657 unfed mosquitoes from 18 species and 320 blood-fed mosquitoes from eight specieswas collected in summer 2008, 2009, and 2010. Three Aedes esoensis that fed on Hokkaido Sika DeerandoneunfedCulexpipiensgroupwere found tobepositive foravianPlasmodiumbypolymerasechainreaction. This is the Þrst report of the detection of avian Plasmodium DNA from mosquitoes distrib-uting in the subarctic zone of Japan. The blood meals were successfully identiÞed to captive or wildanimals, including seven mammalian species, four bird species, and one amphibian species. Theseresults indicated that infected birds with avian Plasmodium inhabited and direct contacts occurredbetween the infected birds and mosquitoes in Kushiro Wetland, Hokkaido, Japan.

KEY WORDS avian Plasmodium, mosquito, blood-source, subarctic zone, Japan

Avian Plasmodium is a vector-borne pathogen trans-mitted by mosquitoes of genus that include Aedes,Culex, Anopheles, and Culiseta (Valkiu�nas 2005). Wildand captive birds and mosquitoes infected with avianPlasmodium have been reported around the worldfrom subarctic to tropical regions (Meyer et al. 1974,Bensch et al. 2000, Perkins and Schall 2002, Ejiri et al.2008, Murata et al. 2008, Kim et al. 2009a). In Japan,�100 mosquito species are distributed in subtropicalto subarctic climatic zone (Tanaka 1979), and avianPlasmodiumDNA has been detected in birds and mos-quitoes in recent studies conducted in central Japan(Murata 2002; Murata et al. 2008; Ejiri et al. 2008, 2009,2011; Kim et al. 2009a, 2009b; Kim and Tsuda 2010).

In Hokkaido, the north island and subarctic zone ofJapan, the mosquito fauna is different from that ofmainland of Japan (Tanaka 1979, Higa et al. 2006), andis characterized by species distributed in subarctic

regions, for example, Aedes dorsalis, Aedes esoensis,Aedes yamadai, Culiseta kanayamensis, and Culisetanipponica (Tanaka 1979, Higa et al. 2006). Some of thesubarctic mosquitoes inhabiting North Americaand/or Europe, such as Ae. dorsalis and Culiseta an-nulata, Culiseta melaneura, and Culiseta morsitans,were reported to be vectors of avian Plasmodium spe-cies, including Plasmodium circumflex and Plasmo-dium relictum (Meyer et al. 1974, Valkiu�nas 2005,Becker et al. 2010). Although the vector mosquitoeswere not identiÞed, avian Plasmodium infections incaptive birds (king penguin) were reported in Hok-kaido (Shimura 1995). Therefore, it is expected thatdifferent avian Plasmodium lineages from those ofmainland Japan could be detected from mosquitoesinhabiting Hokkaido.

The prevalence of vector-borne pathogens andfeeding patterns of the vector mosquitoes are impor-tant factors determining the transmission dynamicsand the infection risk for the vector-borne diseases(Apperson et al. 2004, Molaei et al. 2006, Kim et al.2009b, Kim and Tsuda 2010, Hamer et al. 2009). Incentral Japan, we investigated the prevalence of avianPlasmodium in mosquitoes and identiÞed the source ofblood meals from blood-fed mosquito samples (Ejiri etal. 2009, 2011; Kim et al. 2009a, 2009b; Kim and Tsuda2010); and have shown that the analysis of blood-fedmosquito samples collected in zoological gardens canprovide valuable information on the presence of avianPlasmodium as well as evidence of direct contact be-tween captive animals and mosquitoes (Ejiri et al.

1 Laboratory of Biomedical Science, Department of VeterinaryMedicine, College of Bioresource Sciences, Nihon University, Ka-meino 1866, Fujisawa, Kanagawa, Japan.

2 Corresponding author: Laboratory of Biomedical Science, De-partment of Veterinary Medicine, College of Bioresource Sciences,Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan(e-mail: sato.yukita@nihon-u.ac.jp).

3 Department of Medical Entomology, National Institute of Infec-tious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, Japan.

