Fish Pathology, 50 (4), 183–191, 2015

R esearch article Paradeontacylix buri n . s p . (T re matoda: Aporocotylidae) from Seriola quinqueradiata cultured in Japan with a description of unidentified Paradeontacylix s p . fro m Slaland . i

Kazuo Ogaw a1*, Kousuke Akiyama2 and Daniel Grabner3

1Meguro Parasitological Museum, Tokyo 153-0064, Japan 2Marine Biological Technology Centre, Nippon Suisan Kaisha, Ltd., Oita 876-1204, Japan 3D epartment of Aquatic Ecology, University of Duisburg-Essen, D -45141 Essen, Germany

(Received May 9, 2015)

ABSTRACT—Paradeontacylix buri n. sp. is described based on specimen s fro m the afferent branchial arteries of the Japanese amberjack Seriola quinqueradiata cultured in Mie and Oita Prefectures, Japan. The new species can be differentiated from congeners by a body of up to 4.15 mm w ith lanceolate tegumental spines of the same size throughout the body, uterus ascending after leaving the oötype and the v ite lla riu m not extending posterior to the ovary. P . b u ri n . s p . is u n iq u e a mong Paradeontacylix species fro m S e rio la spp. in having the same s iz e te g u mental spines throughout the body. A phylogenetic analysis based on the sequences of the internal transcribed spacer 2 and on the 28S rRN A-gene demonstrated that P . b u ri is grouped with other Paradeontacylix species from S e rio la spp., indicating that the enlarged tegumental spines in the posteriormost rows in the other Paradeontacylix s p p . fro m S e rio la spp. are not a morphological feature at the generic level. Another blood fluke, Paradeontacylix sp., is described based on a single specimen fro m the afferent branchial artery of yellow tail ambe rja c k S . la la n d i cultured in Oita Prefecture, Japan. This species may be differentiated fro m th e most simila r P . b u ri n . s p . b y th e c irru s (conical and thick walled in Paradeontacylix s p . vs. spherical and thin-walled in P . b u ri) and by the uterus (extending posteriorly up to the level of the oötype vs. extending posterior to the oötype).

K ey word s : Paradeontacylix buri, blood fluke, Seriola quinqueradiata, Seriola lalandi, Japanese ambe rja c k , y e llo w ta il a mberjack, new species

