Bloodmeal identification and the detection of avian malaria parasite from mosquitoes (Diptera: Culicidae) were carried out by polymerase chain reaction-based methods for field samples collected in coastal areas of Tokyo Bay, Japan, from April to October 2007. The following seven mosquito species were collected: Aedes albopictus (Skuse), Culex pipiens pallens Coquillett, Culex pipiens form molestus Forskal, Culex tritaeniorhynchus Giles, Culex inatomii Kamimura & Wada, Culex bitaeniorhynchus Giles, and Lutzia vorax Edwards. Forty blood-fed mosquitoes were collected and 95% of bloodmeals of Cx. pipiens pallens were avian-derived, whereas only mammalian bloodmeals were identified for Ae. albopictus. Plasmodium DNA was amplified from 65% (15/23) of blood-fed Cx. pipiens pallens and unfed females of Cx. pipiens pallens and Cx. pipiens form molestus with a minimum infection rate of 29.9 and 13.5, respectively. One unfed female of Lt. vorax was also positive for Plasmodium parasites. Five genetically distinct lineages of Plasmodium were identified, with 0.21 to 5.86% sequence divergence. Rinshi-8, the most prevalent lineage at our study site, was identical to the published sequence of Plasmodium relictum-P5.
Avian malaria caused by Hemoproteus sp. and Plasmodium sp. is a vector-borne disease globally distributed among wild birds, and recent molecular-based studies on avian malaria parasites revealed the remarkable diversity of the parasites (Ricklefs and Fallon 2002, Beadell et al. 2004, Valkiūnas 2005). Although there has been no report of devastating impacts of avian malaria parasites on native bird communities in Japan as reported in Hawaii (Van Riper et al. 1986), detrimental effects and deaths of captive birds due to avian malaria infection have been reported previously (Murata 2002, Isobe 2007, Murata et al. 2008). It is important to understand the present situation about transmission of avian malaria parasites in Japanese wild bird communities to differentiate indigenous from novel parasite lineages that could be introduced by exotic birds. Because infected birds can carry the malaria parasites for years as a chronic infection, it is difficult to identify the time and location of transmission for each parasite in the field (Valkiūnas 2005). However, ecological studies on mosquito (Diptera: Culicidae) populations such as seasonal prevalence and feeding behavior could provide essential information in relation to ongoing transmission pathways of avian malaria parasites as demonstrated in other vector-borne diseases (Kilpatrick et al. 2006, Silver 2008). Bloodmeal identification and detection of avian malaria parasites from single blood-fed mosquitoes by a polymerase chain reaction (PCR)-based method were carried out to obtain evidence of direct contact of the mosquito with the blood source animal and pathogen in the field, which is valuable information for identifying possible transmission pathways of vector-borne pathogens (Massey et al. 2007, Ishtiaq et al. 2008).
We focused on mosquitoes inhabiting coastal areas of Tokyo Bay, Japan, because the coastal areas receive many birds that migrate between northern and southern regions along island chains (Yamashina Institute for Ornithology 2002) and are good locations to examine the risk of new introductions of pathogens. Our objectives were to investigate 1) mosquito fauna in the current study site, which serve as a sanctuary for resident and migratory birds; 2) feeding patterns of these mosquito species with respect to their potential as vectors of avian malaria parasites that are transmitted among Japanese wild bird communities; and 3) to conduct a survey for Plasmodium parasites in potential mosquito vectors.
Materials and Methods
From April to October 2007, mosquitoes were collected at Tokyo Port Wild Bird Park (35° 35′ N, 136° 41′ E), which was created out of reclaimed land along Tokyo Bay, Japan, in the southern Tokyo metropolitan area. The park is 24.2 ha and contains freshwater ponds surrounded by shrubs, mud flats, and a tidal basin (Tsuda et al. 2009). Mosquito collections were carried out twice a month using battery-operated dry-ice traps without a light (CDC-like suction trap, Inokuchi-Tekko, Nagasaki, Japan), the BG-sentinel trap (BioGents GmbH, Regensburg, Germany) and sweep nets. Twelve dry-ice traps enhanced with 1 kg of dry ice were fixed to trees at 1.2–2 m from the ground, and one BG-sentinel trap was operated in combination with the BG-Lure and 1 kg dry ice in a small field hut for 24 h. Resting mosquitoes were collected from vegetation under a tree canopy by using a sweep net (36 cm in diameter) by two persons for 1.5 h in three locations. In addition, resting mosquitoes were collected by a sweep net and sucking tube from the wall of a rest room in August, September, and October 2007. The following morning, all trapped mosquitoes were taken to the National Institute of Infectious Diseases for species identification following Tanaka et al. (1979). The PCR-based identification method developed by Kasai et al. (2008) was applied to distinguish Culex pipiens pallens Coquillett and Culex pipiens form molestus Forskal. Unfed mosquito samples were divided and used for West Nile Virus isolation and avian malaria parasite detection. This article deals with the results of avian malaria parasite detection.
