bloodmeal identiﬁcation and feeding habits of mosquitoes

Bloodmeal identification and feeding habits of mosquitoes(Diptera: Culicidae) collected at five islands

in the Ryukyu Archipelago, Japan

Mikako T6B6H=>GD1), 2), Takako TDB61), Kosuke M6CC:C1),Yukiko H><63) and Ichiro M>N6<>1)

1) Laboratory of Medical Zoology, School of Health Sciences, Faculty of Medicine,University of the Ryukyus, Nishihara, Okinawa, 903�0215 Japan

2) Ikari Okinawa Corporation, 185�1 Haebaru, Okinawa, 901�1111 Japan3) Department of Medical Entomology, Institute of Tropical Medicine, Nagasaki University,

1�12�4 Sakamoto, Nagasaki 852�8523 Japan

(Received: 30 August 2010; Accepted: 16 December 2010)

Abstract: To determine the host-feeding patterns of mosquitoes, blood-fed mos-quitoes were collected by 5 di#erent methods from various places such as mountainforests, residential areas and animal sheds of the following 5 islands, Amamioshima,Tokunoshima, Iheyajima, Okinawajima and Iriomotejima in the Ryukyu Archipelago,Japan over a 6 yr period (2005�2010). A total of 975 bloodmeals derived from blood-fed mosquitoes of 35 species representing 11 genera were successfully identified bya polymerase chain reaction (PCR) based method, and 34 vertebrate species wereidentified as the bloodmeal hosts. Our results indicate that mosquitoes of the samegenus show similar host preferences when blood source animals are classified into 5major groups; mammals, birds, reptiles, amphibians and fishes. The genus Anophelesexhibited mammalophilic and Aedes exhibited mammalophilic, but Ae. (Geoskusea)baisasi Knight and Hull fed predominantly on fishes. Host preferences of the genusCulex were somewhat di#erent among subgenera. Subgenus Culex fed on warm-blooded animals, including mammals and birds, whereas other subgenera fed onvarious hosts both warm-blooded and cold-blooded animals. The Uranotaenia specieswere amphibian-feeders and also fed on reptiles and fishes, but not on warm-bloodedanimals. Medical importance of mosquito species is discussed as related to theirfeeding patterns and transmission of mosquito-borne diseases.

Key words: mosquito species, Culicidae, bloodmeal identification, host-feeding pat-tern, Ryukyu Archipelago, Japan

ICIGD9J8I>DC

Information on host-feeding patterns ofmosquitoes in nature is an essential com-ponent for evaluating their vectorialcapacities and for assessing the role ofindividual vertebrates as potential reser-voir hosts involved in maintenance andamplification of zoonotic agents of humanand animal diseases (Molaei et al., 2008;Miyagi et al., 2010). It is a well-known

that mosquitoes obtain their bloodmealsfrom a wide variety of hosts. These in-clude warm-blooded animals such asmammals (humans, cattle, pigs and nu-merous wild mammals) and birds, cold-blooded animals such as amphibians andreptiles (frogs, turtles and snakes), andfishes such as mudskippers (Tempelis,1975). Host-feeding patterns of mos-quitoes vary depending on mosquito spe-cies and availability of host animals. How-ever, detailed information on host-feeding

�Med. Entomol. Zool. Vol. 62 No. 1 p. 53�70 2011�

patterns of mosquito species is largely lim-ited to species belonging to the generaAnopheles, Aedes and Culex (Clements,1999; Tempelis, 1975; Belkin, 1962). Theprimary hosts for most species of theformer 2 genera are mammals and for thelatter are variable. Accumulation of moreinformation about host-feeding patternsof many mosquito species should revealthat mosquito species of the same genusand subgenera tend to have a similar hostpreference. Tempelis (1975) reviewed thehost-feeding patterns of many mosquitospecies. Bloodmeals of mosquitoes havetraditionally been identified by serologictechniques such as the precipitin test andenzyme-linked immunosorbent assay(ELISA). Recent advances in moleculartechniques for bloodmeal analyses byusing polymerase chain reaction (PCR)based assay, permits the identification ofhost species with a higher accuracy thanprevious serologic techniques. These mo-lecular techniques have been used to ex-amine the host-feeding patterns of vectorsof the West Nile virus (WNV) (Molaei et al.,2008; Kent et al., 2009; Sawabe et al.,2010), avian malaria (Ejiri et al., 2008; Kimet al., 2009a, b) and Eastern equine enceph-alitis virus (EEEV) (Cupp et al., 2004).

The mosquito fauna of the Ryukyu Ar-chipelago in Japan is well studied and richin mosquito fauna consisting of 72 speciesof 15 genera (Toma and Miyagi, 1986;Miyagi et al., 1992). Our previous studieson host-feeding patterns were mainly con-cerned with information on several vectormosquitoes that have a special preferencefor human and domestic animals, Anophe-les (Cellia) minimus Theobald, An. (Ano-pheles) sinensis Wiedemann, Culex (Culex)tritaeniorhynchus Giles, Cx. (Cux.) quin-quefasciatus Say, and Aedes (Stegomyia)albopictus (Skuse) (Miyagi and Toma, 1978a, b, 1980; Toma and Miyagi, 1981). Re-cently, bloodmeal hosts of Ae. albopictusand Cx. quinquefasciatus collected fromthe Ryukyus were identified by moleculartechniques (Sawabe et al., 2010). However,reports on bloodmeal identification by mo-

lecular techniques are heretofore lackingfor many mosquito species inhabitingdi#erent areas of the Ryukyus.

The present study reports the blood-meal hosts of 35 mosquito species throughbloodmeal identification by PCR basedmethods applied to a variety of fieldsamples. Our mosquito collections focu-sed on mosquitoes inhabiting forest areasof the large islands of Amamioshima,Tokunoshima, Okinawajima, Iheyajimaand Iriomotejima of the Ryukyu Archipel-ago, because mosquito fauna are rich inthese islands (Toma and Miyagi, 1986).Based on the results of the present study,host-feeding patterns of mosquito speciesare reviewed and host preferences ofgenera and subgenera discussed. Medicalimportance of mosquito species is also dis-cussed as related to host-feeding patternsand transmission of mosquito-borne dis-eases.

