Establishment of Ochlerotatus japonicus (Diptera: Culicidae) in Ontario, Canada

SAMPLING, DISTRIBUTION, DISPERSAL Establishment of Ochlerotatus japonicus (Diptera: Culicidae) in Ontario, Canada AYNSLEY THIELMAN AND FIONA F. HUNTER Brock University, Department of Biological Sciences, St. Catharines, Ontario, L2S 3A1, Canada J. Med. Entomol. 43(2): 138Ð142 (2006) ABSTRACT Ochlerotatus (Finlaya) japonicus (Theobold) is newly established in Ontario, Canada. It was Þrst discovered in 2001 during the province-wide West Nile virus mosquito surveillance program implemented by the Ontario Ministry of Health and Long Term Care. Although the numbers of adults trapped in CDC light traps were low in 2001 and 2002, they increased during the 2003 and 2004 surveillance seasons. Oc. japonicus larvae also have been collected in large numbers in the Niagara Peninsula in a variety of natural and artiþcial containers. The number of health units with records for Oc. japonicus has increased over the 4 yr of surveillance, illustrating the ability of this species to rapidly extend its range. As a potential arboviral bridge vector, its establishment in Ontario requires further study and should be considered a public health concern. KEY WORDS Ochlerotatus japonicus, distribution, Þrst record, breeding sites, Ontario Ochlerotatus (Finlaya) japonicus (Theobold) is an East Asian species native to Japan, Taiwan, China, and Korea (Tanaka et al. 1979). It has been detected outside its native range in New Zealand (Laird et al. 1994) and France (Schaffner et al. 2003); in the United States, including Georgia and South Carolina (Reeves and Korecki 2004), Maryland (Sardelis and Turell 2001), Connecticut (Andreadis et al. 2001), New Jersey (Peyton et al. 1999), New York (Falco et al. 2002), Ohio (Darsie and Ward 2005), Pennsylvania (Fonseca et al. 2001), and Washington (Roppo et al. 2004); and in Quebec (Savignac et al. 2002) and now in Ontario, Canada. Peyton et al. (1999) suggest that the method of introduction for this species may be via the international transport of used tires. In their native range, Oc. japonicus larvae inhabit a variety of natural and artiþcial containers, with their preferred habitat being rock holes (Tanaka et al. 1979). They have been shown to feed on avian and mammalian hosts in the laboratory (Miyagi 1972), although they are thought to be reluctant to bite humans (Tanaka et al. 1979). In North America, surveys have revealed additional larval habitats, including surface water pools, wooden tubs, metal containers, bird baths, and catch basins (Andreadis et al. 2001). They have been collected from used tires exposed to shade, partial, and full sun, and a wide range of water temperatures (Oliver et al. 2003). Adults have been found in light traps, gravid traps, and mouth-aspirated adult landing collections. However, in the United States, although Oc. japonicus is known to feed on chickens in the laboratory (Sardelis et al. 2001, 2002a, 2003), all bloodmeals analyzed from wild-caught Oc. japonicus were of mammalian origin (Apperson et al. 2004). In August 2001, an adult mosquito surveillance program was established in Ontario because of growing concerns regarding the spread of West Nile virus. Beginning with 14 health units in southern Ontario, the program expanded to include most of the 37 health units in the province from 2002 to 2004. Mosquitoes were collected from May through November by using CDC light traps and gravid traps at a variety of sampling sites, including both rural and urban areas. In addition, larval collections were made at a variety of sites in the Niagara region during 2003 and 2004. Materials and Methods Adult Collections. Adult mosquitoes were collected by various public health units across the province as part of the Ontario Ministry of Health and Long Term CareÕs (OMOHLTC) West Nile Virus mosquito surveillance program. Of 37 health units in Ontario, we processed adult traps from 14 Health Units in 2001, 29 in 2002, 36 in 2003, and 31 in 2004. Health unit personnel were trained regarding proper operation of gravid traps and CO 2 -baited CDC light traps. Gravid traps were only used in 2002 and 2004. In 2004, the OMOHLTC suggested that health units use a gravid trap infusion made of hay, yeast, beer, and water, which was to be left to sit for 4Ð5 d. Trap collections were shipped live to Brock University where the mosquitoes were freeze-killed, sorted, labeled (date of collection, health unit, and site code), and the adult females were identiþed. Voucher specimens of Ontario-collected Oc. japonicus adults have been deposited in the Canadian National Collection, Ottawa. 0022-2585/06/0138Ð0142$04.00/0 2006 Entomological Society of America