Preparation of Genomic DNA for PCR Assay.
For blood-fed specimens, each mosquito was individually processed: the head was removed to eliminate an inhibitory effect on PCRs (Arez et al. 2000), and the rest of the body was separated into thorax and abdomen by using microscissors under a microscope. Genomic DNA was extracted from the thorax and abdomen separately, by using a REDExtract-N-Amp Tissue PCR kit (Sigma, St. Louis, MO). For unfed mosquito specimens, 1–20 (average, 10.5) individuals, of which the head was removed, were pooled by species, and DNA was extracted using DNeasy Blood & Tissue kits (QIAGEN, Valencia, CA) as described by the manufacturer.
Parasite Detection by Nested PCR.
For genetic analysis of parasite DNA, we followed the nested-PCR protocol described by Waldenström et al. (2004) to amplify a fragment of 478 nucleotides of the parasite cytochrome b (cyt b) gene, with slight modifications. Final reaction volumes were adjusted to 20 μl instead of 25 μl and the initial PCR was performed over 25 cycles instead of 20 cycles. All amplified fragments were purified using the QIAEX II-Gel Extraction kit or QIAquick PCR Purification kit following the manufacturer’s instructions (QIAGEN). Purified DNA fragments were directly cycle-sequenced in both directions using the ABI PRISM BigDye Terminator cycle sequencing kit version 1.1 (Applied Biosystems, Foster City, CA) and the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Sequencing analyses were performed using the program GENETYX-WIN version 8 and compared with published sequences in the GenBank database (National Center for Biotechnology Information website, http://www.ncbi.nlm.nih.gov/BLAST/).
Genomic DNA extracted from the abdomen of blood-fed mosquitoes was used for PCR-based assay with primers designed by a multiple alignment of the cyt b sequences of mitochondrial DNA and 16S rDNA region obtained from GenBank or published primer sequences (Ngo and Kramer 2003, Molaei et al. 2006) to identify the vertebrate host on which mosquitoes had fed. DNA templates were initially screened with two pairs of primers to detect avian- and mammalian-derived bloodmeals, which are Avian-3 [5′-GACTGTGAYAAAATYCCMTTCCA-3′] and Avian-8 [5′-GYCTTCAITYTTTGGYTTACAAGAC-3′] and Mammalian-1 [5′-TGAYATGAAAAAYCATCGTTG-3′] and Mammalian-2 [5′-TGTAGTTRTCWGGGTCKCCTA-3′]. When no PCR products were obtained from a specimen, an additional universal primer set VerU-1 (5′-AAGACGAGAAGACCCYATGGA-3′) and VerU-2 (5′-CCTGATCCAACATMGAGGTCGTA-3′), which detects all types of vertebrate-derived bloodmeals, including avian, mammalian, and amphibian-derived, was applied. The PCR cycling conditions were as follows: initial denaturation at 94°C for 2 min, then 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s and 90 s for avian and mammalian primers, respectively, followed by 4-min final extension at 72°C. The thermal profile for the primer set of VerU-1/VerU-2 was the same as that for avian primers, except that the annealing temperature was 58°C. All PCR products were used for the sequencing analysis described above and identified to species with >99% sequence identity.
Results and Discussion
The following seven mosquito species were collected in this study: Aedes albopictus (Skuse), Cx. pipiens pallens, Cx. pipiens form molestus, Culex tritaeniorhynchus Giles, Culex inatomii Kamimura & Wada, Cx. bitaeniorhynchus Giles, and Lutzia vorax Edwards (Table 1). Species composition of the samples differed between the three collection methods. Culex pipiens group was dominant and made up 67 and 46% of all females in the BG-sentinel trap and dry-ice trap collections, respectively, whereas Ae. albopictus made up 98% of all females in sweeping collection. The four other species comprised 8% of the total collections.