C arangid fish of the genus S e rio la a re c o mposed have been reported in Paradeontacylix; P. odhneri of nine species, which are distributed in low and (L a y man, 1930) (as Aporocotyle odhneri) fro m Takifugu middle latitude areas worldwide (Froese and Pauly, porphyreus (Tetraodontidae) (as Spheroides borealis) 2015). Seven species of blood flukes of the genus (L a y man, 1930), P . s in e n s is L iu, 19 9 7 fro m Takifugu Paradeontacylix have been recorded from three S e rio la oblongus (Tetraodontidae) (as Fugu oblongus) (Liu, spp., namely , P. sanguinicoloides fro m y e llo w ta il a mbe r- 1997) and P . megalaspium Lakshmi and Madhavi, ja c k Seriola lalandi (s e e McIntosh, 1934; Hutson and 2007 from Megalaps is c o rd y la (Carangidae) (Lakshmi Whittington, 2006) and Samson fish S. hippos (s e e and Madhavi, 2007). Of th e m, P. sinensis Liu, 1997 Hutson and Whittington, 2006); P. grandispinus, P . w as recently moved to Psettarium a s P. sinense (nec P . kampachi, P. ibericus and P. balearicus fro m greater sinensis) (Oré lis -R ib e iro et al., 2 0 1 4 ), w h ic h w ill b e re d e - ambe rja c k S . d u merili (s e e Ogaw a and Egusa, 1986; scribed elsewhere in more detail (Ogaw a and Liu, in R epullés-Albelda et al., 2008); and P. godfreyi fro m y e l- preparation). lo w ta il a mberjack (Hutson and Whittington, 2006) and S e rio la species are highly valuable fish that are Paradeontacylix sp. fro m y e llo wta il a mberjack and cultured in the Far East, Europe and Oceania. Samso n fish (Hutson and Whittington, 2006). F is h Paradeontacylix species are know n as serious patho- common names accord with those in FishB ase gens of cultured S e rio la s p p . P. grandispinus and P . (http://fishbase.org/h o me.h tm) throughout this article. kampachi caused mass mortalities in greater amberjack Three more species other than those from S e rio la s p p . in Japan (Ogawa and Egusa, 1986; Ogawa and Fukudome, 1994). C respo et al. (1992) reported mass * Corresponding author mortality of greater amberjack juveniles cultured in E-mail: o g a w a k @kiseichu.org Spain associated with unidentified blood flukes. Low - 184K. Oga w a , K . A k iy a ma and D. Grabner le v e l mor ta litie s a mon g y e llo w ta il a mberjack juveniles in species were examined: P. grandispinus, P . k a mpachi, New Zealand were caused by an unidentified P. balearicus and P. ibericus deposited in the Meguro Paradeontacylix (see Diggles and Hutson, 2005). In Parasitological Museum, Tokyo, Japan (MPM C o ll. N o . Japan, blood fluke infections have been reported 19415, 19416, 18871 and 18872, respectively). F o r P . not only in greater amberjack but also in cultured sanguinicoloides, digital images of the type specimen Japanese amberjack Seriola quinqueradiata (Ogaw a w ere obtained from the United States National Parasite and Yokoyama, 1998). Kumon et al. (2002) reported C o lle c tio n (U S N P C ), B e lts v ille , Mary la n d , U S A (U S N P C that blood fluke infection caused death in the latter fish N o. 34329). and suggested that the infection promote s mor ta lity o f S. quinqueradiata co-infected with the pathogenic bacte- Molecular data and phylogeny riu m Lactococcus garvieae. However, the species D N A w a s e x tra c te d fro m two specimens obtained infecting S. quinqueradiata has yet to be identified. In fro m Japanese ambe rja c k fro m Miyazaki using an this paper, morphological and phylogenetic analyses Agencourt DNAdvance Kit (Beckman Coulter, Inc.) based on partial sequences of the ribosomal DNA according to the manufacturer’s instructions. The revealed that the blood fluke is a new species of the e x tra c te d D N A w a s a mplifie d b y P C R w ith th e p rimers genus Paradeontacylix. In addition, a single specimen 3S /ITS2.2 and U 178/L1642 to obtain the comple te o f Paradeontacylix s imilar to this new species found in sequences of the internal transcribed spacer 2 (ITS2) y e llo w ta il a mberjack cultured in Japan is also described and a partial sequence of the large subunit of the ribo- in this paper. somal RN A gene (28S rDN A) as described in Ogaw a e t al. (2 0 1 1 ), w ith s lig h t modifications. All PC R reactions w ere performed in 20 m L reactions containing TaKaR a Materials and Methods Ex Taq (TAKARA BIO, In c .), 1 x E x T a q B u ffe r, 0 .8 mM C ollection of parasite specimens dN TP-mix , 0 .5 m M o f e a c h P C R p rimer and PC R grade One -y e a r-o ld Japanese amberjack S e rio la w ater to 20 m L. The denaturation of DN A (95°C for 2 quinqueradiata, which were wild caught and cultured in min ) w a s fo llow e d b y 3 0 c yc les o f a mplific a tio n : 9 5 ° C Mie , Oita and Miyazaki Prefectures, and 1-year-old yel- for 30 s, 56°C for 30 s, 72°C for 30 s for ITS2 and 95°C lo wta il a mberjack Seriola lalandi, whic h were wild fo r 3 0 s , 5 0 °C fo r 3 0 s , 7 2 °C fo r 1.5 min fo r 2 8 S rD N A caught and cultured in Oita Prefecture were examined and a final extension at 72°C for 10 min. DNA w a s for blood fluke infection. The gills