New Geographical Records of Three Gill Monogeneans ( Platyhelminthes ) Parasitic on Pseudorasbora parva ( Cypriniformes : Cyprinidae ) in Japan

Three species of monogenean, Dactylogyrus squameus Gussev, 1955, Ancyrocephalus pseudorasborae Achmerow, 1952, and Bivaginogyrus obscurus (Gussev, 1955), were collected from the gills of the cyprinid Pseudorasbora parva (Temminck and Schlegel, 1846) in Japan. Dactylogyrus squameus and A. pseudorasborae were collected in Nara, Tottori, and Ibaraki prefectures, and in Ibaraki and Okayama prefectures, respectively, and are redescribed as new country records from Japan. Bivaginogyrus obscurus has been reported from only Ibaraki and Nagano prefectures, and its discovery in Nara and Okayama prefectures as represents as new locality records. Although D. squameus and B. obscurus are known as alien parasites in Europe, all three monogeneans found in this study are considered to be native to Japan.


Introduction
Pseudorasbora parva (Temminck and Schlegel, 1846) (Cypriniformes: Cyprinidae) is natively distributed in Far-East Asia, from south-eastern Russia and regions west of the Kantō District of Honshū, Japan, in the north to northern Vietnam in the south (Hosoya 2013).The fish has spread into Europe as an invasive species along with two dactylogyrid monogeneans, Dactylogyrus squameus Gussev, 1955 and Bivaginogyrus obscurus (Gussev, 1955) (Ondračkova et al. 2004;Galli et al. 2007;Gozlan et al. 2010;Anonymous 2012).The monogenean fauna of this fish is poorly known in Japan, where only B. obscurus has been reported from Ibaraki Prefecture (Nitta and Nagasawa 2014).In this paper, we examined Pseudorasbora parva collected from natural habitats in Japan and report Dactylogyrus squameus and Ancyrocephalus pseudorasborae Achmerow, 1952 as new country records and Bivaginogyrus obscurus as new prefectural records.

Materials and Methods
Seventeen specimens of Pseudorasbora parva (standard length 25.5-52.7 mm) were collected by a hand net or angling from four sites in Japan: two specimens from the Tomio River (34°41′50″N, 135°43′55″E), a tributary of the Yamato River system at Tomio-gawa-nishi, Nara city, Nara Prefecture on 22 July 2012; four specimens from the Kitakata River (35°21′26″N, 133°19′00″E), a tributary of the Hino River system at Kitakata, Nanbu town, Saihaku County, Tot-tori Prefecture on 6 August 2013; ten specimens from Lake Kasumigaura (36°04′05″N, 140°15′23″E) at Okijuku-machi, Tsuchiura city, Ibaraki Prefecture (eight and two specimens on 13 and 16 June 2014, respectively); and one specimen from the Senō River (34°36′16″N, 133°51′52″E), a tributary of the Kurashiki River system, at Uchio, Minami Ward, Okayama city, Okayama Prefecture on 16 March 2015.The fish were brought alive to the laboratory at Hiroshima University, Higashi-Hiroshima city, Hiroshima Prefecture, and examined for gill monogeneans under an Olympus SZ61 dissecting microscope.Monogeneans were collected from the gills by using small needles and forceps, fixed in 70% ethanol, AFA (acetic acid-formalin-alcohol), or APG (ammonium picrate glycerin; in Lim 1991) under coverslip pressure and stained with Heidenhain's iron hematoxylin or alum carmine, but some specimens fixed in APG were not stained.All specimens were dehydrated in a graded ethanol series, cleared in xylene, and mounted in Canada balsam.Drawings were made with the aid of a drawing tube fitted on an Olympus BX51 compound microscope.The hook measurements are presented in Fig. 1.The male copulatory organs and the sclerotized vaginae were measured on images taken by digital camera on an Olympus DP20 microscope at a magnification of ×1,000 using ImageJ software (version 1.48i).Measurements, in micrometers, are expressed as the mean±standard deviation followed in parentheses by the range of structure measurements and the number (n) of specimens examined.The numbering of hook pairs follows Llewellyn (1963).Fish identification was based on Hosoya (2013).Prevalence and intensity of infection are those defined by Bush et al. (1997).Specimens are deposited in the Platyhelminthes collection of the National Museum of Nature and Science (NSMT-Pl), Tsukuba city, Ibaraki Prefecture, Japan.