Avian malaria parasite DNA was amplified from Cx. pipiens pallens and Cx. pipiens form molestus with minimum infection rates (MIRs) of 29.9 and 13.5, respectively. One unfed female Lt. vorax was also positive for avian malaria parasite (Table 1). Seasonal changes in the composition of Cx. pipiens pallens and Cx. pipiens form molestus with the percentage and MIR of unfed Cx. pipiens pallens infected with Plasmodium spp. are shown in Fig. 1. In April and May, Cx. pipiens form molestus made up >78% of Cx. pipiens gr., and the rate decreased markedly to 1.8% from June to September. Females infected with Plasmodium parasites were first collected in July, and the percentage continuously increased until October (Fig. 1). The host-seeking female density and the infection rate of avian malaria parasites in Cx. pipiens pallens were high in summer (Fig. 1), which coincided with the season when a number of susceptible fledglings of residents and summer visitors are present.
Forty blood-fed mosquitoes were collected, of which 10 were collected by dry ice trap, 11 by sweeping, and 19 by the sucking tube in the rest room. The results of bloodmeal identification and detection of avian malaria parasite are summarized in Table 2. Plasmodium DNA was detected from the abdomen, but not the thorax, of blood-fed mosquitoes in this study. The results of bloodmeal identification revealed that Cx. pipiens pallens and Ae. albopictus feed primarily on birds and mammals, respectively. The previous studies on feeding habits of Hawaiian Ae. albopictus revealed that Ae. albopictus fed almost exclusively on mammals, but had an opportunistic feeding behavior and fed on birds where mammalian hosts were scarce (Hess et al. 1968, Tempelis et al. 1970). Although Ae. albopictus is unlikely to be an important vector in the current study site, this mosquito species is a known vector of P. gallinaceum and P. lophurae (Valkiūnas 2005), and Plasmodium DNA has been detected from Ae. albopictus in an oceanic island (Ejiri et al. 2008). Therefore, this mosquito species could play a role as a vector for avian malaria parasites where birds are the dominant blood source animals.
Sequences of the 478-bp fragment of parasite cyt b were compared and five genetically distinct Plasmodium lineages were identified in this study, with 0.21 to 5.86% sequence divergence (Table 2). Rinshi-8, a 100% match to P. relictum-P5, was most prevalent in our study site. This parasite lineage has been detected from blood of carrion crow (Corvus corone) in Japan (Beadell et al. 2006) and detected from blood-fed and unfed Cx. pipiens pallens in this study. Thus, it is likely that transmission of Rinshi-8 is ongoing between Japanese wild birds and this mosquito. Rinshi-7 has been detected from Culex quinquefasciatus Say collected in Japan (Ejiri et al. 2008) but not reported from blood specimens of resident birds in Japan. The detection of Rinshi-7 from Cx. pipiens pallens with a bloodmeal derived from Delichon dasypus (Asian house martin), which is a summer visitor in Japan, demonstrates the possible introduction of this malaria parasite into Japan through the movement of infected migratory birds. Vector competence of the mosquito species for each parasite lineage is not conclusive because of the limitations of the PCR-based approach; infection experiments are required in the future.
Our data provide evidence of direct contact between Cx. pipiens pallens and both resident and migratory birds infected with avian malaria parasites in Japan. Thus, we conclude that Cx. pipiens pallens is probably the principal vector in transmission of avian malaria parasite in Japanese wild bird communities and that resident birds in coastal areas of Tokyo Bay could be exposed to novel avian malaria parasites carried by infected migratory birds, and vice versa.
We thank Kyoko Sawabe, Haruhiko Isawa, Shinji Kasai, Keita Hoshino, and Osamu Komagata (Department of Medical Entomology, National Institute of Infectious Diseases) for help with molecular laboratory work. This work was partly supported by a Grant-in-Aid for Scientific Research of Emerging and Reemerging Infectious Diseases from the Ministry of Health, Labor and Welfare of the Japanese Government (H18-Shinko-009).