and heart were purified from the PC R product using Agencourt AMPu re re mov e d fro m freshly killed fish, placed in physiological (B e c k man Coulter, Inc.), and cycle sequencing of both saline and were examined for flukes under a dissection the forward and reverse strands was performed using a microscope. Live blood flukes were collected from th e B ig Dye Te rminator v3.1 Cycle Sequencing Kit (Life afferent branchial arteries; they were either flattened Technologies Corporation). T h e P C R p rimers a n d tw o under a cover slip and fixed in AFA (mixtu re o f 70 % additional inner primers (fo r 2 8 S rD N A ) w e re u s e d fo r ethanol 20 parts, 37% fo rmaldehyde 1 part, acetic acid cycle sequencing (ABI PRISM 3100-Avant Genetic 1 part) for morphological examination or fixed in 70% Analyzer, Life Technologies Corporation). T o v e rify ethanol for molecular analysis. the identity of the sequences, a BLAST search was conducted (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Morphological data A phylogenetic tree was constructed using the con- Who le mount specimen s fro m ambe rja c k s in Mie catenated ITS2 and 28S rDNA a lig n ment of related and Oita , which were stained with Heidenhain’s iron blood fluke species (see Fig. 8 for accession numbe rs ). haematoxylin, were prepared as described previously A lig n ments were created using the software ClustalX (Ogaw a et al., 2010). All figures were draw n using a v.2.0 (Larkin et al., 2007), and ambiguous sections draw ing tube. Measurements were mad e u s in g a c a li- were manually deleted using the program B io E d it brated ocular mic ro meter and were given in mic ro me- v .7 .2 .0 (Hall, 1999). The jModeltest tool (Posada, ters as the mean plus the range in parentheses. 2008) was used to determine the appropriate substitu- Because the entire body, the vesicula seminalis and the tio n models. Bayesian inference (BI) and maximum cirrus sac were strongly curved, these parts were not lik e lih o o d (ML) phylogenetic analyses were conducted measured using an ocular mic ro meter but were meas- using MrB a y e s v 3 .1 .2 (Ronquist and Huelsenbeck, u re d o n a c o mputer using the ImageJ program (image 2003) and phyML v .3 .0 (Guindon and Gascuel, 2003) processing program available at: http://imagej.nih.gov/ij/) s o ftware, respectively. A general time reversible as described previously (Ogawa et al., 2 0 1 1 ). The model (GTR + G + I) w ith g a mma rate variation across number of specimens of Paradeontacylix b u ri n . s p . sites and several invariant sites were used for the measured for each body part is given in parentheses analysis. P a ra meters for the BI analysis were set to only when not all of the specimens could be measured. 1,000,000 generations, 4 chains and 2 independent The type specimens of the following Paradeontacylix runs. Trees were sampled every 100 generations, and N ew blood fluke from Seriola quinqueradiata 185 the first 35% o f the sa mple s were discarded (burn- ence in the spine arrangement, the phylogenetic analy- in-phase). Gamma dis trib u tio n and substitution sis deduced from the ITS2 and 28S regions of rDNA paramete rs w e re e s timated during the analysis using 4 sequence indicates that the present species is included substitution rate categories. A s ta rtin g tre e was in the clades of other Paradeontacylix species. Thus, constructed using the neighbour-joining method, th e blood flukes fro m Japanese amberjack are and 5 random starting trees were used. A bootstrap- described in this study as new species of the genus ping analysis was conducted with 1,000 replicates. Paradeontacylix. Definition of the genus followed Aporocotyle spinosicanalis was used as an outgroup Smith (2002) with the exception of the distribution of the for both analyses. For BI and ML, a 5 0 % majo rity -ru le v ite lla riu m, a s th e v ite lla riu m o f Paradeontacylix odhneri consensus tree was obtained from the resulting trees. extends posterior to the ovary. The trees were visualised using the program FigTree v .1.4 .0 (h ttp ://tre e .b io .e d .a c .u k /). Aporocotylidae Odner, 1912 Paradeontacylix McIntosh, 1934 Paradeontacylix buri n . s p . R esults D escription (Figs. 1–4) Taxonomy B ased on 14 who le -mounted specimens. Body Blood flukes collected from Japanese amberjack smooth, dorsoventrally flattened, lanceolate, 2,710– share all the morphological characteristics with 4,150 (3,180) long, 300–470 (400) wide, 6.20–10.12 Paradeontacylix s p p . fro m S e rio la spp., except for the (8 .1 5 ) times longer than wide. Tegumental spines on absence of enlarged spines (=both large posterior tegu- lateral margins ventrally, in 476–616 (541) (n=13) row s mental spines, LPTS, and med iu m posterior tegumental in n u mber on either side of body; same size throughout spines, MPTS, according to Hutson and Whittington the body, 5–6 (n=13) long, 9–19 per row, decreasing in [2006]) on the posteriormos t ro w s . D espite the differ- number tow ards both extremities to about 5 in anterior-