Results
Three species of monogeneans were collected from Pseudorasbora parva.Dactylogyrus squameus and Ancyrocephalus pseudorasborae were collected in Nara, Tottori, and Ibaraki prefectures and Ibaraki and Okayama prefectures, respectively.Bivaginogyrus obscurus was found with D. squameus in Nara Prefecture and with A. pseudorasborae in Okayama Prefecture.Furthermore, these three monogenean species concurrently infected four host individuals in Lake Kasumigaura, Ibaraki Prefecture.
Remarks.Dactylogyrus squameus was originally described by Gussev (1955) from the gills of Pseudorasbora parva from the Amur River and Lake Chanka, Far-East Russia, and subsequently reported from the same host in Heilongjiang, Liaoning, Hubei, and Yunnan, China (Ji et al. 1982;Chen 1984;Wu and Wang 1991;Liang 2000).The species was transferred to the genus Neodactylogyrus by Yamaguti (1963), although this genus had been synony-mized with Dactylogyrus by Mizelle and Donahue (1944).The specimens examined in this study almost conform to the descriptions and illustrations of D. squameus by Gussev (1955Gussev ( , 1985)), Bykhovskaya-Pavlovskaya et al. (1962), Wu  va, vagina;van, ventral anchor;vd, vas deferens;vl, vitellaria;vo, vaginal opening. andWang (1991), Liang (2000), Ondračkova et al. (2004), andGussev et al. (2010).The dorsal anchors of our specimens are slightly smaller than those authors' measurements, but this may be due to intraspecific variation because the anchor's shape and the male copulatory organs of our specimens agree with the cited descriptions.The present collection in Japan represents a new country record for Dactylogyrus squameus.This monogenean established populations infecting Pseudorasbora parva in Kazakhstan, Tajikistan, the Kyrgyz Republic, Ukraine, the Czech Republic, Slovak Republic, and Italy after both species were introduced from Far-East Asia into these countries (Gvozdev and Agapova 1977;Gussev 1985;Ondračkova et al. 2004;Galli et al. 2007).Ondračkova et al. (2004) listed the monogenean as occurring in "Uzbekistan" by reference to Gussev (1985), but this was most probably a mistranslation of "Ukraine".
Japanese name.The new Japanese name is a combination of "motsugo" and "yubigata-mushi", which are the Japanese names of P. parva and the genus Dactylogyrus (as "yubigata-mushi-zoku"), respectively.
Material examined.Twenty specimens were used for the description.Soft anatomy was observed in 10 specimens (NSMT-Pl 6180) from Ibaraki Prefecture stained in Heidenhain's iron hematoxylin.Sclerotized structures were measured in eight and two specimens (NSMT-Pl 6180 and 6182) fixed in APG, from Ibaraki and Okayama prefectures, respectively.

Site of infection. Gills.
Prevalence and intensity range (mean).90% (9/10) with no data for intensity in Lake Kasumigaura, Ibaraki Prefecture; seven worms infected one P. parva in the Senō River, Okayama Prefecture.
Japanese names.The new Japanese generic name, "yotsume-ikari" ("zoku" means a genus), refers to the opisthohaptor with four anchors possessed by Ancyrocephalus spp.and means a grapnel anchor in Japanese.Part of the new Japanese name of the species is "motsugo", which is the Japanese name of the host.
Remarks.In Japan this monogenean was first described from Pseudorasbora parva and P. pumila Miyadi, 1930 in Lake Kasumigaura, Ibaraki Prefecture and in ponds of Shinonoi, Nagano Prefecture, respectively by Nitta and Nagasawa (2014).The specimens from Nara and Okayama prefectures correspond well with those from Lake Kasumigaura and ponds of Shinonoi.The present collections constitute new prefectural records and extend the geographical distribution of the species from Ibaraki and Nagano prefectures westward to Nara and Okayama prefectures.
Japanese names.The new Japanese generic name is a combination of "Futa-ana" and "mushi" which mean two holes (i.e., vaginae) and worms, respectively ("zoku" means a genus).Part of the new Japanese name of the species is "motsugo", which is the Japanese name of the host.

Discussion
Two of the three monogeneans found in this study, Dactylogyrus squameus and Ancyrocephalus pseudorasborae, were collected from Pseudorasbora parva in the native habitats of this fish in Japan.Thus, as with Bivaginogyrus obscurus (Nitta and Nagasawa 2014), they are considered to be native to Japan.Recently, P. parva has been recorded in Hokkaidō, the Tōhoku District, and Okinawa Prefecture as a domestic alien freshwater fish (Matsuzawa and Senou 2008), and it has also been suggested that some individuals of the species were introduced from the Eurasian continent into several rivers in Japan (Watanabe et al. 2000;Yoshigou 2013).Two dactylogyrids, D. squameus and B. obscurus, have spread into Europe with P. parva (Ondračkova et al. 2004;Galli et al. 2007;Gozlan et al. 2010;Anonymous 2012), and they might have become established outside of their original range on this fish in Japan as well.Many species of freshwater fish are currently known as domestic alien species in Japan (Matsuzawa and Senou 2008), but no information is available about their monogeneans.It is necessary to clarify the monogenean fauna of such domestic alien freshwater fishes.
At present, five species of Pseudorasbora Bleeker, 1860 are recognized as valid, and three of them, P. parva, P. pumila, and P. pugnax Kawase and Hosoya, 2015, are distributed in Japan (Kawase and Hosoya 2015).The latter two species are endemic to Japan and have been designated as critical endangered species by the Ministry of the Environment of Japan (Sugiyama 2015;Kawamura 2015).To date, Bivaginogyrus obscurus has been reported from P. pumila (Nitta and Nagasawa 2014), but nothing is known about the monogenean fauna of P. pugnax.More study is needed on the monogeneans of these fishes because host-specific parasites of endangered hosts are sometimes threatened with co-extinction (Windsor 1990;Stork 1993;Barus et al. 1997).