March 2006 THIELMAN AND HUNTER: Oc. japonicus IN ONTARIO, CANADA 139 Larval Collections. Sampling for immature mosquitoes was conducted throughout the Niagara region during 2003 and 2004. Collections were made sporadically and at different sites each year. Larvae were collected using standard larval dippers (plastic collection cups attached to long, wooden handles) and turkey basters from various types of habitats, including roadside ditches, marshes, rain puddles, plastic buckets, used tires, outdoor concrete ashtrays, tractor scoops, tree holes, and rock pools. Mosquito Identification. Adult and larval mosquitoes were identiþed using a combination of the keys of Wood et al. (1979), Darsie et al. (1981), and Tanaka et al. (1979) for the description of Oc. japonicus. Results Adults. Our Þrst record of Oc. japonicus in Ontario was an adult female recovered from a CDC light trap on 13 September 2001. Only one specimen was collected during the Þrst year of surveillance, and it was found in the Niagara region (Table 1). During the 2002 season, the number of health units participating in the surveillance program increased from 14 to 29, and the number of specimens collected increased from one to nine (Table 1). Most Oc. japonicus were collected in the Niagara region (eight of nine), but one was found in the Hamilton health unit as well. The maximum number of Oc. japonicus collected per trap in the 2002 season was two. In 2003, 36 health units participated in the surveillance program through Brock University, and the number collecting Oc. japonicus increased from two to nine (Table 1). More than half of the specimens (14 of 22) were trapped in Niagara and Hamilton. The maximum number of Oc. japonicus collected per trap in the 2003 season was two. In 2004, 31 health units participated in the surveillance program, and the number collecting Oc. japonicus increased from nine to 20 (Table 1). The number of specimens collected increased dramatically, from 22 to 460. The Niagara and Hamilton regions continued to collect the majority of specimens (315 of 460). The maximum number of Oc. japonicus collected per trap in 2004 was 25, with numerous traps having between 10 and 15 individuals per trap. In all years of surveillance, most specimens (408 of 492) were collected in August and September (Table 2). In 2001 and 2003, gravid traps were not used by any health units to collect mosquitoes. Gravid traps were used in 2002 but not by the health units in which Oc. japonicus were found. Therefore, all collections of Oc. japonicus during the Þrst 3 yr of surveillance were made in light traps. In 2004, 51 of 460 (11.09%) of the Oc. japonicus adults were collected in gravid traps compared with light traps. The proportion of Oc. japonicus specimens collected to total mosquitoes identiþed was higher in gravid (2.67%) versus light traps (0.14%). Larvae. Oc. japonicus larvae were collected in the Niagara region in 2003 and 2004 from the following natural and artiþcial habitats: small ponds, discarded Table 1. Oc. japonicus specimens collected during adult mosquito surveys from 2001 to 2004 Yr Health unit No. mosquitoes identiþed No. Oc. japonicus collected % trap total 2001 NI 520 1 0.19 Others a 41,497 Totals 42,017 1 0.00 2002 NI 2,883 8 0.28 HM 3,982 1 0.03 Others b 158,742 Totals 165,607 9 0.01 2003 NI 2,562 10 0.39 HM 3,836 4 0.10 HL 19,027 1 0.01 PE 19,430 1 0.01 TO 20,660 2 0.01 BR 1,895 1 0.05 HP 2,466 1 0.04 LG 4,018 1 0.02 EO 4,676 1 0.02 Others c 110,916 Totals 189,486 22 0.01 2004 NI 9,810 65 0.66 HM 18,271 250 1.37 HL 11,278 14 0.12 PE 28,292 19 0.07 TO 31,604 9 0.03 BR 4,593 50 1.09 HP 15,739 4 0.03 LG 10,161 2 0.02 EO 9,097 3 0.03 PD 5,477 2 0.04 OX 4,934 3 0.06 HN 6,823 9 0.13 EG 955 2 0.21 ML 11,779 17 0.14 CK 8,195 1 0.01 DR 10,019 1 0.01 WE 11,491 3 0.03 YK 14,505 3 0.02 HK 4,729 2 0.04 NB 7,182 1 0.01 Others d 70,345 Totals 295,279 460 0.16 Health units that did not collect any Oc. japonicus are combined as ÔOthersÕ and are listed below. Abbreviations are as follows: AL, Algoma; BR, Brant; CK, Chatham-Kent; DR, Durham; EG, Elgin-St. Thomas; EO, Eastern Ontario; GB, Grey Bruce; HK, Haliburton- Kawartha; HL, Halton; HM, Hamilton; HN, Haldimand-Norfolk; HP, Hastings-Prince Edward; HR, Huron; KF, Kingston-Frontenac; LG, Leeds, Grenville, Lanark; LM, Lambton; ML, Middlesex-London; MP, Muskoka-Parry Sound; NI, Niagara; NB, North Bay; NW, Northwestern; OT, Ottawa; OX, Oxford; PC, Porcupine; PD, Perth District; PE, Peel; PT, Peterborough; RE, Renfrew; SB, Sudbury; SM, Simcoe; TB, Thunder Bay; TK, Timiskiming; TO, Toronto; WA, Waterloo; WD, Wellington-Dufferin-Guelph; WE, Windsor-Essex County; YK, York. a CK, DR, EO, HL, HM, HP, LG, LM, ML, PE, TO, WE, YK. b AL, BR, CK, DR, EG, EO, HK, HL, HN, HP, LG, LM, ML, MP, OT, OX, PC, PD, PE, PT, RE, SM, TO, WA, WD, WE, YK. c AL, CK, DR, EG, GB, HK, HN, HR, KF, LM, ML, MP, NB, NW, OX, RE, PC, PD, PT, SB, SM, TB, TK, WA, WD, WE, YK. d AL, GB, HR, KF, MP, NW, PC, SB, SM, TB, TK. tires, rainwater buckets, an outdoor concrete ashtray, tractor scoop, and rock pools along streams and rivers. In May 2003, 29 specimens were reared from two larval dipping attempts from discarded tires found in Niagara Falls, Ontario. In July, four specimens were collected from a plastic garden tub, also in Niagara Falls. In August, 169 of 205 (82.4%) of the specimens collected came from two larval dipping collections in

