A pictorial key to differentiate the recently detected exotic Haemaphysalislongicornis Neumann, 1901 (Acari, Ixodidae) from native congeners in North America

Abstract Until recently, only two haemaphysaline species, Haemaphysalischordeilis (Packard, 1869) and Haemaphysalisleporispalustris (Packard, 1869), were known to occur in the United States, and neither was considered to be of significant medical or veterinary importance. In 2017–2018 established populations of the Asian longhorned tick, Haemaphysalislongicornis Neumann, 1901, were detected in the eastern US for the first time. Haemaphysalislongicornis has the potential to be a significant threat to human and animal health, and the urgent need to determine the full extent of its distribution and host range requires availability of a straightforward and practical guide to differentiate it from native species. We created a pictorial dichotomous key to all stages of Haemaphysalis spp. known to occur in North America with scanning electron photomicrographs of all H.longicornis life stages, including rarely seen males, to aid researchers in differentiating these species. The largely Neotropical species Haemaphysalisjuxtakochi Cooley, 1946, with established populations in Mexico and sporadic detections in the US on migrating birds is also included.


Introduction
Only two native species of Haemaphysalis ticks are known to occur in the United States: the rabbit tick, Haemaphysalis leporispalustris (Packard, 1869) and the bird tick, Haemaphysalis chordeilis (Packard, 1869) (Keirans and Litwak 1989). Haemaphysalis leporispalustris is common and widespread in North America, and is frequently collected from lagomorphs (rabbits and hares) (Bishopp and Trembley 1945). Its full distribution extends from Alaska and Canada southward to Argentina (Kohls 1960, Guglielmone et al. 2003. The agents of tularemia (Francisella tularensis) and of Rocky Mountain spotted fever (Rickettsia rickettsii) have been isolated from this tick (Eremeeva et al. 2018) although its role, if any, as a vector of human disease appears to be minor. Haemaphysalis chordeilis is far less often collected but nonetheless assumed to have a broad distribution in North America, based on sporadic avian records spanning the US and southern Canada (Bishopp and Trembley 1945, Kohls 1960, Lindquist et al. 2016. Because these two species have historically been considered specialists on rabbits and birds, respectively, and are not significant pests of humans or domestic animals, relatively little attention has been paid to their ecology and geographical distribution. In 1993, a single specimen of the Central/South American species Haemaphysalis juxtakochi Cooley, 1946(Haemaphysalis kochi Aragão, 1908 is a junior synonym) was detected in Ohio, USA, on a white-tailed deer at a deer-checking station (Keirans and Restifo 1993). While the current northern limit of this species' distribution appears to be Mexico, immatures may occasionally be brought into the US by northward migrating birds (Mukherjee et al. 2014). At present there is no indication that such encounters are common or that the species has become established in the US. Adult H. juxtakochi are chiefly parasites of deer (Kohls 1960, Guglielmone et al. 2005 and may be able to transmit some species of Rickettsia (Souza et al. 2018).
In 2017, established populations of the East Asian/Australasian species Haemaphysalis longicornis Neumann, 1901, were discovered in New Jersey (Rainey et al. 2018) and subsequently throughout a large part of the eastern US, including Arkansas, Connecticut, Maryland, New York, North Carolina, Pennsylvania, Virginia, and West Virginia (Beard et al. 2018). This species, native to East Asia and invasive in Australia/ New Zealand, is associated with disease transmission to humans in the former region (e.g., Zhuang et al. 2018) and is a serious pest of livestock in the latter (Heath 2016). Invasive populations of this species appear to be parthenogenetic, which may facilitate their establishment and spread (Heath 2013). As a result, there is now much concern over this species' potential effect on human and animal health in the United States, and studies are under way to clarify its current geographic range and preferred host species.
In order to study potential impacts of H. longicornis on North America, a critical first step is being able to differentiate this species from co-occurring Haemaphysalis spp. Here we present scanning electron photomicrographs of all stages of H. longicornis, as well as a simple, usable dichotomous key to differentiate the four Haemaphysalis species that may be encountered in North America: H. leporispalustris, H. chordeilis, H. longicornis, and H. juxtakochi. While the rarity of H. juxtakochi detections in the US does not suggest that this species will often be sympatric with H. longicornis, we feel it is important to include it in our key for three reasons: (1) climate change is predicted to alter the distribution of many tick species (Ostfeld and Brunner 2015), therefore the distribution of H. juxtakochi may one day shift north of Mexico; (2) unlike some of the exotic species imported by birds (Mukherjee et al. 2014), H. juxtakochi could easily find suitable host species in the US; and (3) as it continues its invasion of North America, H. longicornis may eventually be collected farther south, coinciding with H. juxtakochi's existing range.

Scanning electron microscopy (SEM)
Specimens of H. longicornis were obtained from US National Tick Collection archives for imaging. Males, females, and nymphs were sent from a laboratory colony started with specimens collected in Jeju-teukbyeoljachido, Republic of Korea (Accession # RML48803). Larvae originated from a colony started with specimens from Queensland, Australia (Accession # RML58949). Specimens were coated with gold and imaged with a JEOL JSM-6610LV scanning electron microscope (JEOL USA, Inc., Peabody, MA) (Figs 1-4). Larval and nymphal H. juxtakochi were collected by flagging in Guanacaste National Park, Costa Rica, and imaged in the same manner (Accession # USNMENT 986092).

Pictorial dichotomous key
A literature search was conducted and key characters useful for distinguishing the four species were gleaned from the following: Cooley (1946), Kohls (1960), Clifford et al. (1961), Fairchild et el. (1966), Hoogstraal et al. (1968), and Hoogstraal and Kim (1985). Of note, characters chosen to distinguish adult stages are present in both males and females of their respective species.

Conclusions
This key enables researchers to distinguish the four species of Haemaphysalis that may be encountered in North America in all life stages. Previously, readers would have had to peruse keys from several distinct parts of the world in order to compare the morphology of these four species, e.g. US keys containing H. chordeilis (Furman andLoomis 1984, Keirans andLitwak 1989); Old World keys with H. longicornis, including Japan (Yamaguti et al. 1971) and Australia (Roberts 1970, Barker andWalker 2014); and Central and South American keys for H. juxtakochi (Fairchild et al. 1966, Nava et al. 2017. The ability to easily distinguish these four species will contribute to ongoing efforts to map the distribution of Haemaphysalis longicornis in North America and understand the potential risks posed by this recently discovered exotic tick species (Rainey et al. 2018). This tool will also help to improve our understanding of the biology and ecology of native Haemaphysalis spp., which have been relatively poorly studied compared to other native ixodids, and will promote the early detection of any northward expansions of H. juxtakochi. In this manner we can capitalize on the interest generated by the arrival of H. longicornis to augment our understanding of the existing New World haemaphysaline fauna.
However, as Haemaphysalis is the second largest genus in the tick family Ixodidae (so-called hard ticks), with over 160 additional species in the Old World (Petney et al. 2007, Guglielmone et al. 2014, including important disease vectors (de la Fuente et al. 2008), careful monitoring to detect the potential arrival of other members of this genus is encouraged. Should additional Haemaphysalis species establish themselves in North America, this key will require revision.