Figs. 1–4. Paradeontacylix buri n . s p . 1; Whole worm, holotype, ventral view. Scale bar: 0.5 mm. 2; Posterior part of body, paratype, ventral view. Scale bar: 0.2 mm. 3; Posteriormos t ro w s o f te g u mental spines, paratype (arrow : posterior end of body). Scale bar: 0.02 mm. 4; Male terminal genitalia, paratypes, ventral view. Scale bar: 0.03 mm. 186K. Oga w a , K . A k iy a ma and D. Grabner most and posteriormos t fe w ro w s . N e rv e c o mmissure (17.1–24.1% of body length) from posterior end of body. 103–159 (125) from anterior end. Mouth opening V ite lla riu m extending from level between mouth opening ventro-subtermin a lly, 11 – 22 (15) fro m anterior end. and nerve commissure to level of posterior end of ovary. Oral sucker absent. Oesophagus sinuous, gradually U terine eggs ellipsoidal, 20–28 (23) long, 14–23 (18) w idening tow ards its end, surrounded by conspicuous w id e . gland cells posterior to nerve commissure, 520–850 (670) long, 16.9–26.8% (2 1 .0 %) of body length. Intes- Taxonomic s u mmary tine H-like; anterior caeca 56–124 (82) long, unequal; Type-host: S e riola quinqueradiata (Actinopterygii: posterior caeca 1,060–2,200 (1,450) long, 10.2–48.5 Perciformes: Carangidae). times longer than anterior pair. Localities and dates: Ohka, Kihokucho, Kitamuro-gun, Te ste s in two ro ws in intercaecal and extending Mie Prefecture (34°14Ń, 136°15É) (type-locality) on slightly into extracaecal field of posterior caeca, 43–83 July 26, 1985; K amiura, Saiki, Oita P ref. (33°02Ń, in n u mber (n=11), each rounded or ellipsoidal. Testicu- 131°55É) on March 15, 1994. lar zone 970–1,870 (1,230) long, 32.5–52.3% (40.3%) Sites in host: Afferent branchial arteries. of body length. Vas deferens originating in anterior Type-material: Holotype and 13 paratypes depos- te s te s , medial, running posteriorly, dorsal to ovary, turn- ited in the Meguro Parasitological Museum, Tokyo, MPM ing sinistrally at its distal end, leading to seminal vesicle. C oll. No. 20998 (holotype), 20,999–21,000 (paratypes). Vesicula seminalis 162–327 (247) long, 22–57 (40) E ty mology: Refers to the Japanese name o f th e w ide, curved sinistrally at its proximal end, then dex- host, “buri”. tra lly in middle and then sinistrally towards its distal end, leading to pars prostatica. Vesicula seminalis and pars Remark s prostatica enclosed in cirrus sac, 168–335 (258) long, The new species can be differentiated from th e 28–62 (46) wide. Pars prostatica sh o rt, 10 – 17 (13 ) nine congeners (P. sinense not included) by the combi- long (n=10). Prostate gland cells at distal portion of cir- nation of the follow ing morphological characters: 1) a rus pouch. C irrus spherical, thin-walled, 21–34 (27) in body of up to 4.15 mm, 2) body length-width ratio of d ia meter, protruded from cirrus sac. Male genital pore 6.20–10.12, 3) lanceolate tegumental spines of identi- dorsal, 37–77 (54) from sinistral body margin, 410–720 cal size throughout the body, 4) distance between end (540) from posterior end of body. of ovary and end of body being 20.4–28.8% of body Ovary shield- or heart-shaped, having small ante- length, 5) uterus ascending after leaving the oötype, rior lobes, medial or slightly dextral, posterior to testes, then descending to level of posterior end of oötype, rela- ventral to vas deferens, 172–294 (231) long, 213–320 tive position of the ovary and 6) the vitellarium not (273) wide, 570–950 (780) (=20.4–28.8% of body extending posterior to the ovary. Among the nine con- length) from its posterior end to body end. Oviduct geners, P. sanguinicoloides is most simila r to P . b u ri n . originating at posterior end of ovary, slightly on its dex- sp. but different from the new species in 1) the enlarged tral side, passing posteriorly. Posterior part of oviduct posterior spines and 2) distribution of the uterus, in expanding to form oviducal seminal receptacle, w hich it extends posterior to the oötype. 