Introduction

The monotypic Furcohaptor Bijukumar & Kearn, 1996 was erected for Furcohaptor cynoglossi Bijukumar & Kearn, 1996, a gill parasite of cynoglossids, Cynoglossus macrostomus Norman, and C. puncticeps (Richardson), from India, and assigned to the Ancyrocephalinae Bychowsky, 1937 in the Dactylogyridae Bychowsky, 1933 (see Bijukumar & Kearn, 1996). This genus is characterised by two long haptoral arms; one pair of typical anchors; one pair of rod-shaped hooks; five pairs of typical-shaped marginal hooks; and one pair of marginal hooks which are reduced in size and have no heels (Bijukumar & Kearn, 1996). Subsequently, Boeger et al. (2015) mentioned that the genus is most likely a member of the Diplectanidae Monticelli, 1903 based on the morphological features. However, the type-specimens of F. cynoglossi were in poor condition to make a detailed observation of their internal anatomy (Bijukumar & Kearn, 1996) and no further specimens of the species have been reported since the original description; thus its taxonomic position is still undetermined. During a survey of the parasites of Cynoglossus robustus Günther in the Seto Inland Sea off Hiroshima Prefecture, Japan, we collected specimens form gills identifiable as a species of Furcohaptor. This paper describes this material as a new species of Furcohaptor and amends the diagnosis of the genus to accommodate the new species.

Materials and methods

In total, 56 fresh specimens of Cynoglossus robustus were purchased at the Yokota fishery port, Fukuyama, Hiroshima Prefecture, Japan, on 25 January 2015 (n = 8; standard length, SL, 220.1–258.9 mm), 1 February 2015 (n = 17; SL 132.5–324.1 mm), 28 February 2015 (n = 8; SL 243.2–275.0 mm), 6 December 2015 (n = 10; SL 248.7–306.5 mm), 10 January 2016 (n = 10; SL 283.5–328.0 mm), and 27 March 2016 (n = 8, 236.0–272.0 mm). These fish were commercially caught by trawling in the Seto Inland Sea off Yoko-shima (34°21.5′N, 133°16.5′E), Hiroshima Prefecture, Japan. They were brought on ice to the laboratory of Hiroshima University and examined for gill parasites under the dissecting microscope. Specimens of monogeneans were picked up using small needles and forceps, and flattened between a slide and a coverslip. One specimen was identified under slight coverslip pressure and preserved in 99% ethanol for molecular analysis. Some specimens were fixed in ammonium picrate glycerin (Lim, 1991), while others were fixed in 70% ethanol or acetic acid-formalin-alcohol and stained in Heidenhain’s iron hematoxylin.

All fixed specimens except the one for molecular study were dehydrated through 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 microscope. The method of measuring sclerotised structures is presented in Fig. 1. The penises were measured on images taken by a digital camera on an Olympus DP20 microscope at a magnification of ×1,000 using ImageJ software (version 1.48i). All measurements are in micrometres, and are given as the range followed by the mean and the number (n) of specimens examined in parentheses. The numbering of hook pairs follows Mizelle (1936). Fish identification was based on Yamada & Yagishita (2013). The type-specimens are deposited in the Platyhelminthes collection of the National Museum of Nature and Science (NSMT-Pl), Tsukuba, Ibaraki Prefecture, Japan.

Fig. 1
figure 1

Measurements of hard parts. A, Anchor; B, Bar; C, Marginal hook; D, Male copulatory organ. Abbreviations: al, accessory piece length; atl, anchor total length; bl, bar length; bw, bar width; drl, deep root length; ln, length to notch; mhl, marginal hook length; pel, penis length; pl, point length; srl, superficial root length