4 Laboratory of Wildlife Science, Department of Animal ResourceSciences, College of Bioresource Sciences, Nihon University, Ka-meino 1866, Fujisawa, Kanagawa, Japan.

5 Institute for Raptor Biomedicine Japan, 2-2101 Hokuto, Kushiro,Hokkaido, Japan.

6 Kushiro Zoo, 11 Ninishibetsu, Akan, Kushiro, Hokkaido, Japan.

0022-2585/11/0904Ð0908$04.00/0 � 2011 Entomological Society of America

2011). Kim et al. (2009a) showed that molecular anal-ysis of blood-fed mosquitoes collected in sanctuaryareas for resident and migratory birds can provideessential information to evaluate the risk for introduc-tion of novel vector-borne pathogens. Therefore, weselected the following two study sites for collection ofblood-fed mosquitoes: Kushiro Zoo and Kushiro Wet-land, a sanctuary for resident and migratory birds, inHokkaido, Japan. The purpose of this study was toexamine the prevalence of avian Plasmodium and theblood-feeding patterns of the mosquitoes for collect-ing basic information to evaluate the risk for avianPlasmodium in the subarctic zone of Japan.

Materials and Methods

Mosquitoes were collected with sweep nets andCenters for Disease Control and Prevention traps on28Ð29 July 2008, 3Ð4 August 2009, and 6Ð8 July 2010at Kushiro Zoo and the Institute for Raptor Biomed-icine Japan in Hokkaido, Japan. Kushiro Zoo (E 144.14,N 43.05) is �47.8 ha in area and exhibits a total of 31mammalian species and 35 avian species. The Institutefor Raptor Biomedicine Japan (E 144.17, N 43.04) islocated on the western side of Kushiro Wetland andkeeps protected birds of Japan, including White-tailedSea Eagle (Haliaeetus albicilla), StellerÕs Sea Eagle(Haliaeetus pelagicus), and BlakistonÕs Fish Owl(Ketupa blakistoni), providing care for injuries andrehabilitation.

Mosquito collections were made using Centers forDisease Control and Prevention traps without lightenhancement with 1 kg of dry ice, and sweeping nets.Twenty dry-ice traps were distributed in areas withshade trees and operated continuously for 2Ð3 d, andmosquito samples were collected every morning.Sweeping collections were made for 20Ð30 min in theevening in a shaded area of vegetation. All capturedmosquitoes were classiÞed into two groups, as follows:blood fed (freshly fed, half gravid, gravid) and unfed,as described previously (Ejiri et al. 2011). The col-lected mosquitoes were identiÞed by species accord-ing to morphologic keys (Tanaka 1979, Toma andMiyagi 1986) and kept at �20�C until DNA extraction.

All collected mosquito body was separated into twoparts, the head-thorax and abdomen, with microscis-sors under a microscope. One to Þve unfed mosquitoesof the same species were pooled with the head-thoraxand abdomen, respectively, whereas the blood-fedmosquitoes were processed individually. DNA wasextracted separately from the head-thorax and abdo-men with a REDExtract-N-Amp Tissue polymerasechain reaction (PCR) kit (Sigma-Aldrich, St. Louis,MO), as described previously (Kim et al. 2009b). Par-asite DNA was ampliÞed by nested PCR for the partialcyt b gene of the avian malaria mitochondrial genome,as described previously (Ejiri et al. 2008). In brief,after DNA extraction, we used DW2 and DW4 primers(Perkins and Schall 2002) for the Þrst PCR, andHAEMF (5�-ATG GTG CTT TCG ATA TAT GCATG-3�) and HAEMR2 (5�-GCA TTA TCT GGA TGTGAT AAT GGT-3�) primers for the second. HAEMF

and HAEMR2 primer set could amplify various avianmalaria lineages, including Plasmodium (Bensch et al.2000). PCR products were subsequently sequenced inboth directions with BigDye terminator mix (AppliedBiosystems, Foster City, CA). Phylogenetic analysisand determination of the sequence similarity of theampliÞed sequences were performed by the neighbor-joining method with the PAUP program (http://paup.csit.fsu.edu/), as described previously (Ejiri etal. 2011), and by Clustal W software, respectively.