170–310 (250) long, 35–71 (48) wide. Oviducal semi- C oncerning the tegumental spines, there is incon- nal receptacle passing posteriorly and then antero-sin- sistency in the description of the large posterior tegu- is ta lly , jo in ing vitelline duct before forming oötype. mental spines (LPTS) (Hutson and Whittington, 2006) Oötype sinistral to oviducal seminal receptacle, direct- o f s o me Paradeontacylix species from S e rio la sp p . ing antero-sinistrally, anterior to junction of oviduct and R e -e x a mination of the tegumental spines of the type vitelline duct, 34–62 (53) long (n=13), 30–52 (45) wide specimens of the Paradeontacylix spp. in question was (n=13), surrounded by Mehlis’ gland. V ite llin e d u c t, mad e in th is stu d y (T a b le 1 ). In P . k a mpachi, pres- 16–31 (21) in maximum w idth (n=12), passing posteri- ence of LPTS (11–15 m m lo n g v s 4 – 7 m m long for mar- orly, sinistral to oviduct. U terus occupying post-ovar- ginal tegumental spines [MT S]) was confirmed a s in th e ia n z o n e , s in is tra l to v ite llin e d uct, after ascending short original description (Ogaw a and Egusa, 1986). In addi- distance, descending until 21–112 (62) anterior to level tion, anterior to the rows o f LP T S , se ve ra l ro ws o f of posterior end of oötype, then taking a counterclock- med iu m posterior tegumental spines (MPTS) (Hutson w ise turn to direct anteriorly and forming ascending por- and Whittington, 2006) were present. T h e n u mber of tion of uterus, coiling three times before forming row s of LPTS plus MPTS varied in the range of 16–30 descending portion of uterus, leading to metraterm. (T a b le 1 ). H u ts o n and Whittington (2006) observed no Metraterm straight or slightly curved sinistrally, 51–102 large spines in P . k a mpachi sensu Montero et al., 2003, (79) long, 14–32 (22) wide. Female genital pore dor- w hich was later re-identified with P. ibericus based on sal, opening in midline, antero-dextrally to male genital th e morphological and molecular data (Repullés-Albelda pore, 107–196 (146) from sinistral body margin, et al., 2008). How ever, enlargement of the posterior 95–159 (127) from male genital pore, 480–760 (630) spines for P. ibericus (two paratypes; LPTS of holotype N ew blood fluke from Seriola quinqueradiata 187 n s . p . 14 none none none Japan (n–13) Seriola Seriola 5–6 long 5–6 this study this P . b u ri quinqueradiata rili . 3 et al et end”] (2008) (n = 2 ) (n = 2 ) (n = 3 ) diterranean 6–7 long 6–7 5 ro w s ? ] – o s row 2–3 [4–6 long] [4–6 P. ibericus P. –2rws row 9–32 [“gradually [13 (8–18)] [13 decreasing 12–14 long 12–14 [3–5 spines/ [3–5 Me Seriola du me Seriola tow ards anterior ards tow R epullés-Albelda ittington (2006). ittington Wh r ili . 2 et al et (2008) 2 ro w s (n = 2 ) (n = 2 ) (n = 2 ) diterranean 2 ro w s ? ] 4–6 long 4–6 [2–5 long] [2–5 –4rws row 8–14 [“gradually 17–22 long 17–22 2–3 spines/ 2–3 [2–3 spines/ [2–3 [22–24 long] [22–24 P. balearicus P. decreasing in decreasing anterior end”] anterior Me eghtwards tow length Seriola du me Seriola R epullés-Albelda 2 [– ] [24] ittington (2006) 4 ro w s ] [5 ro w s ] A u s tra lia [3 spines/ [3 Wh H utson and H utson Seriola hippos Seriola Seriola lalandi, Seriola ntal spines according to Hutson and Hutson to according spines ntal Paradeontacylix sp. Paradeontacylix D ata in square brackets are those in the original descriptions original the in those are brackets square in D ata ns. me 8 [– ] [33] ittington (2006) 4 ro w s ] A u s tra lia P . g o d fre i [4 spines/ [4 46rws] row [4–6 Wh H utson and H utson me tegu posterior Seriola lalandi Seriola m rili d iu spp. type speci type spp. me achi me 10 mp aw a and aw a Japan (n = 7 ) (n = 8 ) (n = 9 ) (n = 7 )] (n = 9 )] (n = 9 )] (n = 1 0 ) 4–7 long 4–7 [6–7 long [6–7 –2rws row 5–12 11–15 long 11–15 Og 2–7 spines/ 2–7 [13–17 long [13–17 63 o s row 16–30 P . k a “a fe w ” ro w s [2–5 spines/ spines/ [2–5 Egusa (1986) Egusa [data not given] not [data S e rio la d u rili Paradeontacylix ntal spines plus plus spines ntal 13 aw a and aw a Japan 3 ro w s ] (n = 1 1 ) (n = 1 3 ) (n = 1 2 ) (n = 1 3 ) 4–7 long 4–7 – o s row 2–3 [6–7 long] [6–7 21–27 long 21–27 Og 12 o s row 11–23 2–3 spines/ 2–3 [2–4 spines/ [2–4 [21–26 long] [21–26 Egusa (1986) Egusa [no data given] data [no P. grandispinus P. Seriola du me Seriola ntal spines of of spines ntal n. me 1 1 * 1 1 * * * 1 2 ro w s ] 9 long 9 2 ro w s [15 long] [15 3 spines/ 3 12 long 12 [3 spines/ [3 Intosh (1934) Intosh ≧ 7 ro w s me tegu of nation Seriola lalandi Seriola F lo rid a , U .S .A . [no data given] data [no given] data [no Mc mi P. sanguinicoloides P. me tegu posterior large of s row of er 2 * ges of the type speci type the of ges R eexa . ma m) ns

