The occurrence of the non-native tapeworm Khawia japonensis ( Yamaguti , 1934 ) ( Cestoda ) in cultured common carp in the Czech Republic confirms its recent expansion in Europe

Invasive parasites represent a serious problem due to their capacity to threaten local populations of native (often endemic) hosts, and fishes in breeding facilities. Tapeworms (Cestoda) are extremely adapted (they lack any gut and circulatory system) parasitic flatworms some of which have colonised new geographical regions as a result of unintentional transfer of hosts infected with these parasites. The highest number of invasive parasites within this host-parasite system is among tapeworms parasitizing common carp (Cyprinus carpio L.), which has also been introduced globally. In the present study, we report another record of the Asian non-native fish tapeworm Khawia japonensis (Yamaguti, 1934) (Cestoda: Caryophyllidea) from common carp in Europe. Previous records of this cestode from Italy (Po River basin) and Slovakia (Danube River basin) and its present finding in the Czech Republic (Elbe River basin) confirms recent expansion of the parasite in Europe. Potential impacts of this non-native parasite on common carp in commercial breeding fisheries should be carefully studied.


Introduction
Tapeworms (Cestoda) are frequent and widely distributed intestinal parasites of cultured and wild fish globally (Williams and Jones 1994;Scholz and Kuchta 2017).Among fish tapeworms, caryophyllideans are the most common parasites of common carp, Cyprinus carpio Linnaeus, which is the most important fish in freshwater aquaculture in temperate Europe and Asia (Froese and Pauly 2018).Four of these tapeworms, Atractolytocestus huronensis Anthony, 1958;Khawia sinensis Hsü, 1935;K. japonensis (Yamaguti, 1934), and possibly Caryophyllaeus fimbriceps Annenkova-Chlopina, 1919, have spread from their original distribution and colonised new regions including new continents (Oros et al. 2004(Oros et al. , 2009(Oros et al. , 2011;;Scholz et al. 2011a).Two of these species, C. fimbriceps and K. sinensis, are potentially lethal pathogens of carp fry (Bauer et al. 1973;Mitterpák and Huňady 1984); and A. huronensis, which has not been connected with mortality or any obvious decline of host fitness, induces serious local histopathological lesions on intestine mucosa (Molnár et al. 2003;Kappe et al. 2006;Gjurcevic et al. 2012).These three species can thus be considered as invasive parasites.In contrast, the effect of K. japonensis on its definitive fish host is still unknown.
The Asian fish tapeworm Khawia japonensis was described as Caryophyllaeus japonensis from common carp in Japan (Figure 1), and its original distribution included eastern and south eastern Asia (Japan, Amur River basin in Russia, and Vietnam) (Yamaguti 1934;Demshin 1978;Protasova et al. 1990;Scholz et al. 2001Scholz et al. , 2011a)).Just recently, this rather neglected carp parasite that had not previously attracted the attention of veterinarians, appeared in Europe: in common carp from a small irrigation canal in the Po River basin, northern Italy (Scholz et al. 2011b), and from farmed common carp in breeding fishponds in the Danube River basin, eastern Slovakia (Oros et al. 2015).In the present report, we present the third record of this non-native parasite in another principal river basin of Europe, which indicates the potential for K. japonensis to invade new geographical regions.