M6I:G>6AH 6C9 M:I=D9H

Study areasThe Ryukyu Archipelago lies in a

curved chain between Taiwan andKyusyu District of Japan, extending about1,000 km from 24� to 28�N, and 122� to132�E, and consisting of approximately160 islands (Fig. 1). The principal island isOkinawajima (OKI) located near themiddle of the Archipelago with an area ofabout 1,207.66 km2. Mosquito collectionswere carried out mainly 2 islands, Okina-wajima and Iriomotejima (IRI, 289 km2).Additional study areas included Amami-oshima (AMA, 712 km2), Tokunoshima(TOK, 248 km2) and Iheyajima (IHE, 20km2) (Fig. 1).

All of these islands are mountainousand heavily forested throughout exceptfor the business areas. The climate is sub-tropical with an annual temperature of23.0� for the past 30 years on Okina-wajima (meteorological data based on theonline database of the Japan Meteorologi-cal Agency). The coldest month is Janu-ary with an average temperature of

Med. Entomol. Zool.54

16.9�, and the hottest month is July withan average temperature of 28.9�. Theaverage annual rainfall is 2,002.8 mm.

Wild mammals include the indigenouswild boar, Sus scrofa riukiuanus, the wildcat, Prionailurus bengalensis iriomotensis,the shrew, Suncus murinus temmincki, andfruit bat, Pteropus dasymallus, on theRyukyus. There are many indigenoussnakes, turtles, lizards and frogs and manydomestic animals and birds such as cattle,pigs, goats, dogs, cats and fowl on all ofthe islands. The zoological garden ex-amined in this study has many imported

large animals, such as gira#es, Gira#acamelopardalis, white rhinoceros, Cerato-therium simum, and ostrich, Struthiocamelus.

Mosquito collectionsFrom 2005 to 2010, mosquitoes were

collected during the mosquito breedingseason from a variety of habitats in thestudy islands. The following 5 collectionmethods (Toma et al., 2007) were adopted:(1) light traps with 30 W fluorescent BLBlamp (BLB trap) were used to collect fromdomestic animal sheds (cattle, goat, pig

Fig. 1. Map showing the location of the 5 islands of the Japan Ryukyu Archipelago examined in thisstudy.

Vol. 62 No. 1 2011 55

and fowl), animal shed (gira#e) of the zoo-logical garden, and residential areas; (2)back-pack type battery-operated CDC-likesuction traps with 4 W BL lights (BL trap)were used to collect mainly from forestsand residential areas of the islands; (3) frogcall battery-operated CDC-like suctiontraps (frog call trap) were used to collectfrom the forests (Toma et al., 2005); (4)ordinary sweep net collections includingbattery-operated handy vacuum cleanercollections (sweeping) were from theforest and mangrove areas; and (5) con-ventional human-landing collections werefrom the residential, mangrove and forestareas. All traps, BLB trap, BL trap andfrog call trap were hung on branches oftrees and under eaves about 1.5 m aboveground and were operated from 18 : 00 to7 : 00 the next morning. Sweep net collec-tions were made in resting or harboragenatural habitats of mosquitoes, such asclefts of stream banks, around holes offresh and brackish water crabs, and fromvegetation beside animal passes. Thehuman-landing collections were madeduring the course of the daytime surveywork.

The blood-fed mosquitoes capturedwere identified morphologically with thetaxonomic keys of Tanaka et al. (1979)and Toma and Miyagi (1986). Recentstudies have made significant generic andsubgeneric changes in the classificationof the Aedini tribe (Reinert et al., 2004;Reinert and Harbach, 2005; Harbach,2007). However, to avoid confusion inspecies names, we used the traditionalnames following the genus Aedes ofKnight and Stone (1977) except for Ver-rallina (Neomacleaya) atriisimilis (Tanakaand Mizusawa) and Ve. (Verrallina) ir-iomotensis (Tanaka and Mizusawa) fol-lowed by Reinert and Harbach (2005) andLutzia (Metalutzia) vorax Edwards byTanaka (2003). We also used PCR basedmolecular identification for some speci-mens that could not be identifiedmorphologically, such as mosquitoes be-longing to the Cx. vishnui subgroup (Toma

et al., 2000) and the An. sinensis groupwith primers developed in our laboratory(unpublished data). Blood-fed mosquitoescollected were preserved in 99� ethanoluntil used for the bloodmeal analyses.

DNA extractionOne blood-fed mosquito was carefully

homogenized in 10 ml of 0.9� NaCl and240 ml of preheated (100� in heatingblock) 5� Chelex 100 (Bio-Rad Labo-ratories, CA, USA) was added and mixed.After heating for 10 minutes, the samplewas centrifuged at 6,000 rpm for 5 min(Tsuzuki et al., 2001). The supernatantwas utilized directly as a template in PCRor was stored at �20� until PCR wascarried out.

Amplification of 16S rRNA region frommosquito bloodmeals

Bloodmeals were identified by PCR am-plification and DNA sequencing of a frag-ment of the vertebrate mitochondrial 16Sribosomal RNA (rRNA) region. PCRprimers, VerU-1 and VerU-2, designed bySawabe et al. (2010) were used with anamplified product size of about 280 bp.The primers were able to detect verte-brate-derived bloodmeals from any spe-cies. Amplification cycles for theseprimers consisted of initial denaturation at94� for 2 min, followed by 30 cycles of94� for 30 s, 57� for 30 s, and 72� for 90s. The final 72� extension was 4 min. Ifthe above sets of primers failed to yieldPCR products, nested PCR was conductedwith the forward primer 16s-FW2 (5�-GACGAGAAGACCCYATGGA-3�), desi-gned at position 2 inside of the 5� se-quence of the VerU-1 primer, and reverseprimer, VerU-2. Ten pmol of each forwardand reverse primer were used for cock-tails. Three microliters of DNA from eachextracted bloodmeal was used as a tem-plate in 20 ml each reaction. DNA amplific-ations were completed on a PCR ThermalCycler SP TP400 (Takara Bio, Shiga,Japan). Amplification products were sepa-rated on a 1.5� agarose gel in the pres-


ence of ethidium bromide.