m ntal me Table 1 Table m) rginal nt e r o ro f w s m ma ntal spines ntal ntal spines: ntal ntal spines ntal er of speci of er ned mi me tegu rginal mb Based on digital i mb nu the digital on Based to Equivalent 1 2 Authors exa Nu Parasite species Parasite Locality rgto etsd frws) row of side left or (right me tegu H ost species H ost those of of those me Arrange * Length (in (in Length spines: Length (in (in Length spines: than larger spines of rgto etsd frws): row of side left or (right me tegu Ma mb nu Total Large posterior Large me tegu Large posterior Large * 188K. Oga w a , K . A k iy a ma and D. Grabner not clearly visible and unmeasurable) and P. balearicus that P. odhneri is not a Paradeontacylix. A redescrip- (holotype and a paratype) from Spanish S . d u merili w a s tion based on newly collected specimens is desired again confirmed in this study, though the measuremen ts because the type specimens have been lost. P . b u ri is were different from those in the original description different from P . megalaspium in the body length-width (Repullés-Albelda et al., 2008). Thus, all the seven ratio, 6.20–10.12 vs. about 20, and uterus first directing know n Paradeontacylix species from Seriola s p p . h a v e anteriorly vs. directing posteriorly. enlarged tegumental spines (LPTS and MPT S ) in th e few posterior rows (T a b le 1 ). P . b u ri n . sp . is thu s Paradeontacylix sp. unique among Paradeontacylix species from S e rio la D escription (Figs. 5–7) spp. in having uniformly sized spines throughout the Based on a single who le -mounted specimen. body. The two Paradeontacylix species from fish other General morphology simila r to P . b u ri n . sp . Body than S e rio la spp. have no enlarged spines in the posteri- smooth, dorsoventrally flattened, lanceolate, 3,430 long, ormos t ro ws. P. odhneri is unique in having a large 380 wide, 9.03 times longer than wide. Tegumental body (9.8 mm in body length; more than twice as large spines on lateral margins ventrally, 6–7 long, approxi- as P . b u ri), posteriorly positioned ova ry (11 % of body mately 20 spines per row, with decreasing number of length from the end of ovary to the body end measured spines tow ards body ends. N one or only a few spines fro m the original figure vs. 20.4–28.8% in P . b u ri) and observed on sinistral side posterior to uterus. Numbe r th e v ite lla riu m extending posterior to the ovary of spine row s not determined. N e rv e c o mmissure 111 (L a y man, 1930). The original description by Layman fro m anterior end. Mou th opening ventro-subtermin a lly , w a s s imple, and a detailed morphological comparison 1 2 fro m anterior end. Oral sucker absent. Oesopha- w ith P . b u ri was not possible in this study. The excep- gus sinuous, gradually widening tow ards its end, sur- tional distribution pattern of the vitellarium may indicate rounded by conspicuous gland cells posterior to nerve