Methods
Tapeworms were found in the anterior intestine of one of two common carp, Cyprinus carpio (host code TS-2976; total length of 45 cm) from Velký Tisý fishpond (49º3′42″N; 14º42′55″E) near Třeboň examined on 20 October 2016; and one of eight carp (host code TS-2982; total length of 50 cm) from Horusický fishpond (49º09′23″N; 14º40′37″E) near Veselí nad Lužnicí (both fishponds in South Bohemia, Czech Republic; Elbe River basin) on 26 October 2017.Tapeworms were gently rinsed in a saline solution and the specimen found in 2016 was divided into three parts: the anterior and posterior regions were fixed in hot (almost boiling) 4% formaldehyde solution, whereas the middle part was placed in moleculargrade 99% ethanol.The anterior and posterior parts of the first specimen, i.e. the hologenophore (Figure 2), and the entire, gravid specimen found in 2017, were stained with Mayer's carmine, mounted in Canada balsam and deposited in the helminthological collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice (Coll.No. IPCAS C-348/1).
Genomic DNA was extracted from the middle body part of the 2016 specimen (field no.TS-2976a) using the DNeasy ® Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer´s instructions.The target D1-D3 region of the large rDNA subunit (lsrDNA) was amplified by using LSU5 and 1500R primers (Littlewood et al. 2000;Olson et al. 2003) expecting a length of approximatelly 1,600 bp.The PCR amplification was initiated by denaturation for 5 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C, 2 min at 72 °C, and terminated by 10 min at 72 °C.The purification of PCR products was described in detail by Brabec et al. (2012).Sanger sequencing was performed at GATC Biotech (Cologne, Germany) using both PCR primers and internal primer 900F (Olson et al. 2003).Contiguous sequences were assembled and inspected for ambiguous and erroneously sequenced bases using the software platform Geneious ® (Kearse et al. 2012).The BLAST algorithm was employed to compare the originally acquired sequence (acc.no.MH237608) with respective sequences of the congeneric species available in GenBank: specifically K. armeniaca from Armenia (JN004257), K. cf.armeniaca from Portugal (JN004263), K. japonensis from Japan and Slovakia (JN004258  (1959), respectively, in fact represent Khawia iowensis (syn.K. japonensis) (see Calentine andUlmer 1961 andMackiewicz 1970).Only first records of K. japonensis occurrence in a state are pointed out, for further geo-referenced data see Supplementary material Table S1.and KP790244), K. parva from Russia (JN004267), K. rossittensis from Japan and Slovakia (JN004259 and JN004260), K. saurogobii from China (JN004262), and K. sinensis from China, Japan, Slovakia and United Kingdom (JN004265, JN004264, JN004261, and EU343740).A sequence of Khawia baltica (JN004266) was not included in the comparison because this species in fact belongs to the genus Caryophyllaeus (see Barčák et al. 2017).

Results
The tapeworms found in common carp from two fishponds in South Bohemia possess morphological characteristics typical of K. japonensis: an afossate scolex lacking apical and lateral structures, with frilled anterior margin and wedge-shaped projections, only slightly wider than the neck, extensive vitelline follicles separated to preovarian and postovarian groups, with a few follicles present alongside the ovarian arms, and the testes reaching anteriorly at approximately same level as anteriormost vitelline follicles (Figure 2).Moreover, morphometrical comparison of both specimens from the Czech Republic with vouchers of K. japonensis from Asia (Japan and Vietnam), North America (USA) and Europe (Italy and Slovak Republic) showed no obvious differences.Species identification was also confirmed by molecular data.

DNA analysis
The partial sequence (D1-D3 regions) of lsrDNA was 1,568 bp long and 100% identical with both available sequences of K. japonensis from Lake Onuma, Hokkaido, Japan and a fish breeding facility near Brzotín village, Slovakia.Unfortunately, no molecular data are available for K. japonensis from the type locality: Lake Biwa, Honshu, Japan.Pairwise sequence identity between the originally obtained sequence presented herein and the remaining species of Khawia ranged from 92.1 % (K.armeniaca from Armenia) to 97.4 % (both specimens of K. rossittensis from Japan and Slovakia).