For blood meal identiÞcation of mosquitoes, weampliÞed vertebrate DNA extracted from the abdo-mens of the blood-fed mosquitoes with primers de-signed by multiple alignment of the vertebrate mito-chondrial DNA, as described previously (Kim et al.2009b, Ejiri et al. 2011, Sawabe et al. 2010). We usedprimer sets, which amplify the vertebrate 16S ribo-somal RNA region, as follows: VerU-1 (5�-AAG ACGAGA AGA CCC YAT GGA-3�) and VerU-2 (5�-CCTGAT CCA ACA TMG AGG TCG TA-3�), and whichamplify the cyt b sequence, as follows: Avian-3 (5�-GAC TGT GAY AAA ATY CCM TTC CA-3�) andAvian-8 (5�-GYC TTC AIT YTT TGG YTT ACA AGAC-3�) for birds; Mammalian-1 (5�-TGA YAT GAA AAAYCA TCG TTG-3�) and Mammalian-2 (5�-TGT AGTTRT CWG GGT CKC CTA-3�) for mammals.

PCR products were sequenced in both directionswith the BigDye terminator mix (Applied Biosys-tems). Furthermore, the vertebrate species were de-termined by BLASTn searches against the GenBanknucleic acid sequence database (http://www.ncbi.nlm.nih.gov/BLAST/). When matched DNA se-quences were not obtained from the database, wecollected blood samples of captive animals and ob-tained the DNA sequences. The most similar hostspecies was identiÞed by considering sequence iden-tity and a list of vertebrate species available to bitingmosquitoes in the study area.

Results and Discussion

A total of 5,977 mosquitoes from 17 species wascollected in this study (Table 1). Ae. esoensis and Cs.nipponica,which were distributed in subarctic regions(Tanaka 1979), were the dominant species both in2008 and 2009, and higher species compositions ofAedes vexans nipponii, Cs. kanayamensis, Ae. yamadai,andAedes excrucians characterized the results of mos-quito collection in 2009.Culex pipiens group, includingCx. pipiens pallens, which is an important vector ofavian Plasmodium in central Japan, was collected insmall numbers only.

Three blood-fed Ae. esoensis collected in 29 July2008 and one unfedCx. pipiens group collected during6Ð8 July 2010 were positive for avian malaria by PCR(Table 2). This is the Þrst report of detection of avianPlasmodium DNA from Þeld-collected mosquitoes inHokkaido, subarctic Japan. Result of avian Plasmo-dium parasite DNA positive from both the head-tho-rax and abdomen of a single mosquito could suggestthat the mosquito species is a vector of the avianPlasmodiumparasite and transmits the parasite to their

July 2011 EJIRI ET AL.: STUDY OF AVIAN MALARIA IN SUBARCTIC ZONE OF JAPAN 905

blood-source birds (Kim et al. 2009b, Kim and Tsuda2010, Ejiri et al. 2011). In this study avian Plasmodiumis positive only in the head-thorax or the abdomen(Table 2). Therefore, results of this study are notstrong enough to demonstrate the actual transmissionof avian Plasmodium in the study area; however, it isevident that infected birds inhabited in the study areaand direct contacts had occurred between the in-fected birds and some mosquitoes.

On the basis of the difference in cyt b gene se-quence (361 bp), three and one genetically distinctlineages were identiÞed from blood-fed Ae. esoensis(Kushiro-1, Kushiro-2, and Kushiro-3) and unfedCx. pipiens group (Kushiro-4), respectively. TheKushiro-3 lineage was identical to Plasmodium galli-naceum, and the remaining three lineages had a highsequence homology (0.995Ð0.997) to P. gallinaceum(Table 2). However,Cx. pipiens pallens andCx. p. formmolestus, Cx. pipiens group mosquito species distrib-uted in Hokkaido (Tanaka 1979), andAe. esoensiswerenot reported as vector of P. gallinaceum (Valkiu�nas

2005). Furthermore, P. gallinaceum infection in wildbirds has not been reported in Japan. Additional in-vestigations, including prevalence of P. gallinaceumfrom birds and vector competence of Ae. esoensis,willbe necessary to demonstrate the avian Plasmodiumtransmission in study area.