Figs. 5–7. Paradeontacylix s p . fro m y e llo w ta il a mberjack Seriola lalandi. 5; Whole worm, ventral view. Scale bar: 0.5 mm. 6; Posterior part of body, ventral view. Scale bar: 0.2 mm. 7; Male terminal genitalia, ventral view. Scale bar: 0.03 mm. N ew blood fluke from Seriola quinqueradiata 189 commis s u re , 6 6 0 lo n g , 1 9 .2 % of body length. Intestine times before forming descending portion of uterus, lead- H-like; anterior caeca both 92 long, with short side ing to metraterm. Metraterm c u r v e d s in is tra lly in mid- branches; posterior caeca 1,620 and 1,650 long, 17.6 d le, 12 0 lon g , 38 w ide . Female genital pore dorsal, and 17.9 times longer than anterior pair. opening in midline, antero-dextrally to male genital pore, T e s te s in tw o ro w s , a b o u t 7 0 in n u mbe r. Testicular 127 from sinistral body marg in , 11 7 fro m male genital zone 1,820 long, 53.1% of body length. Vas deferens pore, 530 (15.5% of body length) from posterior end of medial, running posteriorly, dorsal to ovary, turning sinis- body. V ite lla riu m extending along body from level of trally at its distal end, leading to vesicula semin a lis . nerve commissure to anterior portion of ovary. U terine Vesicula seminalis, 236 long, 38 wide, curved sinistrally eggs ellipsoidal, 21–28 (24) long (n=5), 15–18 (17) a t its p ro x imal end, then formin g a le tte r C to w a rd s its w ide (n=5). distal end, leading to pars prostatica. V esicula seminalis and pars prostatica enclosed in cirrus sac, 253 long, Taxonomic s u mmary 43 wide. Prostate gland cells at distal portion of semi- H o s t: Seriola lalandi (Actinopteryg ii: P e rc ifo r mes : nal vesicle. Pars prostatica short, 17 long. Cirru s C arangidae). conical and thick-walled, 30 long, 20 wide at base, 14 Locality and date: Saiki, Oita P ref. (32°57´N, w ide at its opening. Male genital pore dorsal, 26 from 131°53´E) on March 14, 1991. sinistral body margin, 450 from posterior end of body. Site in host: Afferent branchial artery. Ovary shield-shaped, having small anterior lobes, Specimen: deposited in the Meguro Parasitological posterior to testes, ventral to vas deferens, 259 long, Museum, T o k y o , MPM C oll. No. 21001. 306 wide. Oviduct originating at posterior end of ovary, slightly on its dextral side, passing posteriorly. Remark s Posterior part of oviduct expanding to form oviducal This species appears to be differentiated morpho- seminal receptacle, 237 long, 56 wide. Oviducal semi- lo g ic a lly fro m th e most simila r P . b u ri b y th e c irru s a n d nal receptacle passing posteriorly and then antero-sin- by the uterus. T h e c irru s in th e fo rmer is conical and istally, joining vitelline duct before forming oötype. thick-walled, whereas that of the latter is spherical and Oötype sinistral to oviducal seminal receptacle, direct- th in -walle d . The uterus extends posteriorly up to ing anteriorly, anterior to junction of oviduct and vitelline the level of the oötype in P . b u ri, whereas it extends duct, 61 long, 45 wide, surrounded by Mehlis’ gland. posterior to the oötype in Paradeontacylix s p . Until V ite llin e d u c t, 1 5 in maximum w idth, passing posteriorly, additional specimens are collected for more accurate s in is tra l to o vidu ct. Uterus occupying post-ovarian morphology and for molecular analysis, this species zone, sinistral to vitelline duct, ascending first and soon re mains as an unidentified species of Paradeontacylix. descending until 82 posterior to level of posterior end of oötype, then taking a clockw ise turn to direct anteriorly Molecular analysis and form ascending portion of uterus, coiling three The two ITS2 (accession no. AB904153) and 28S