Discussion
In contrast to another Asian carp tapeworm, Schyzocotyle acheilognathi (Yamaguti, 1934) (syn.Bothriocephalus acheilognathi; order Bothriocepha-lidea), which is invasive and able to infect an extremely wide spectrum of unrelated fishes (Scholz et al. 2012;Kuchta et al. 2018), all invasive or potentially invasive caryophyllideans of carp have a strict (oioxenous) or narrow (stenoxenous) host specificity (Scholz et al. 2011a;Scholz and Oros 2017).As tapeworms are entirely dependent on the presence of both definitive and intermediate hosts, a narrow host spectrum may limit their ability to colonise larger areas.However, caryophyllidean cestodes of carp use similar species of globally occurring naidids as intermediate hosts (Mackiewicz 1972;Demshin 1978;Scholz 1991), and common carp has also been distributed worldwide for aquaculture, game fishing, or as ornamental pond fish (i.e.japanese "koi" or "nishikigoi").These tapeworms can therefore easily complete their life cycle in newly colonised areas.
Caryophyllidean cestodes of carp differ markedly from each other in their colonisation success.Caryophyllaeus fimbriceps, described from the Caspian Sea region, has been reported only from central and eastern Europe and may be autochthonous there (Barčák et al. 2017).Interestingly, this species may have disappeared completely because there have been no reliable records of its occurrence for several decades (see Barčák et al. 2017).Khawia sinensis, described from China, has successfully colonised Europe, including the British Isles, and has also been reported from North America (Scholz et al. 2011a).However, its expansion does not seem to be continuing, unlike Atractolytocestus huronensis which is currently the most frequent cestode in common carp, with reports from a number of European countries (Oros et al. 2011).This tapeworm had been imported to North America together with its specific host from Eastern Asia, most probably before or around the middle of the 20th century; it was originally described from common carp from an impoundment of the Huron River (Michigan, USA) by Anthony (1958).
Khawia iowensis was originally found in Cyprinus carpio in Kansas (USA) (Wilson 1957), but was misidentified as Caryophyllaeus sp.(see Mackiewicz 1970).Calentine and Ulmer (1961) provided a description of a new species, K. iowensis, from the same host in Iowa (USA), which was later synonymised with K. japonensis by Scholz et al. (2011a).Therefore, records of tapeworms designated as K. iowensis in North America (USA -California, Illinois, Iowa, Kansas, Minnesota, Nebraska, North Dakota, Oklahoma, Oregon, Tennessee, South Dakota, Wisconsin, Canada -Manitoba, Ontario, Mexico -Rio Grande basin; see Supplementary material Table S1) represent the first evidence of non-natural expansion of K. japonensis to a new continent.
Recently, this cestode has colonised three major river basins in Europe, having escaped from fish cultures to free waters (Scholz et al. 2011b), which makes its effective control and eradication problematic.Moreover, pathogenicity of K. japonensis, perhaps intensity-dependent as in congeneric species K. sinensis (Bauer et al. 1973), is thus far unknown.Calentine and Ulmer (1961) recorded a maximum intensity of infection of 624 (immature) specimens, but provided no information about the impact on host health.Such intensities in a fish appears to be rare since 9 other fish from the same collection sites and over a nine-month period had a maximum mean intensity of only 35 worms.However, considering the situation of heavily infected farmed young carp, potentially burdened by low hygiene and poor nutrition as in the case of Caryophyllaeus fimbriceps (see Mitterpák and Huňady 1984), the impact of any introduced parasite is unpredictable.
Since K. japonensis has recently been found in regions where intensive research on fish parasites has been carried out, it is unlikely that the parasites have been there for longer but at a low prevalence.It is therefore alarming that this parasite has colonised three distinct watersheds in Europe within a few years (Figure 1).

Figure 1 .
Figure 1.Distribution of the non-native Khawia japonensis (Yamaguti, 1934) in A -North America, B -Europe, C -Japan (only the type locality is shown).Number indicates year of first occurrence in a given country, or year of publication of such finding if it is in parentheses.*, ** Specimens misidentified as Caryophyllaeus sp. by Wilson (1957) and C. laticeps by Rehder(1959), respectively, in fact represent Khawia iowensis (syn.K. japonensis) (seeCalentine andUlmer 1961 andMackiewicz 1970).Only first records of K. japonensis occurrence in a state are pointed out, for further geo-referenced data see Supplementary material TableS1.