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DNA was extracted from one specimen using the DNeasy blood and tissue kit (Qiagen, Valencia, USA) in accordance with the manufacturer’s instructions. The DNA was amplified by polymerase chain reaction (PCR) using the primer pair C1 (5′-ACC CGC TGA ATT TAA GCA T-3′) and D2 (5′-TGG TCC GTG TTT CAA GAC-3′) to amplify partial 28S rDNA (Hassouna et al., 1984). A total of 25 μl PCR reaction consisted of 1 μl of DNA template, 1× ExTaq Buffer (TaKaRa, Kusatsu, Japan), 0.2 mM of each dNTP, 1 μM of each primer, and 2.5 units of TaKaRa Ex Taq DNA Polymerase (TaKaRa). PCR was carried out with the following protocol: 94°C for 30 s followed by 35 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 2 min, and 10 min of final extension at 72°C. PCR products were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) and sequenced with a 3130X Genetic Analyzer (Applied Biosystems, Foster City, USA) with the primers used in the PCR. The newly generated 28S rDNA sequence was aligned with sequences for 27 diplectanid species, one pseudomurraytrematid species, and two dactylogyrid species retrieved from the GenBank database. Alignment was performed with ClustalW using the default parameters. The phylogenic tree was constructed with the maximum likelihood (ML) method under the GTR + G model, which was determined to be the best-fit model using the Bayesian information criterion, with the phylogeny tested by 1,000 bootstrap repeats using MEGA 6 (Tamura et al., 2013).


Family Diplectanidae Monticelli, 1903

Subfamily Diplectaninae Monticelli, 1903

Genus Furcohaptor Bijukumar & Kearn, 1996

Furcohaptor brevis n. sp.

Type-host: Cynoglossus robustus Günther, 1873 (Pleuronectiformes: Cynoglossidae).

Type-locality: The Seto Inland Sea, off Yoko-shima (34°21.5′N, 133°16.5′E), Fukuyama, Hiroshima Prefecture, Japan.

Type-material: Holotype stained in Heidenhain’s iron hematoxylin (NSMT-Pl 6294 collected on 1 February 2015), and four and one paratypes stained in Heidenhain’s iron hematoxylin (NSMT-Pl 6296–6297) and fixed in ammonium picrate glycerin (NSMT-Pl 6295), respectively.

Site in host: Gill filaments.

Prevalence and intensity: 16.1% (9/56) [25% (2/8) on 25 January 2015; 29.4% (5/17) on 1 February 2015; 25% (2/8) on 27 March 2016], and a total of nine monogeneans infected nine fish.

Representative DNA sequence: Submitted to DNA Data Bank of Japan (DDBJ) under the accession number LC270915.

ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN, 2012), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) for Furcohaptor brevis n. sp. is urn:lsid:zoobank.org:act: 80258204-E048-4183-BF50-75074A203E9C.

Etymology: The specific name of “brevis” means short in Latin and refers to the short arms of the bifurcate haptor.

Description (Figs. 2–3)

Body (Fig. 2A) elongate, length including haptor 405–709 (588; n = 6), width at mid-body 149–222 (189, n = 6). Three pairs of cephalic glands open on each anterolateral side of anterior extremity. Two pairs of eye-spots present. Pharynx subspherical (38–65 × 33–49 (51 × 41) (n = 6); oesophagus short; intestinal caeca extend to haptor. Testis pyriform, posterior to ovary. Vas deferens arising from anterior margin of testis, extending intercaecally, forming seminal vesicle. Single saccate prostatic reservoir located left to seminal vesicle. Male genital pore opening ventrally posterior to male copulatory organ. Male copulatory organ located posterior to oesophagus (Fig. 2K), consisting of penis and accessory piece. Penis tapered, curved, with flared proximal end, length along curve 58–66 (61, n = 6). Accessory piece rod-shaped, 29–39 (36, n = 6) long, its point growing wider, curved and covering tip of penis, non-articulated with penis. Ovary in mid-body, tubular, pretesticular, encircling right intestinal cecum dorsoventrally, then forming oviduct. Oviduct receiving duct from seminal receptacle, continuing as oötype surrounded by Mehlis’ gland. Vagina (Fig. 2L) sclerotised, curved tubular, entering anterior side of seminal reservoir; vaginal pore funnel-shaped, opening posterior to male genital pore on left side of ventral body surface. Vitellarium approximately co-extensive with intestinal ceca.