Paradeontacylix ibericus AM489598/AM489593 57.7 / 0.70 Paradeontacylix kampachi AM489599/AM489595 99.0 / 1.00 Paradeontacylix buri n. sp. ex Seriola quinqueradiata AB904153/AB904154 60.7 / 0.69 Paradeontacylix godfreyi AM489602/AM489597

51.4 / 0.81 Paradeontacylix balearicus AM489600/AM489594 86.2 / 0.99 Paradeontacylix grandispinus AM489601/AM489596

Psettarium sinense EU082007/EU368853 (formerly Paradeontacylix sinensis) 54.3 / 0.58 Plethorchis acanthus AY465875/AY222178

Aporocotyle spinosicanalis -- /AY222177

0.04 F ig . 8 . Phylogenetic tree of the genus Paradeontacylix based on concatenated ITS2 and 28S rDN A sequences constructed with Bayesian and maximum likelihood inferences. N ode labels show bootstrap support/posterior probabilities. A ccession num- bers of sequences are provided behind species names (ITS2/28s). Aporocotyle spinosicanalis was used as an outgroup. 190K. Oga w a , K . A k iy a ma and D. Grabner rDNA (accession no. AB904154) sequence isolates clades, although these species all parasitize the same obtained from each of the two blood fluke specimens host species, S . d u merili. w ere 100% identical. The phylogenetic analysis clearly The authors of the latter study discussed that the show s the placemen t o f Paradeontacylix buri n . s p . w ith in ancestral species of P . k a mpachi/P. ibericus and P . the genus Paradeontacylix (F ig . 8 ). Paradeontacylix grandispinus/P. balearicus groupings already existed b u ri n . sp . fo rms a clade together with P. godfreyi, P . before the geographical separation of the ho s t balearicus and P. grandispinus fo rmin g th e s iste r c la de populations. The results of the present study show to P. ibericus and P . k a mpachi. How ever, node sup- that both P. godfreyi and P . b u ri n . sp . (fro m S. lalandi port is rather low, therefore the exact position of P . b u ri and S. quinqueradiata, respectively) are more closely n . sp . w ith in th e Paradeontacylix-tre e might change if related to the sib lin g species P. balearicus/P . further sequence information regarding other species grandispinus th a n to P. ibericus/P . k a mpachi. T h is becomes a v a ila b le . Paradeontacylix sinensis (n o w result might indicate that host switching plays a role in Psettarium sinense) does not cluster with the other spe- the evolution of marine blood fluke species; therefore, cies of Paradeontacylix b u t is in a b a s a l p o s itio n to th e more data regarding the host specificity of the Paradeontacylix clade, as show n previously (Holzer e t Paradeontacylix spp. would be of interest to clarify this al., 2008; Cribb et al., 2011), together with Plethorchis issue. acanthus. P . b u ri n . s p . may b e s p e c ific to S. quinqueradiata because this blood fluke has never been found in other S e rio la sp p . Compa re d w ith S. quinqueradiata, S . D iscussion lalandi, the host of Paradeontacylix sp., has been less Paradeontacylix buri n . sp . an d Paradeontacylix frequently examined for parasites, and it is uncertain sp. described in the present study are unique among w hether this blood fluke is a common and specific para- Paradeontacylix species from S e rio la spp. in the site of S. lalandi. arrangement of the body spines. In P . b u ri n . s p ., th e spines in the posteriormost row s were uniformly s iz e d A cknowledgmen ts with no enlarged spines observed in the posterior region, whereas in Paradeontacylix s p ., o n ly a fe w o r The present authors would like to thank Dr. no spines are present in the sinistral row s posterior to Y u k io Maeno of the National Research Institute of the uterus. These atypical morphological features sug- Aquaculture, Mie Prefecture, Japan and Dr. Yutaka gest that the two new species have developed a mod i- Fukuda of the Oita Prefectural Agriculture, Forestry and fied attachment or locomotion in the host vascular Fisheries Research Center, Oita Prefecture, Japan system. P . b u ri n . s p . fo rms a clade together with P . for providing opportunities to examine infected fish. godfreyi, P. balearicus and P. grandispinus. A ll th e la t- They also thank Dr. P a tric ia P ilitt fo r p ro v id in g d ig ita l ter three species have enlarged body spines in the pos- images of the type specimen o f Paradeontacylix te rio r fe w ro w s . sanguinicoloides deposited at the Unite d S ta te s Paradeontacylix sinensis was assigned to N ational Parasite Collection, Beltsville, Maryland, USA. Psettarium based on its morphological and molecular The study was partially funded to KO b y a Gra n t-in -A id features (Repullés-Albelda et al., 2008; Ogaw a et al., fro m the Japan Ministry of Education, Science and 2011; Ogaw a et al., 2007; Holzer et al., 2008; Cribb e t C ulture (No. 25292120). al., 2 0 1 1 ; Ore lis -R ib e iro et al., 2014) and also the present findings substantiate the placement outside of Paradeontacylix. N o molecular data were availa b le fo r R eferences Paradeontacylix sp. in the present study, because a sin- C re s p o , S ., A . Grau and F. Padros (1992): Sanguinicoliasis in gle specimen obtained was used for the morphological the cultured amberjack S e rio la d u merili Risso, from th e description. It would be desirable to construct a phylo- Spanish Mediterranean area. Bull. E u r. A s s o c . F is h genetic tree including this species to improve the resolu- Pathol., 12, 157–159. tion within the genus Paradeontacylix. C rib b , T . H ., R . D . A d la rd , C . J . H a y w a rd , N . J . B o tt, D . E llis , D . Greater amberjack S . d u merili harbours four spe- E v a n s a n d B . F . N o w a k (2 0 11 ): T h e life c y c le o f C ardicola fo rs te ri (T re matoda: Aporocotylidae), a pathogen of cies of Paradeontacylix, viz. P. grandispinus and P . ranched southern bluefin tuna, Thunnus maccoyi. In t. J . kampachi fro m Japan and P. ibericus and P. balearicus Parasitol., 41, 861–870. fro m th e Mediterranean. The present study found Diggles, B. and K. S. Hutson (2005): Diseases of kingfish results similar to that of Repullés-Albelda et al. (2008), (Seriola lalandi) in Australasia. Aquacult. Health Int., 3, which, based on sequences of the partial 28S rDNA, 12 – 14. Froese, R. and D. Pauly, Editors (2015): FishBase. World ITS2 and partial mitochondrial cytochrome o x id a s e 1 - Wide Web electronic publication. www.fishbase.org gene (C O1), show ed that P . k a mpachi/P . ib e ric u s and (02/2015). P. grandispinus/P. balearicus are placed in different Guindon, S. and O. Gascuel (2003): A simple, fast and accu- N ew blood fluke from Seriola quinqueradiata 191

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