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Jpn. J. Infect. Dis., 63, 401-404, 2010 Original Article Laboratory Colonization of Aedes japonicus japonicus (Diptera: Culicidae) Collected in Narita, Japan and the Biological Properties of the Established Colony Keita Hoshino, Haruhiko Isawa, Yoshio Tsuda*, and Mutsuo Kobayashi Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan (Received May 17, 2010. Accepted August 23, 2010) SUMMARY: A laboratory colony of the mosquito Aedes japonicus japonicus, which has recently in- vaded the United States and is recognized as a highly competent vector of West Nile virus, was estab- lished from larvae collected in Narita, Japan. The mosquitoes were maintained with induced insemina- tion, blood-feeding on humans, and oviposition in water provided from the original collection site during the first few generations, then the colony was transferred to a large cage (40 × 40 × 100 cm in height) and adapted to conditions in which specimens were allowed to mate freely. White mice were provided as the blood source, and deionized water was available for oviposition. Approximately 185 eggs, most of which were tolerant to desiccation for at least 1 month, with some surviving for up to 2.5 months, were obtained per female following a single blood-feeding. The rate of successful emergence was nearly 90z, although this rate decreased significantly at high larval densities. The colony has been maintained for 5 years, and developmental profiles of the species have been obtained during that time. tablishment of a laboratory colony of Ae. j. japonicus INTRODUCTION will therefore contribute to both basic and applied Aedes japonicus (Theobald) is a mosquito distributed research associated with the vector competence and the primarily in eastern Asia, including Japan, Korea, reproductive ecology of this species. A colony of an in- China, Russia, and Taiwan (1). Aedes j. japonicus,the vasive population of Ae.j.japonicusfrom New Jersey original subspecies of this species, was first found to has been established recently (14), although no similar haveinvadedtheeasternUnitedStatesin1998(2)and colony of the original population of Ae.j.japonicusex- has since expanded to other areas including Canada, ists to date. Herein we report the establishment and France, and Belgium (3–6). Ae. j. japonicus has also maintenance of a laboratory colony of Ae. j. japonicus been confirmed to be an important vector of West Nile from Japan, together with descriptions of the coloniza- virus (WNV) in North America, based on experimental tion methodology and some basic biological properties analysis of its vector competence and field isolations of obtained from this colony. These findings may prove WNV (7,8). Additionally, it was reported that Ae. j. useful for the characterization of this species, including japonicus is also susceptible to Japanese encephalitis invasive populations, and provide the information re- virus (9), St. Louis encephalitis virus (10), Eastern quired to design laboratory experiments using this equine encephalomyelitis virus (11), and La Crosse virus colony, such as infection experiments with mosquito- (12), indicating that this species is a potential vector for borne pathogens. these viruses. Ae. j. japonicus is a common inhabitant of wooded environments, including residential areas, in MATERIALS AND METHODS palearctic Japan, whereas two other subspecies, Ae. japonicus amamiensis (Tanaka, Mizusawa & Saugstad) Origin of mosquito colony: The various instar larvae and Ae. japonicus yaeyamaensis, (Tanaka, Mizusawa & of Ae. j. japonicus (approximately 500 individuals) were Saugstad), are distributed sporadically only in the collected from the stone basin of a shrine in Narita Amami and Ryukyu Archipelago, respectively, at the (35938?27!N, 140928?52!E), Chiba Prefecture, Japan, southernmost area of Japan (1,13). Despite this, in June and July 2004, and they were used to establish however, very few experimental data are available for the laboratory colony. Ae. j. japonicus in Japan due to the difficulties in estab- Maintenance of mosquitoes: Thelarvaewerereared lishing a laboratory colony, although the bionomics of at a density of approximately 10 larvae per 100 mL and this subspecies as regards distribution, host preferences, fedwithfishfoodTetraMin(fortheearlyinstarlarvae; and larval habitats have been sporadically reported. Es- Tetra, Melle, Germany) and SWIMMY BABY (for the late instar larvae; Nippon Pet Food, Tokyo, Japan) in a white plastic tray (W:L:D ＝ 30 × 20 × 5cm).Anap- *Corresponding author: Mailing address: Department of propriate amount of larval food (10–20 and 30–50 Medical Entomology, National Institute of Infectious mg/day during the 1st to 3rd and 4th larval stages, re- Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, spectively) was supplied on the basis of the number and Japan. Tel: ＋81-3-5285-1111, Fax: ＋81-3-5285-1147, developmental stage of the larvae. Additional food was E-mail: tsudayso＠nih.go.jp provided once the blocks of larval food had been