DNA sequencing and mosquito bloodmealidentification

PCR products were purified using aQIAquick PCR Purification Kit (Qiagen,Valencia, CA, USA). Vertebrate PCR prod-ucts from mosquito bloodmeals weresequenced in both directions with theBigDye Terminator version 1.1 CycleSequencing Kit (Applied Biosystems,Foster City, CA, USA) with the sameprimers used in the first PCR. PCR-amplified products were purified by etha-nol purification and sequenced directly incycle sequencing reactions with thesequencer ABI PRISM 310 geneticanalyzer (Applied Biosystems).

Sequences of the PCR amplified DNAfragments were then used to performBLAST searches with the GenBank nucle-ic acid database (NCBI, 2010) to determinethe identities of the blood host species.Positive identification and host species as-signment were made when exact or nearlyexact matches (�98�) were obtained.When the results were indicated as pig orwild boar, the host was decided based onthe environment where the mosquitoeswere collected.

R:HJAIH

Mosquito collectionsA total of 35 species in 11 genera were

collected from 5 islands in this study(Table 1). Among the blood-fed mos-quitoes, 581 (13 species) were collected byBLB traps, 196 (12 spp.) by human-landingcollections, 89 (17 spp.) by BL traps, 84 (11spp.) by sweeping and 25 (4 spp.) by frogcall traps (Table 2).

Host-feeding patterns of mosquitoesBloodmeal hosts were successfully

identified from 975 mosquitoes with 13mammalian, 5 bird, 3 reptile, 11 amphibi-an, including 2 unknown frogs, and 2 un-known fish species identified (Table 3).The most common bloodmeal hosts for

mosquitoes in this study were cattle(43.6�), and humans (17.7�) were second.

A total of 799 (81.9� of 975) field-collected mosquitoes fed on mammals(Table 4). Bloodmeals from mosquitoesof the genera Anopheles (4 species), Heiz-mannia (1 sp.), Verrallina (2 spp.), Ficalbia(1 sp.) and Mansonia (1 sp.) were allof mammalian origin. Of the 371 Aedesmosquitoes, 80.1� fed on mammals, 5.1�reptiles, 1.1� amphibians and 13.5�fishes. Of the 302 Culex mosquitoes, 88.1�fed on mammals, 9.9� birds and 1.7� am-phibians. Amphibian-derived bloodmealscomprised 9.3� of all the analyzed blood-meals from Aedes, Culex, Mimomyia andUranotaenia mosquitoes. Reptile-derivedbloodmeals were uncommon and iden-tified only in Ae. (Stg.) riversi Bohartand Ingram, Cx. (Culiciomyia) ryukyensisBohart and Uranotaenia (Pseudoficalbia)jacksoni Edwards. Bloodmeals of fishorigin were identified from Ae. (Geoskusea)baisasi Knight and Hull and Ur. (Pfc.)ohamai Tanaka et al.

Among 13 mosquito species, includingAe. albopictus, Cx. quinquefasciatus, Cx. tri-taeniorhynchus and Armigeres (Armigeres)subalbatus (Coquillett) that fed on humans,8 species fed exclusively on mammals, 2species fed on mammals and birds, 1 spe-cies fed on mammals, birds and amphibi-ans, and 2 species fed on mammals andreptiles suggesting potential pathways ofzoonotic pathogens from wild animals tohuman (Table 4).

Mosquitoes feeding on warm-blooded ani-mals

Bloodmeals derived from warm-bloodedanimals were identified in 21 species of 9genera (Table 5). Cattle-derived blood-meals were most common (46.7�)followed by human-derived bloodmeals(25.1�). Of the 25 bloodmeals of An.(Ano.) lesteri Baisas and Hu collectedaround animal sheds, 72.0� fed on cattleand 20.0� on goats. In the bloodmeals ofAn. (Ano.) saperoi Bohart and Ingram col-lected in the forest, 81.1� were human-

Vol. 62 No. 1 2011 57

and 18.9� were Sus scrofa-derived blood-meals. Because An. saperoi were collectedfrom breeding places of wild boars, weconcluded that they fed dominantly onwild animals. Anopheles sinensis fed pre-dominantly on large domestic animals,

such as cattle (72.1�), goats (16.4�) andwater bu#alos (9.8�). Two An. minimuscollected in the forest of Iriomotejima fedon cattle. Aedes (Finlaya) aureostriatusokinawanus Bohart collected in the forestof Okinawajima fed exclusively on

Table 1. Mosquito species and number of blood-fed mosquitoes collected from 5 islands in the RyukyuArchipelago.

SpeciesIslands mosquitoes collected� Total no.

exam.OKI IHE IRI AMA TOK

An. (Ano.) lesteri 24 0 1 0 0 25An. (Ano.) saperoi 37 0 0 0 0 37An. (Ano.) sinensis 25 29 7 0 0 61An. (Cel.) minimus 0 0 2 0 0 2

Ae. (Fin.) a. okinawanus 5 0 0 0 0 5Ae. (Fin.) j. amamiensis 0 0 0 0 1 1Ae. (Stg.) albopictus 53 0 0 0 11 64Ae. (Stg.) f. miyarai 0 0 1 0 0 1Ae. (Stg.) riversi 22 0 4 0 10 36Ae. (Aed.) v. nipponii 190 13 6 2 0 211Ae. (Geo.) baisasi 9 0 44 0 0 53

Ar. (Arm.) subalbatus 53 4 7 4 0 68

Hz. (Hez.) kana 0 0 0 0 24 24

Ve. (Nma.) atriisimilis 0 0 8 0 0 8Ve. (Ver.) iriomotensis 0 0 6 0 0 6

Cx. (Cux.) fuscocephala 0 0 4 0 0 4Cx. (Cux.) mimeticus 1 0 0 0 0 1Cx. (Cux.) tritaeniorhynchus 158 16 42 20 0 236Cx. (Cux.) pseudovishnui 12 0 0 0 0 12Cx. (Cux.) vishnui 10 0 0 0 0 10Cx. (Cux.) bitaeniorhynchus 4 0 0 0 0 4Cx. (Cux.) quinquefasciatus 27 0 1 0 0 28Cx. (Cui.) ryukyensis 2 0 1 0 0 3Cx. (Lop.) infantulus 0 0 3 0 0 3Cx. (Eum.) h. ryukyuanus 0 0 1 0 0 1