A total of 320 blood-fed mosquitoes of the followingeight species was collected in this study (Table 3).Among the 320 blood-fed mosquitoes, the sources of287 blood meals were successfully identiÞed as sevenmammalian species (268/287), four bird species (19/287), and one amphibian species (1/287) (Table 3).There was only one sample from which two differentblood-source animals were identiÞed; Bos taurus andan unidentiÞed bird DNA were ampliÞed from a fresh-ly-fed Ae. esoensis. Four captive animals in KushiroZoo, three protected birds in the Institute for RaptorBiomedicine Japan, and Þve pastured and wild animalsin Kushiro Wetland were identiÞed as the bloodsources of the collected mosquitoes. The proportion ofavian blood meals from birds was high in Culiseta

Table 1. A list of mosquito species and total numbers collected using sweeping nets and dry-ice traps in Kushiro Wetland, Japan

Mosquito species2008 2009 2010

TotalSweeping Trap Sweeping Trap Trap

Aedes esoensis 305 265 387 2,064 22 3,043Culiseta nipponica 2 113 12 1,535 36 1,698Ae. vexans nipponii 0 2 307 123 0 432Ae. punctor/hokkaidensis 26 35 156 15 65 297Cs. kanayamensis 0 1 170 34 0 205Ae. excrucians 0 2 14 89 1 106Ae. yamadai 0 0 86 12 1 99Ae. japonicus 41 1 3 4 1 50Culex pipiens group 1 0 2 4 12 19Anopheles sinensis 1 3 3 1 0 8Cx. orientalis 2 1 0 1 1 5Ae. nipponicus 0 0 2 2 0 4Cx. inatomii 0 0 0 3 0 3Cx. vagans 0 0 1 2 0 3Ae. flavopictus 0 1 0 1 0 2Ae. bekkui 0 0 1 0 0 1Ae. galloisi 0 0 0 1 0 1Culex spp. 0 0 0 1 0 1Total 378 424 1,144 3,892 139 5,977

Table 2. Mosquito species examined, blood-source animals, and sequence homologies of avian Plasmodium species detected in thisstudy

Avian Plasmodium linage detected

Kushiro-1 Kushiro-2 Kushiro-3 Kushiro-4

Mosquito examined Ae. esoensis Ae. esoensis Ae. esoensis Cx. pipiens gr.

Plasmodium detection

From head-thorax � � � �From abdomen � � � �

Blood-source animalidentiÞed

Cervus nippon yesoensis Unfed female

Sequence homology

Kushiro-2 99.5Kushiro-3 99.7 99.7Kushiro-4 99.2 99.2 99.5P. gallinaceuma 99.7 99.7 100 99.5

a P. gallinaceum (AB250690).

906 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 4

species,Cs. nipponicus (0.48 � 12/25) andCs. kanaya-mensis (1.0 � 1/1), whereas no or few avian bloodmeals were identiÞed fromAedes species,Ae. esoenseis(0.02 � 4/257) and Aedes punctor/hokkaidensis(0.08 � 1/13).

In this study, we demonstrated that blood mealhosts of mosquitoes distributed in subarctic area ofJapan and also detected avian Plasmodium from mos-quitoes there for the Þrst time, providing a part of basicinformation for considering the risk assessment ofavian malaria there. Additional systematic and long-termcollectionofmosquitoes andPCR-basedanalysesof the samples will be necessary to clarify the trans-mission cycle of avian Plasmodium in the KushiroWetland in future studies.

Acknowledgments

This work was supported in part by a Grant-in-Aid forScientiÞc Research (21580406) and a Grant-in-Aid for JapanSociety for the Promotion of Science Fellows (226290) fromthe Japan Society for the Promotion of Science; researchfellowships were from the Japan Society for the Promotionof Science for Young Scientists (22-4605); the Strategic Re-search Base Development Program “International Researchon Epidemiology of Zoonoses and Training for Young Re-searchers” and the High-Tech Research Center Project forPrivate Universities from the Ministry of Education, Culture,Sports, Science, and Technology of Japan; a Grant-in-Aid forScientiÞc Research of Emerging and Reemerging InfectiousDiseases from the Ministry of Health, Labor and Welfare of

the Japanese Government (H18-Shinko-009 and H20-Sinkou-ippan013) and Nihon University research grants.