con- 401 sumed and disappeared completely. Once the pupae ap- eggs to dry in an airtight container for between 0 and 11 peared, they were carefully transferred into a cup filled weeks after oviposition. The survival rate of eggs was with deionized water and placed inside a cage with stain- determined by counting the number of hatched larvae less supports covered with nylon-mesh net (20 × 30 × and confirming the completely collapsed non-hatching 20 cm) for eclosion. The emerging adults were supplied eggs by optical microscopy in November 2005. with cotton soaked in an approximately 3z sucrose so- To examine the developmental period of the colony, lution. The adults were reared in a 20 × 30 × 20 cm hatched larvae were reared individually in 20 mL of cage with frequent opportunities to feed, which in- pure water with 5 mg of larval food, which were volved introducing a human arm into the cage up to the changed daily, and their developmental stage recorded wrist as the first generation of mosquitoes was not at- twice a day at the beginning and end of the light period. tracted to white mice. Artificial mating was subsequent- The developmental and survival effects of the density ly performed as described previously (15,16). A glass of immature stages were determined by measuring the dish (110 mm in diameter, 60 mm in depth) was lined adult emergence rate and the mortality of immature with Advantech filter paper Grade 2 (Toyo Roshi stages at four different initial densities of hatched larvae Kaisha, Tokyo, Japan) positioned with the rough side with a fixed amount (25 mg per day) of larval food. A facing the inside for oviposition. This dish was filled to glass dish (110 mm in diameter, 60 mm in depth) filled the halfway mark with water and placed stably in the with 100 mL of water was used, and both the water and cage of approximately 50 gravid females for 1 week. food were changed daily. Water from the original larval habitat was used to Adult survivorship and longevity of the established stimulate oviposition. The eggs deposited on the filter colony were investigated by recording the number of paper were submerged in a tray filled with deionized deaths every day for three experimental cohorts of ap- water. A number of adults which had developed from proximately 20 pairs of newly emerged adults reared larvae in the original habitat were subsequently collect- with a 3z sucrose solution in a cage. ed and placed into the cage along with the second gener- ation in July 2004. The next five generations of the RESULTS AND DISCUSSION colony were maintained under the same conditions de- scribed above. Then approximately 3,000 adults from Colony maintenance: To improve free mating, the the following generation were transferred into 40 × large cage was designed to have a wide vertical space 40 × 100 cm cage, the top of which was covered with with no direct light other than a few beams of light, pro- aluminum foil containing a few small holes. The adults duced by covering the top of the cage with aluminium were allowed to mate freely for a period of 3–4 weeks foil containing several small holes, to simulate sun- after emergence. Adult females were allowed to take a beams passing through tree leaves in the mosquito's blood meal from anesthetized white mice (Japan SLC, natural wooded habitat. We observed active flight with Hamamatsu, Japan) for 2 h in the latter half of the light phototaxis toward the light beam from the top of the period in order to mimic the blood-feeding habit of this cage. Such environmental conditions have been suggest- species in its natural environment. Additionally, the ed to be essential for the successful mating of some blood-feeding activity on mice was enhanced for the Aedes and Toxorhynchites mosquitoes in their native first few generations by exhaling over the mice for a few habitat (woodland) (16,17). Deposited eggs were found minutes, since these mosquitoes exhibited a low prefer- on the filter paper 5 days after blood-feeding. These ence for white mice. Deionized water was used for eggs subsequently hatched into neonate larvae. The oviposition. All developmental stages of mosquitoes hatching process was observed within 2 days. The eggs were maintained in a controlled insectary at 50–70z rel- on the filter paper were submerged in the water and any ative humidity, a temperature of 259C, and a light/dark air bubbles attached to them were removed by pipetting photoperiod of 16/8 h, with no dawn or dusk light dim- the water. A number of eggs hatched, indicating that ming. they were fertilized and deposited by females which had Basic biological properties of the established colony: mated successfully under our laboratory conditions. We The numbers of eggs per female, the hatching rate, the confirmed that the hatched larvae grew and metamor- tolerance of eggs to desiccation, the larval development, phosed into adults, with the next generation being ob- the density effects of immature stages, and the adult tained in the same way.

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