140 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 43, no. 2 Table 2. Seasonal abundance of adult Oc. japonicus collections in light and gravid traps in Ontario Mo No. Oc. japonicus adults collected/yr (no. traps) 2001 2002 2003 2004 Total May 0 (0) 0 (6) 0 (138) 2 (43) 2 (187) June 0 (0) 0 (285) 0 (629) 26 (1,173) 26 (2,087) July 0 (0) 1 (720) 2 (975) 53 (1,787) 56 (3,482) Aug. 0 (115) 6 (1,043) 14 (1,041) 168 (2,340) 188 (4,539) Sept. 1 (487) 2 (711) 6 (1,112) 211 (1,706) 220 (4,016) Oct. 0 (246) 0 (322) 0 (149) 0 (304) 0 (801) Nov. 0 (49) 0 (0) 0 (0) 0 (0) 0 (49) Total 1 (897) 9 (3,087) 22 (4,044) 460 (7,353) 492 (15,381) a plastic bucket used to collect rainwater in Pelham, 30 were collected from one attempt from an outdoor concrete ashtray in St. Catharines, and 11 were collected from a plastic container in a backyard in St. DavidÕs. In 2004, the number of specimens collected increased from 238 to 1211. The earliest collection of Oc. japonicus larvae occurred on 2 May 2004 with 12 specimens collected from a plastic-lined garden pond in Stevensville. Numerous specimens continued to be collected throughout the season from a variety of larval breeding sites. In May, 26 larvae were collected from two discarded tires, one in a residential yard in the city of Thorold and one on a farm in Niagara Falls. In June, 13 were collected from the same Niagara Falls farm tire and 18 were from a rain water-þlled bucket in a residential backyard in Thorold. In July, 16 more were collected from the bucket in Thorold. In August, 345 specimens were collected, some from previously known sites, although most (248 of 345) came from a large plastic tub used to collect rainwater on a farm in St. Catharines. There were also 56 larvae collected from rock pools at Balls Falls in Vineland. In September, a total of 610 larvae were collected from numerous old and new locations, including rock puddles in the Whirlpool area of the Niagara Gorge, a tractor scoop and tires on a different farm in St. Catharines, and from a tire in a residential backyard in Fort Erie. In October, there were 153 collected in St. Catharines. The last two collections (22 larvae) were made in November from a tire on the Brock University campus. In 24 of 38 collections (63.2%) in 2004, Oc. japonicus was the only species present. However, they were found on 14 occasions to be associated with other species, the most common being Culex restuans Theobald, Culex pipiens L., Ochlerotatus triseriatus (Say) and Ochlerotatus atropalpus (Coquillett) (Table 3). Discussion It is clear that Oc. japonicus has become established in southern Ontario and that it is expanding its range throughout the province (Fig. 1). Niagara and Hamilton both saw large increases in the number of adults collected after a couple years of relatively low numbers, demonstrating the ability of this species to become quickly established in an area. In the Niagara region, extremely large numbers of Oc. japonicus larvae were observed breeding in single containers with very little organic matter to support such large populations. Within the rearing chambers in the laboratory, specimens were very hardy, requiring very little care to ensure adult emergence. Larval collections of this species in artiþcial habitats such as outdoor ashtrays and extremely dirty tire water suggest a potential tolerance to environmental pollutants. These characteristics of Oc. japonicus may contribute to its ease of establishment. Larvae have been collected from a variety of natural and artiþcial habitats, representing a potential problem in both urban and rural areas. In the current study, their absence in previously reported habitats, such as tree holes (Scott et al. 2001) and catch basins (Andreadis et al. 2001), is probably an artifact of lack of sampling effort in these habitats. The mosquito species found commonly associated with Oc. japonicus larvae in Canada are similar to those found in previous studies in Connecticut and New York (Andreadis et al. 2001, Oliver et al. 2003). Only one association not found in Canada is noteworthyñthe absence of associated Anopheles punctipennis (the most abundant species found associated with Oc. japonicus in artiþcial containers, used tire casings, and surface water pools in Connecticut). All larval collections of An. puncti- Table 3. Other mosquito species found associated with Oc. japonicus in 2004 and the corresponding larval habitat and frequencies of co-occurrences Associated species Habitat Occurrence Frequency Cx. pipiens Rock pool 1 0.026 Cx. pipiens, Tractor Scoop 1 0.026 Oc. triseriatus Cx. pipiens/restuans Tire 1 0.026 Cx. restuans Tire 2 0.053 Cx. restuans, Tire 1 0.026 Oc. triseriatus Oc. atropalpus Rock Pool 6 0.158 Oc. atropalpus, An. Rock Pool 1 0.026 quadrimaculatus Oc. triseriatus Tire 1 0.026 None Bucket 6 0.158 None Garden Pond 2 0.053 None Rock Pool 6 0.158 None Tire 9 0.237 None Tractor Scoop 1 0.026 38 1.000