Lt. (Mlt.) vorax 0 0 1 0 0 1

Fi. ichiromiyagii 0 0 1 0 0 1

Mi. (Eto.) elegans 0 0 1 0 0 1Mi. (Eto.) luzonensis 3 0 0 0 0 3

Ma. (Man.) uniformis 6 0 1 0 0 7

Ur. (Pfc.) jacksoni 4 0 0 2 0 6Ur. (Pfc.) nivipleura 0 0 4 0 0 4Ur. (Pfc.) ohamai 0 0 14 0 0 14Ur. (Pfc.) yaeyamana 0 0 3 0 0 3Ur. (Ura.) macfarlanei 2 0 29 0 0 31

Total no. exam. 647 62 192 28 46 975

�OKI, Okinawajima; IHE, Iheyajima; IRI, Iriomotejima; AMA, Amamioshima; TOK, Tokunoshima.


mammals that consisted of wild boars(40.0�), fruits bats (40.0�) and humans(20.0�). Also Ae. albopictus fed exclusive-ly on mammals, 96.9� fed on humans,1.6� fed on a shrew and a rabbit. Of the68 blood-fed Ar. subalbatus collected fromvarious habitats, such as animal sheds,

residential areas, zoological garden andforest areas, 94.1� fed on various domes-tic animals and humans, and 5.9� fed onbirds (ostrich and goose). Although Ar.subalbatus is a mammalophilic feeder, thisspecies fed opportunistically on birds atthe residential areas and the zoological

Table 2. Blood-fed mosquito species collected by di#erent collection methods from 5islands in the Ryukyu Archipelago.

SpeciesCollection methods

BLB trap Human-landing BL trap Sweeping Frog call trap

An. lesteri 25 0 0 0 0An. saperoi 0 36 1 0 0An. sinensis 60 0 1 0 0An. minimus 2 0 0 0 0

Ae. a. okinawanus 0 5 0 0 0Ae. j. amamiensis 0 1 0 0 0Ae. albopictus 2 59 0 3 0Ae. f. miyarai 0 1 0 0 0Ae. riversi 0 36 0 0 0Ae. v. nipponii 191 0 20 0 0Ae. baisasi 0 0 0 53 0

Ar. subalbatus 56 11 1 0 0

Hz. kana 0 24 0 0 0

Ve. atriisimilis 0 8 0 0 0Ve. iriomotensis 0 6 0 0 0

Cx. fuscocephala 1 0 3 0 0Cx. mimeticus 0 0 1 0 0Cx. tritaeniorhynchus 213 0 23 0 0Cx. pseudovishnui 7 0 5 0 0Cx. vishnui 10 0 0 0 0Cx. bitaeniorhynchus 0 0 4 0 0Cx. quinquefasciatus 4 7 13 4 0Cx. ryukyensis 0 2 0 1 0Cx. infantulus 0 0 0 3 0Cx. h. ryukyuanus 0 0 0 1 0

Lt. vorax 0 0 0 1 0

Fi. ichiromiyagii 0 0 1 0 0

Mi. elegans 0 0 1 0 0Mi. luzonensis 0 0 3 0 0

Ma. uniformis 6 0 1 0 0

Ur. jacksoni 0 0 1 5 0Ur. nivipleura 0 0 0 0 4Ur. ohamai 0 0 2 7 5Ur. yaeyamana 0 0 0 1 2Ur. macfarlanei 4 0 8 5 14

Total no. exam. 581 196 89 84 25

Vol. 62 No. 1 2011 59

garden. Heizmannia (Heizmannia) kanaTanaka et al. in the forest of Tokunoshimafed only on humans (100�). Two endemicspecies Ve. atriisimilis and Ve. iriomotensiscollected by human-landing collection fedon humans.

Four Cx. (Cux.) fuscocephala Theobald

collected around cattle sheds in Iri-omotejima fed on cattle. A total of 236blood-fed Cx. tritaeniorhynchus were col-lected from various habitats such as forestareas, animal sheds, residential areas andzoological garden. This species fed pre-dominantly on mammals (96.1�) e.g.,

Table 3. Host animals for blood-fed mosquitoes collected from 5 islands in the Ryukyu Archipelago.

Host

selehpo

nA

sede

A

seregimr

A

ain

namzie

H

anillarre

V

xelu

C

aiztu

L

aiblaciF

aiymo

miM

ainos

naM

aineato

narU

Total no.exam.

Human (Homo sapiens) 30 80 11 24 14 14 0 0 0 0 0 173Cat (Felis catus) 0 0 0 0 0 3 0 0 0 0 0 3Dog (Canis lupus familiaris) 1 0 0 0 0 2 0 0 0 0 0 3Shrew (Suncus murinus) 0 1 0 0 0 0 0 0 0 0 0 1Cattle (Bos taurus) 64 154 11 0 0 194 0 0 1 1 0 425Pig (Sus scrofa) 1 12 14 0 0 1 0 0 0 0 0 28Goat (Capra hircus) 15 38 23 0 0 17 0 0 0 6 0 99Bu#alo (Bubalus arnee) 7 0 0 0 0 2 0 0 0 0 0 9Rabbit (Oryctolagus cuniculus) 0 1 0 0 0 0 0 0 0 0 0 1Wild boar (Sus scrofa) 7 2 0 0 0 3 0 1 0 0 0 13Fruit bat (Pteropus dasymallus) 0 3 0 0 0 0 0 0 0 0 0 3Gira#e (Gira#a camelopardalis) 0 1 1 0 0 3 0 0 0 0 0 5White rhinoceros (Ceratotherium simum) 0 5 4 0 0 27 0 0 0 0 0 36

Fowl (Gallus gallus) 0 1 0 0 0 25 0 0 0 0 0 26Goose (Anser anser) 0 0 1 0 0 0 0 0 0 0 0 1Ostrich (Struthio camelus) 0 0 3 0 0 4 0 0 0 0 0 7Sparrow (Turdus ruficollis) 0 0 0 0 0 1 0 0 0 0 0 1Sparrow (Parus major) 0 0 0 0 0 0 1 0 0 0 0 1