Fig. 2
figure 2

Furcohaptor brevis n. sp. ex Cynoglossus robustus. A, Holotype (NSMT-Pl 6294), whole mount (ventral view). B–L, Paratype (NSMT-Pl 6295). B, Ventral anchors; C, Ventral bars; D, Marginal hook of pair I; E, Marginal hook of pair II; F, Marginal hook of pair III; G, Marginal hook of pair IV; H, Marginal hook of pair V; I, Marginal hook of pair VI; J, Marginal hook of pair VII; K, Male copulatory organ; L, Vagina. Scale-bars: A, 50 μm; B–L, 10 μm

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Fig. 3
figure 3

Left half of haptor of Furcohaptor brevis n. sp. ex Cynoglossus robustus Holotype (NSMT-Pl 6294), ventral view. Abbreviations: gc, gland-cells; ms, muscle; r, reservoir. Scale-bar: 20 μm

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Haptor bifurcates posteriorly to form 2 short haptoral arms. Dorsal anchor absent. Ventral anchor (Fig. 2B) with reduced superficial root, total length 30–32 (31, n = 5), deep root length 11–13 (12, n = 5), superficial root length 1–2 (1, n = 5), point length 16–19 (17, n = 5). Dorsal bar absent. One pair of ventral bars (Fig. 2C) with longitudinal groove, rod-shaped, associating with root of each ventral anchor, length 22–28 (26, n = 6), width 2–3 (3, n = 6). Marginal hooks in 7 pairs, pairs II, III, IV, VI, VII with typical marginal hook shape for diplectanids: pair I (Fig. 2D) small, lacking heel of sickle, protruding from haptor ventrally, 11–12 (11, n = 5) long; pair II (Fig. 2E) 17–19 (18, n = 6) long; pair III (Fig. 2F) 17–20 (19, n = 6) long; pair IV (Fig. 2G) 17–21 (19, n = 6) long; pair V (Fig. 2H) long, lacking heel of sickle, length 36–43 (40, n = 6), pair VI (Fig. 2I) 18–19 (18, n = 5) long; pair VII (Fig. 2J) 17–20 (18, n = 6) long. Pair of pyriform reservoirs located at tip of haptoral arm ventrally, ducts open at extrusion of ventral anchors. (Fig. 3). Pair of gland-cells located at mid-length of haptor dorsally associated with marginal hooks V (Fig. 3).

Phylogenetic analysis

The newly generated 28S rDNA sequence for Furcohaptor brevis n. sp. was 880 nt long. Three species, Pseudomurraytrema sp. (Pseudomurraytrematidae), Dactylogyrus gotoi Gussev, 1965 and Dactylogyrus lamellatus Achmerow, 1952 (Dactylogyridae) were used as the outgroup. The phylogenetic analysis based on 28S rDNA sequences showed that the diplectanids separated into two clades, the Diplectaninae and Lamellodiscinae Oliver, 1969 (Fig. 4). The new species was assigned to the Diplectaninae in the Diplectanidae, and exhibited affinity with Diplectanum veropolynemi Nagibina, 1976 (Fig. 4). The sequences reported as Pseudorhabdosynochus latesis [sic] (AY553621) and Laticola lingaoensis Yang, Kritsky, Sun, Zhang, Shi & Agrawal, 2006 (DQ054825) should be referred to as Laticola latesi (Tripathi, 1955) and L. seabassi (Wu, Li, Zhu & Xie, 2005), respectively (Chotnipat et al., 2015).

Fig. 4
figure 4

Phylogenetic tree based on 28S rDNA partial sequences. The tree was constructed using the Maximum Likelihood method. The numbers along the branches indicate bootstrap values based on 1,000 bootstrap pseudoreplications

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Discussion

Furcohaptor brevis n. sp. resembles F. cynoglossi described by Bijukumar & Kearn (1996) in most of the morphological characters including a divided haptor; a pair of typical anchors with reduced superficial root; a pair of bars; a pair of rod-shaped hooks; five pairs of typical-shaped marginal hooks; a pair of marginal hooks reduced in size and lacking heels; and a penis with a flared proximal extremity. However, the new species differs from F. cynoglossi by the shape of the accessory piece and the length of the haptoral arms.