References Cited

Apperson, C. S., H. K. Hassan, B. A. Harrison, H. M. Savage,S. E. Aspen, A. Farajollahi, W. Crans, T. J. Daniels, R. C.Falco, M. Benedict, et al. 2004. Host feeding patterns ofestablished and potential mosquito vectors of West Nilevirus in the eastern United States. Vector Borne ZoonoticDis. 4: 71Ð82.

Becker, N., D. Petric, M. Zgomba, C. Boase, M. Madon, C.Dahl, and A. Kaiser. 2010. Mosquitoes and Their Con-trol, 2nd ed. Springer, New York, NY.

Bensch, S., M. Stjernman, D. Hasselquist, O. Ostman, B.Hansson, H.Westerdahl, and R. T. Pinheiro. 2000. HostspeciÞcity in avian blood parasites: a study of Plasmodiumand Haemoproteus mitochondrial DNA ampliÞed frombirds. Proc. Biol. Sci. 267: 1583Ð1589.

Ejiri, H., Y. Sato, E. Sasaki, D. Sumiyama, Y. Tsuda, K. Saw-abe, S.Matsui, S.Horie, K.Akatani,M.Takagi, et al. 2008.Detection of avianPlasmodium spp. DNA sequences frommosquitoes captured in Minami Daito Island of Japan. J.Vet. Med. Sci. 70: 1205Ð1210.

Ejiri, H., Y. Sato, R. Sawai, E. Sasaki, R.Matsumoto,M. Ueda,Y. Higa, Y. Tsuda, S. Omori, K.Murata, et al. 2009. Prev-alence of avian malaria parasite in mosquitoes collectedat a zoological garden in Japan. Parasitol. Res. 105: 629Ð633.

Ejiri, H., Y. Sato, K. Kim, T. Hara, Y. Tsuda, T. Imura, K.Murata, and M. Yukawa. 2011. Entomological study ontransmission of avian malaria parasites in a zoological

Table 3. Blood-source animals identified from blood-fed mosquitoes collected during 2008–2010 in Kushiro, Hokkaido, Japan

Studysite

Blood-source animalidentiÞed

Mosquito species examined

TotalAe.esoensis

Ae.excrucians

Ae.p/ha

Ae.vexans

Ae.yamadai

Cs.kanayamensis

Cs.nipponica

Cx.orientalis

Zoo MaHomo sapiens 1Bison bison 1 1Cervus nippon yesoensis 3 3Bos taurus 1 2 1 4Phoca vitulina 1 1

AvAix galericulata 1 1Ketupa blakistoni 1 1

UnidentiÞed 1 4 5Wetland Ma

Cervus nippon yesoensis 194 8 12 214Bos taurus 26b 1 2 1 1 7 38Clethrionomys rufocanusbedfordiae

1 1 2

Homo sapiens 2 2Vulpes vulpes 2 2

AvCygnus cygnus 1 1Haliaeetus pelagicus 2 1 10 13Haliaeetus albicilla 1 1Ketupa blakistoni 1 1UnidentiÞed 1b 1

AmHyla japonica 1 1

UnidentiÞed 23 1 1 2 1 28Total 257 2 13 17 4 2 25 1 321

Ma, Av, and Am indicate mammalian, avian, and amphibian, respectively.a Ae. p/h indicates Ae. punctor or Ae. hokkaidensis.b Bos taurus DNA and unidentiÞed bird DNA were detected from one Ae. esoensis.

July 2011 EJIRI ET AL.: STUDY OF AVIAN MALARIA IN SUBARCTIC ZONE OF JAPAN 907

garden in Japan: blood-meal identiÞcation and detectionof avian malaria parasite DNA from blood-fed mosqui-toes. J. Med. Entomol. (in press).