Ground gecko (Goniurosaurus kuroiwae) 0 0 0 0 0 0 0 0 0 0 1 1Lizard (Takydromus smaragdinus) 0 0 0 0 0 1 0 0 0 0 0 1Tortoise (Geoemyda japonica) 0 19 0 0 0 0 0 0 0 0 0 19

Newt (Cynops ensicauda) 0 0 0 0 0 0 0 0 0 0 1 1Indian rice frog (Fejervarya limnocharis) 0 1 0 0 0 1 0 0 0 0 0 2Sakishima rice frog

(Fejervarya sakishimensis)0 2 0 0 0 1 0 0 0 0 34 37

Namie’s frog (Limnonectes namiyei) 0 0 0 0 0 0 0 0 0 0 4 4Rana sp. 0 0 0 0 0 0 0 0 0 0 2 2Okinawa green tree frog

(Rhacophorus viridis viridis)0 0 0 0 0 0 0 0 0 0 1 1

Rhacophorus sp. 0 0 0 0 0 3 0 0 0 0 3 6White-lipped tree frog

(Polypedates leucomystax)0 0 0 0 0 0 0 0 1 0 0 1

Ei$nger’s tree frog (Kurixalus ei$ngeri) 0 0 0 0 0 0 0 0 0 0 6 6Ryukyu kajika frog (Buergeria japonica) 0 0 0 0 0 0 0 0 2 0 1 3Narrow-mouthed toad (Microhyla okinavensis) 0 1 0 0 0 0 0 0 0 0 4 5

Goby (suborder Gobioidei order Perciformes) 0 37 0 0 0 0 0 0 0 0 1 38Snake eel (order Anguilliformes) 0 13 0 0 0 0 0 0 0 0 0 13


cattle and humans in animal sheds, whiterhinoceroses and gira#es in zoologicalgarden. Of 201 the Cx. tritaeniorhynchuscollected around animal sheds, 96.5� fedon domestic animals and 3.5� fowls. Of12 the Cx. (Cux.) pseudovishnui Colless col-

lected around animal sheds, 58.3� fed oncattle and 41.7� fowls. Of the 10 Cx.(Cux.) vishnui Theobald collected aroundhuman habitations, such as animal shedsand zoological garden, 60.0� fed onmammals (cattle and white rhinoceroses),

Table 4. Number of bloodmeals by host class identified from 35 species of mosquitoes collected from 5Islands in the Ryukyu Archipelago, 2005�2010.

Species Human Mammals Birds Reptiles Amphibians Fishes Total no. exam.

An. lesteri � 25 0 0 0 0 25An. saperoi � 37 0 0 0 0 37An. sinensis � 61 0 0 0 0 61An. minimus � 2 0 0 0 0 2

Ae. a. okinawanus � 5 0 0 0 0 5Ae. j. amamiensis � 1 0 0 0 0 1Ae. albopictus � 64 0 0 0 0 64Ae. f. miyarai � 1 0 0 0 0 1Ae. riversi � 17 0 19 0 0 36Ae. v. nipponii � 209 1 0 1 0 211Ae. baisasi 0 0 0 3 50 53

Ar. subalbatus � 64 4 0 0 0 68

Hz. kana � 24 0 0 0 0 24

Ve. atriisimilis � 8 0 0 0 0 8Ve. iriomotensis � 6 0 0 0 0 6

Cx. fuscocephala � 4 0 0 0 0 4Cx. mimeticus 1 0 0 0 0 1Cx. tritaeniorhynchus � 227 9 0 0 0 236Cx. pseudovishnui � 7 5 0 0 0 12Cx. vishnui � 6 4 0 0 0 10Cx. bitaeniorhynchus � 2 2 0 0 0 4Cx. quinquefasciatus � 17 10 0 1 0 28Cx. ryukyensis � 2 0 1 0 0 3Cx. infantulus 0 0 0 3 0 3Cx. h. ryukyuanus 0 0 0 1 0 1

Lt. vorax 0 1 0 0 0 1

Fi. ichiromiyagii 1 0 0 0 0 1

Mi. elegans 0 0 0 1 0 1Mi. luzonensis 1 0 0 2 0 3

Ma. uniformis � 7 0 0 0 0 7

Ur. jacksoni 0 0 1 5 0 6Ur. nivipleura 0 0 0 4 0 4Ur. ohamai 0 0 0 13 1 14Ur. yaeyamana 0 0 0 3 0 3Ur. macfarlanei 0 0 0 31 0 31

Total no. exam. 799 36 21 68 51 975

�� mosquitoes fed on human in this study.�� based on Toma and Miyagi (1986), Tanaka et al. (1979) and Miyagi et al. (1992).

Vol. 62 No. 1 2011 61

Table 5. Number of warm-blooded animal-derived bloodmeals identified from 21 mosquito speciescollected from 5 islands in the Ryukyu Archipelago, 2005�2010.

Species Is.�

��statiba

H

Sources of bloodmeals

Totalno. exam.

MammalsMammals BirdsBirds

nam

uH

go

D

werh

S

elttaC

giP

tao

G

ola#

uB

tib

baR

rao

B

tab

tiur

F

e#ari

G

.o

nih

R

lw

oF

eso

oG

hcirtsO

worra

pS

An. lesteri OKI A 0 0 0 18 1 5 0 0 0 0 0 0 0 0 0 0 24IRI A 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1

An. saperoi OKI F 30 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 37

An. sinensis OKI A 0 1 0 14 0 9 0 0 0 0 0 0 0 0 0 0 24IHE A 0 0 0 29 0 0 0 0 0 0 0 0 0 0 0 0 29IRI A 0 0 0 1 0 0 6 0 0 0 0 0 0 0 0 0 7OKI R 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1

An. minimus IRI F 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2

Ae. a. okinawanus OKI F 1 0 0 0 0 0 0 0 2 2 0 0 0 0 0 0 5

Ae. j. amamiensis TOK F 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

Ae. albopictus OKI F 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3TOK F 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11OKI M 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4OKI A 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1OKI R 44 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 45