March 2006 THIELMAN AND HUNTER: Oc. japonicus IN ONTARIO, CANADA 141 Fig. 1. Distribution of Oc. japonicus in Ontario from 2001 to 2004. Health unit abbreviations are as in Table 1. pennis in the current study were made from woodlot habitats and none were collected from used tires or artiþcial containers. The seasonal abundance of adult Oc. japonicus collections is also similar to that reported for Connecticut (Andreadis et al. 2001), with the majority of specimens collected in August and September. However, in New York, an earlier peak in July also was seen (Falco et al. 2002). Each year, Oc. japonicus comprised 1% of the total number of mosquitoes collected through the surveillance program. This could be because of the inability of health units, because of time constraints, to allow the traps to continue collecting during the day, when we found Oc. japonicus to be biting. In 2004, the only year in which a comparison could be made of the effectiveness of gravid versus light traps for collecting Oc. japonicus, a higher proportion of specimens was collected using gravid traps. Oc. japonicus comprised 2.67% of the total number of adult females collected in gravid traps, compared with 0.14% in light traps. Other studies also have concluded that gravid traps are superior for the collection of Oc. japonicus (Scott et al. 2001). Falco et al. (2002), using a rabbit chow infusion versus standard sod grass or hay infusions, found that they were able to collect an even higher proportion of Oc. japonicus with gravid versus light traps (18.1 versus 1.5%). Apart from the potential to become nuisance mosquitoes in this country, Oc. japonicus are potential vectors of arboviruses such as West Nile virus (family Flaviviridae, genus Flavivirus, WNV) and eastern equine encephalitis (family Togaviridae, genus Alphavirus, EEE) virus. Studies in the United States have shown Oc. japonicus to be a competent laboratory vector of WNV (Sardelis and Turell 2001), La Crosse virus (Sardelis et al. 2002b), EEE virus (Sardelis et al. 2002a), and St. Louis encephalitis virus (Sardelis et al. 2003). Because WNV-infected specimens were kept at 26 C for an incubation period to allow the infection to disseminate and the individuals to become infective in these studies, similar outdoor temperatures may be required before Oc. japonicus becomes a potential risk to the public. Although wild-caught adult specimens have tested positive for WNV in the United States (White et al. 2001), they have yet to test positive for the virus in Canada. Although the numbers of adult Oc. japonicus collected in light traps increased in 2003 and 2004, the fact that average daily temperatures in both seasons were relatively low compared with previous years may be responsible for the lack of WNV-positive individuals to date. Because temperature is known to have an effect on the transmission of arboviral diseases (Gubler et al. 2001), it is possible that Oc. japonicus requires relatively high temperatures to become an efþcient vector of WNV. Therefore, should this species continue to extend its range and achieve large population sizes, seasons in which the daily temperatures reach highs of 26 C or more should be watched carefully to determine whether this is indeed the case. Because Oc. japonicus has not been shown to bite birds in the wild in North America to date (Apperson et al. 2004), it may not acquire WNV as readily as other established vectors of the disease. However, until

142 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 43, no. 2 more is known about the feeding habits and vectorial capacity of this species, it should be watched carefully in future epidemic years. Because Oc. japonicus blood feed during the day and are collected in greater numbers in gravid traps, surveillance methods could be changed to include more gravid traps and to allow CDC-light traps to run during the day to increase the probability of collecting this species. If Oc. japonicus becomes an efþcient vector of WNV or other arboviruses, public awareness campaigns (which target dusk and dawn as times to avoid the outdoors to reduce the risk of contracting the virus) also will need to be modiþed to reßect the potential increased risk of disease transmission by this species during the day. 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