Furcohaptor has so far been assigned to Dactylogyridae, but based on the morphological features of F. brevis n. sp. (e.g. the ovary looping the right intestinal cecum; the distal part of the penis directed posteriorly; and the ventral bar having a longitudinal groove) and the phylogenetic analysis of the 28S rDNA sequences (Fig. 4), this genus evidently belongs to the Diplectaninae in the Diplectanidae. The sequences of F. brevis n. sp. and Diplectanum veropolynemi Nagibina, 1976 formed a sister group with a high bootstrap value (100%). Diplectanum veropolynemi was regarded as a species incertae sedis (Domingues & Boeger, 2008), and Kritsky & Diggles (2015) suggested that this monogenean is reassigned to Acanthocercodes Kritsky & Diggles, 2015. Further molecular data for species of Acanthocercodes will clarify the generic position of D. veropolynemi and show the affinities of Furcohaptor and Acanthocercodes.

Bijukumar & Kearn (1996) and Boeger et al. (2015) discussed the origin of typical anchors and rod-shaped hooks found in Furcohaptor spp. Based on the specimens of F. brevis n. sp., both the typical anchors and bars are identified as the ventral anchors and bars, respectively. This is supported by the bars located most ventrally on the typical anchors, the muscles running ventrally attached to the deep root of the typical anchors, and the duct of the reservoirs located ventrally opening the typical anchors (Fig. 3). Many monogeneans possess reservoirs of the haptor (e.g. dactylogyrids, tetraonchids and diplectanids), and most of these reservoirs are associated with the blades of the anchors (e.g. Lim, 1995; Lim & Gibson, 2009; Lim et al., 2010). The rod-shaped hook is identified as a fifth marginal hook because of its location on the ventral side of the tip of the haptor, and because it is enveloped by the ducts of the gland-cells (Fig. 3). The fifth marginal hook is commonly located ventrally at the tip of the haptor (e.g. Mizelle, 1936; Lim, 2015), and the gland-cells are associated with their handle of the marginal hooks in Bravohollisia Bychowsky & Nagibina, 1970 (see Wong et al., 2008). As Bijukumar & Kearn (1996) mentioned, the study on the ontogenetic development of Furcohaptor spp. reinforces the designation of the identification of haptoral structures.

Genus Furcohaptor Bijukumar & Kearn, 1996 (amend.)

Diagnosis

Diplectanidae, Diplectaninae. Body elongate. Two pairs of eye-spots present or absent. Cephalic glands open on each anterolateral side. Pharynx, oesophagus and bifurcate intestinal caeca present. Testis pyriform, posterior to ovary. Vas deferens arising from anterior margin of testis, extending intercaecal, forming seminal vesicle. Single saccate prostatic reservoir present. Male genital pore opening ventrally posterior to male copulatory organ. Male copulatory organ consisting of penis and accessory piece. Penis tapered, curved, with flared proximal end. Accessory piece rod-shaped. Ovary in mid-body, tubular, pretesticular, encircling right intestinal caecum dorsoventrally, then forming oviduct. Oviduct receiving duct from seminal receptacle, continuing on as oötype surrounded by Mehlis’ gland. Vagina, sclerotised, curved tubular, entering anterior side of seminal reservoir; vaginal pore funnel-shaped, posterior to male genital pore. Vitellaria approximately co-extensive with intestinal caeca. Haptor bifurcate posteriorly to form haptoral arms. Squamodiscs or lamellodiscs absent. Dorsal anchor and dorsal bar absent. Ventral anchor with reduced superficial root. One pair of ventral bars with longitudinal grooves. Seven pairs of marginal hooks present; pair I small, lacking heel of sickle; pair V long, lacking heel of sickle; other pairs typical marginal hook shape for diplectanids. On gills of cynoglossid fishes. Type-species: Furcohaptor cynoglossi Bijukumar & Kearn, 1996. Other-species: Furcohaptor brevis n. sp.