Ae. f. miyarai IRI F 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

Ar. subalbatus IRI F 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1OKI A 0 0 0 0 14 23 0 0 0 0 0 0 0 0 0 0 37IHE A 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2IRI A 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2AMA A 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 4OKI R 6 0 0 1 0 0 0 0 0 0 0 0 0 1 1 0 9IHE R 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2IRI R 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4OKI Z 0 0 0 0 0 0 0 0 0 0 1 4 0 0 2 0 7

Hz. kana TOK F 24 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 24

Ve. atriisimilis IRI F 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8

Ve. iriomotensis IRI F 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6

Cx. fuscocephala IRI A 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 4

Cx. mimeticus OKI F 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1

Cx. tritaeniorhynchus OKI F 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1IRI F 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1OKI A 1 0 0 102 1 17 0 0 0 0 0 0 3 0 0 0 124IHE A 0 0 0 12 0 0 0 0 0 0 0 0 4 0 0 0 16IRI A 0 0 0 39 0 0 2 0 0 0 0 0 0 0 0 0 41AMA A 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 20OKI R 0 0 0 1 0 0 0 0 0 0 0 0 2 0 0 0 3OKI Z 0 0 0 0 0 0 0 0 1 0 3 26 0 0 0 0 30


and 40.0� birds (fowls and ostriches).The endemic species Ficalbia ichiro-

miyagii Toma and Higa from Iriomotejimafed on a wild boar in the forest.

Mosquitoes feeding on cold-blooded animalsMosquitoes feeding exclusively on cold-

blooded animal hosts consisted of 1 spe-cies of Aedes, 2 Culex, 1 Mimomyia and 5Uranotaenia (Table 6). Of 116 mosquitoesthat fed on cold-blooded animals, 54.3�were frog-derived. Of the 53 bloodmealsof Ae. baisasi, 94.3� fed on fishes (gobyand snake eel) and 5.7� on the frogFejervarya sakishimensis. Mimomyia (Etor-leptiomyia) elegans (Taylor) collected in theforest area of Iriomotejima fed on a frogBuergeria japonica. Five species of Urano-taenia fed predominantly on frogs (94.8�),a ground gecko (1.7�), a newt (1.7�) and afish (1.7�), none of them fed on warm-blooded animals. They fed on 10 frogspecies, such as F. sakishimensis andMicrohyla okinavensis (Table 3). TheUranotaenia species fed notably on frogsbut had no particular preferred species.

Mosquitoes feeding on warm- and cold-blooded animals

Mosquitoes feeding on both warm- andcold-blooded animals consisted of 2 spe-cies of Aedes, 2 Culex and 1 Mimomyia(Table 7). Of 36 Ae. riversi collected fromthe forest on 3 islands, 52.8� fed on rep-tiles (tortoises) and 47.2� on mammalssuch as human and fruit bat. The host-feeding pattern of this species was di#er-ent from other species belonging to thesame subgenus Stegomyia. Aedes (Aedimo-rphus) vexans nipponii (Theobald) fed pre-dominantly on large animals such ascattle and goats around animal sheds, andwhite rhinoceroses in the zoologicalgarden with 99.1� of 211 analyzed blood-meals. The remaining 2 fed on a fowl anda frog F. limnocharis.

Although most of Cx. quinquefasciatuswere collected from human habitations,the host-feeding patterns of this specieswere somewhat di#erent from thesamples collected in di#erent places. Ofthe 9 Cx. quinquefasciatus collected at theanimal sheds, 22.2� fed on humans and77.8� fed on fowls, while of the 17 at the

Table 5. Continued

Species Is.�

��statiba

H


Totalno. exam.

MammalsMammals BirdsBirds

nam

uH

go

D

werh

S

elttaC

giP

tao

G

ola#

uB

tib

baR

rao

B

tab

tiur

F

e#ari

G

.o

nih

R

lw

oF

eso

oG

hcirtsO

worra

pS

Cx. pseudovishnui OKI A 0 0 0 7 0 0 0 0 0 0 0 0 5 0 0 0 12

Cx. vishnui OKI A 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 5OKI R 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1OKI Z 0 0 0 0 0 0 0 0 0 0 0 1 0 0 3 0 4

Cx. bitaeniorhynchus OKI F 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2OKI A 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 2

Lt. vorax IRI F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1

Fi. ichiromiyagii IRI F 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1

Ma. uniformis OKI A 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 5IRI A 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1OKI R 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1

� Same as Table 1.�� A, Animal sheds; F, Forest; R, Residential area; M, Mangrove forest; Z, Zoological garden.

Vol. 62 No. 1 2011 63

residential areas, 88.2� fed on mammalsincluding humans, 5.9� on a fowl and5.9� a frog. Three bloodmeals of Cx.ryukyensis were human, wild boar andlizard origins.

Three bloodmeals of Mi. (Etorleptiomyia)luzonensis (Ludlow), 2 from forest areasfed on frogs (Polypedates leucomystax andBuergeria japonica) and the other from aresidential area fed on cattle.

D>H8JHH>DC

The host-feeding patterns of mosquitoesare governed by a number of factors suchas innate tendencies (host preferences),flight behavior and feeding periodicity ofmosquitoes as well as availability, spatial

distribution and defensive behavior ofhost animals (Clements, 1999). Therefore,it is important to clarify host preferencesfor the large numbers of blood-fedmosquitoes including many species col-lected by various methods at di#erenthabitats throughout the year. The presentstudy provides information on the feedinghabits of 35 species collected by 5methods from 5 islands of the Ryukyus,Japan during 2005�2010.

Twenty-one species including 6 speciesof Culex, 4 Anopheles and 4 Aedes fed en-tirely on warm-blooded animals suggest-ing potential vectors of diseases amonghumans as well as wild warm-bloodedanimals. Five species that fed on bothwarm- and cold-blooded animals are con-

Table 6. Number of cold-blooded animal-derived bloodmeals identified from 9 mosquito speciescollected from 3 islands in the Ryukyu Archipelago, 2005�2010.

Species Is.�

��statiba

H


Totalno. exam.