Hamer, G. L., U.D. Kitron, T. L. Goldberg, J. D. Brawn, S. R.Loss, M. O. Ruiz, D. B. Hayes, and E. D. Walker. 2009.Host selection byCulex pipiensmosquitoes and West Nilevirus ampliÞcation. Am. J. Trop. Med. Hyg. 80: 268Ð278.

Higa, Y., K. Hoshino, Y. Tsuda, and M. Kobayashi. 2006.Dry-ice trap and human bait collection of mosquitoes inthe eastern part of Hokkaido, Japan. Med. Entomol. Zool.57: 93Ð98.

Kim, K., and Y. Tsuda. 2010. Seasonal changes in feedingpattern of Culex pipiens pallens govern transmission dy-namics of multiple lineages of avian malaria parasite inJapanese wild bird community. Mol. Ecol. 19: 5545Ð5554.

Kim, K., Y. Tsuda, and A. Yamada. 2009a. Blood meal iden-tiÞcation and detection of avian malaria parasite frommosquitoes (Diptera: Culicidae) inhabiting coastal areasof Tokyo Bay, Japan. J. Med. Entomol. 46: 1230Ð1234.

Kim, K., Y. Tsuda, T. Sasaki, M. Kobayashi, and Y. Hirota.2009b. Mosquito blood-meal analysis for avian malariastudy in wild bird communities: laboratory veriÞcationand application to Culex sasai (Diptera: Culicidae) col-lected in Tokyo, Japan. Parasitol. Res. 105: 1351Ð1357.

Meyer, C. L., G. F. Bennett, and C. M. Herman. 1974. Mos-quito transmission ofPlasmodium(Giovannolaia) circum-flexum Kikuth, 1931, to waterfowl in the TantramarMarshes, New Brunswick. J. Parasitol. 60: 905Ð906.

Molaei, G., T. G. Andreadis, P.M. Armstrong, J. F. Anderson,and C. R. Vossbrinck. 2006. Host feeding patterns ofCulex mosquitoes and West Nile virus transmission,northeastern United States. Emerg. Infect Dis. 12: 468Ð474.

Murata, K. 2002. Prevalence of blood parasites in Japanesewild birds. J. Vet. Med. Sci. 64: 785Ð790.

Murata, K., R. Nii, E. Sasaki, S. Ishikawa, Y. Sato, K. Sawabe,Y. Tsuda, R. Matsumoto, A. Suda, and M. Ueda. 2008.Plasmodium (Bennettia) juxtanucleare infection in cap-tive white eared-pheasant (Crossoption crossoptilon) at aJapanese zoo. J. Vet. Med. Sci. 70: 203Ð205.

Perkins, S. L., and J. J. Schall. 2002. A molecular phylogenyof malarial parasites recovered from cytochrome b genesequences. J. Parasitol. 88: 972Ð978.

Sawabe,K.,H. Isawa,K.Hoshino,T. Sasaki, S.Roychoudhury,Y. Higa, S. Kasai, Y. Tsuda, I. Nishiumi, N. Hisai, et al.2010. Host-feeding habits of Culex pipiens and Aedes al-bopictus (Diptera: Culicidae) collected at the urban andsuburban residential areas of Japan. J. Med. Entomol. 47:442Ð450.

Shimura, Y. 1995. AvianPlasmodium sp. infection of captiveking-penguin (Aptenodytes patagonica) J. Jpn. Assoc.Zool. Gardens and Aquarium 36: 92 (in Japanese).

Tanaka, K. 1979. A revision of the adult and larval mosqui-toes of Japan (including the Ryukyu Archipelago and theOgasawara Islands)andKorea(Diptera:Culicidae).Con-trib. Am. Entomol. Inst. 16: 1Ð981.

Toma, T., and I. Miyagi. 1986. The mosquito fauna of theRyukyu Archipelago with identiÞcation keys, pupal de-scriptions and notes on biology, medical importance anddistribution. Mosq. Syst. 18: 1Ð109.

Valkiu�nas, G. 2005. Avian Malaria Parasites and Other Hae-mosporidia. Taylor and Francis Publishing, London,United Kingdom.

Received 19 March 2011; accepted 15 May 2011.

908 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 4