ReptilesReptiles AmphibiansAmphibians FishesFishes

okce

gd

nu

orG

sg

orF

twe

N

��sehsi

F

Ae. baisasi OKI M 0 0 0 9 9IRI M 0 3 0 41 44

Cx. infantulus IRI F 0 3a 0 0 3

Cx. h. ryukyuanus IRI F 0 1a 0 0 1

Mi. elegans IRI F 0 1 0 0 1

Ur. jacksoni OKI F 1 3 0 0 4AMA F 0 2b 0 0 2

Ur. nivipleura IRI F 0 4 0 0 4

Ur. ohamai IRI F 0 13 0 1 14

Ur. yaeyamana IRI F 0 3 0 0 3

Ur. macfarlanei OKI F 0 3 1 0 4IRI F 0 27 0 0 27

� Same as Table 1. �� Same as Table 5.�� Fishes include goby (suborder Gobiodei of order Perciformes, with 91�92� sequence identity)

and snake eel (order Anguilliformes, with 89�95�).a Two specimens of Cx. infantulus and 1 of Cx. h. ryukyuanus showed 97 and 96� sequence identity,

respectively, to Rhacophorus moltrechti which does not occur in Iriomotejima.b Two specimens showed 91� sequence identity to Rana pleuraden which does not occur in

Amamioshima.


sidered to be general feeders and havenumerous opportunities to contact a widevariety of wild animals. In contrast, 9species that fed entirely on cold-bloodedanimals are special feeders and have thepotential to be an e$cient vector of spe-cific host animals.

The Culex species exhibited di#erenthost-feeding patterns between subgeneraand fed on mammals, birds, a reptile andamphibians. Genera of Aedes, Anopheles,Armigeres, Heizmannia, Verrallina andMansonia were mammalophilic feeding,most species of these genera exceptAnopheles and Mansonia fed on humans.The 5 Uranotaenia species were mainlyfrog-feeders.

Culex is a large genus consisting of var-ious subgenera including several speciesof medical importance, namely Cx. tri-

taeniorhynchus that transmits Japanese en-cephalitis virus (JEV), and the Cx. pipiensgroup that transmits West Nile virus(WNV) and human filariasis. The host-feeding patterns of Culex are known for alimited number of species that aremosquito-borne disease vectors. Thesespecies fed on various hosts. For somespecies the primary hosts are mammals,for others birds, while certain species feedregularly on both mammals and birds. Asmall percentage of Culex species feed onamphibians or reptiles (Clements, 1999).The patterns were somewhat di#erent be-tween subgenera. Of the 20,522 Cx. tri-taeniorhynchus of subgenus Culex collect-ed near animal sheds on Okinawajima,more than 80� fed on cattle and pigs(Pennington and Phelps, 1968). In ourstudy, the feeding habits of Cx. tri-

Table 7. Number of warm- and cold-blooded animal-derived bloodmeals identified from 5 mosquitospecies collected from 5 islands in the Ryukyu Archipelago, 2005�2010.

Species Is.�

��statiba

H


Totalno. exam.

MammalsMammals BirdsBirds Reptil.Reptil. AmAm

nam

uH

taC

go

D

elttaC

giP

tao

G

rao

B

tab

tiur

F

e#ari

G

.o

nih

R

lw

oF

hcirtsO

worra

pS

draziL

esiotr

oT

sg

orF

Ae. riversi OKI F 1 0 0 1 0 0 0 1 0 0 0 0 0 0 19 0 22IRI F 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4TOK F 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10

Ae. v. nipponii OKI A 0 0 0 130 12 38 0 0 0 0 1 0 0 0 0 1 182IHE A 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 13IRI A 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 6AMA A 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2OKI R 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1OKI Z 0 0 0 1 0 0 0 0 1 5 0 0 0 0 0 0 7

Cx. quinquefasciatus OKI M 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1OKI A 2 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 9OKI R 9 3 2 0 0 0 0 0 0 0 1 0 0 0 0 1 16IRI R 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1OKI Z 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1

Cx. ryukyensis OKI F 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1IRI F 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1OKI M 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1

Mi. luzonensis OKI F 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2OKI R 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1

� Same as Table 1. ��Same as Table 5. Am; Amphibians.

Vol. 62 No. 1 2011 65

taeniorhynchus and Cx. vishnui were mam-malophilc, but they occasionally fed onbirds and humans at the animal sheds andthe zoological garden. These species are apotential bridge vector for WNV in theRyukyus. In the 1970’s many blood-fedfemales of Cx. tritaeniorhynchus were col-lected from a cattle shed, pigsty and hen-house in Okinawajima (Iha, 1971), and JEVwas isolated from Cx. tritaeniorhynchus andCx. pseudovishnui on this island (Ura, 1976;Ura et al., 1981). Localized persistence ofJEV in Okinawajima under suitable condi-tions was suggested in a previous study(Hayashi et al., 1978), and we suggestthese 2 species play a role in bird tomammal and mammal to mammal trans-mission of JEV. The susceptibility of Cx.pseudovishnui to JEV is lower than Cx.tritaeniorhynchus under laboratory condi-tions (Doi et al., 1977). Although Cx.pseudovishnui is not common in theRyukyus (Toma and Miyagi, 1986), thisspecies fed on cattle and fowls near theanimal sheds in the present study,suggesting it could become a potentialbridge vector for WNV, and play a role inthe transmission for JEV as well as Cx.tritaeniorhynchus and Cx. vishnui. In Thai-land, Cx. fuscocephala is considered to bean e$cient vector of JEV (Muangman etal., 1972). This species is very common onthe Yaeyama Islands including Iriomo-tejima, but has never been found onOkinawajima and Amamioshima (Tomaand Miyagi, 1986). Because of themammalophilic feeding habit of Cx.fuscocephala, this species could also be avector in the mammal to mammal trans-mission of JEV in the Yaeyama Islands.

Irby and Apperson (1988) reported thatCx. quinquefasciatus fed primarily on birdsin natural habitats near farms in NorthCarolina. Sawabe et al. (2010) reportedthat of the blood-fed Cx. quinquefasciatuscollected around residential areas inOkinawa, 62.5� fed on mammals and29.2� on birds. In our study, this speciesfed on birds rather than mammals at theanimal sheds, but fed on mammals rather

than birds at the residential areas. Al-though, this species is basically orni-thophilic, it is also an opportunistic feederthat feeds readily on mammals includinghumans in residential areas. This speciesis regarded as an important vector forWNV (Molaei et al., 2007; Turell et al.,2005), and we suggest it is a candidatebridge vector for WNV in the Ryukyus.Culex ryukyensis of subgenus Culiciomyiafed on mammals (human and wild boar)and a reptile (lizard) in this study. Culex(Cui.) sasai Kano et al. of the same subge-nus fed predominantly on birds in urbanTokyo (Kim et al., 2009a). In the presentstudy, Cx. (Lophoceraomyia) infantulus Ed-wards from Iriomotejima fed on frogs, in-cluding F. sakishimensis and Rhacophorusspecies. This species collected in NagasakiCity fed readily on frogs, snakes, turtles,chicks and mice under laboratory condi-tions (Miyagi, 1972). In the absence ofpreferred hosts, this mosquito may beforced to feed on other available hosts.Culex (Eumelanomyia) hayashii ryukyuanusTanaka et al. from Iriomotejima fed on afrog in this study. The feeding habits ofthe subspecies are similar to Cx. (Eum.)hayashii Yamada collected in NagasakiCity (Miyagi, 1973).

The genus Aedes includes many speciesand subgenera. Some species, such as Ae.(Stg.) aegypti (Linnaeus) and Ae. albopictus,are medically important as vectors. Theresults of bloodmeal identification ob-tained in this study suggest that mammalsare the primary hosts for most Aedes spe-cies, but most of the available data is limit-ed to several species of the subgeneraOchlerotatus and Stegomyia (Tempelis,1975; Clements, 1999). In Japan, Ae.albopictus, Ae. riversi and Ae. v. nipponiiwere reported to feed predominantly onmammals (Sawabe et al., 2010; Toma andMiyagi, 1981, 1986; Takahashi et al., 1971;Kim et al., 2009b). In our study, the Aedesspecies, except Ae. baisasi that fed onfishes, exhibited mammalophilic feedinghabits. Okudo et al. (2004) reported thatthe bloodmeals from Ae. baisasi were fish-


derived blood as identified by ELISA andobservations of mosquitoes fed on fishunder laboratory conditions. The hostpreference of Ae. baisasi is di#erent fromthe other members of the subgenusGeoskusea.

Epidemics of dengue fever have beenreported from time to time in the Ryukyus(Ura, 1978), and Ae. aegypti was consideredan important vector at that time (Miyao,1931). Recently, Ae. aegypti were notfound in spite of extensive surveillancesby ovi-traps in the Ryukyus (Higa et al.,2007). In our study, Ae. albopictus ex-hibited a highly anthropophilic feedinghabit. As mentioned in previous studies(Sawabe et al., 2010), this species may playan important role in mammal-to-mammaltransmission of viruses such as denguevirus and Chikungunya virus in theRyukyus.

Anopheles is a large genus and widelydistributed throughout the world. This isthe most extensively investigated genus,because it includes many species of medi-cal importance. Bloodmeal analyses haveshown that many species of the genus feedalmost exclusively on mammals, this mayprimarily be because the samples analyzedwere collected mainly in and aroundhuman habitations (Clements, 1999; Belkin,1962; Tempelis, 1975). In the Ryukyus, 7species of Anopheles are recorded fromdi#erent islands (Toma and Miyagi, 1986).In the previous studies of host-feeding pat-terns by human- and cattle-baited net-trapcollections in Ishigakijima, the Ryukyus,many adult females of An. sinensis and An.lesteri were collected by cattle-baited net-traps, and many An. minimus were collect-ed by both methods from sunset to mid-night (Miyagi and Toma 1978a). In thepresent studies, An. minimus, An. sinensisand An. lesteri fed predominantly oncattle, but An. saperoi collected from theforest fed on humans and wild boars. Theavailability of humans as a blood source inthe forest is low, so the primary host ofAn. saperoi may be the Ryukyu wild boar(S. s. riukiuanus), which is very common in

the mosquito’s breeding areas onOkinawajima and Iriomotejima. Theseresults indicate that Anopheles is attractedto large mammals or humans dependingon the main availability of host animals.

In the past An. minimus was the mostimportant malaria vector in the Ryukyus(Kuroshima, 1960; Miyagi et al., 1996), andthis species is still an important malariavector in Southeast Asia (Trung et al.,2004, 2005; Kiszewski et al., 2004). Inrecent years the population of An. mini-mus in Ishigakijima has been increasing(Toma et al., 2002). Although An. minimusfed on cattle in the previous study, it is ananthoropophilic as well as zoophilic spe-cies (Miyagi and Toma, 1978a). Anophelessaperoi may have played an important rolein the vivax malaria epidemics thatoccurred in the forests of the northernpart of Okinawajima from 1946 to 1948(Nakamatsu, 1942; Toma and Miyagi,1981).

A8@CDLA:<B:CIH

We thank Ms. Y. Endo, Ms. T. Higa andMs. T. Kinjyo of our laboratory, Mr. S.Fujiwara and Mr. S. Omi of Amami CityHall, Ms. H. Nakamura of TokunoshimaHealth Center for performing laboratorywork and assisting with the collection ofmosquitoes from the field. We would alsolike to thank Dr. Yong Hoi Sen, UniversityMalaya for kindly reviewing the English,and Mr. A. Nakamoto director of IkariOkinawa Corporation for his kind supportand encouragement in this study. Weextend special thanks to the sta#members of the Subtropical Field ScienceCenter, University of Ryukyus, IriomoteWildlife Center and Amami WildlifeCenter of Ministry of the Environment forproviding facilities during the fieldstudies. This contribution was partiallysupported by Grant for Research (2009)from Uruma Foundation, for ExploratoryResearch from the Ministry of Education,Culture, Sports, Scientific and Technology(MEXT), 2005�2007, and for Scientific Re-

Vol. 62 No. 1 2011 67

search on Emerging and Re-emerging In-fectious Diseases (2003�2008) from theJapanese Ministry of Health, Labor andWelfare, Japan.

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