Neohexostoma gymnosardae n. sp. (Monogenea, Hexostomatidae), a gill parasite of Gymnosarda unicolor (Valenciennes) (Teleostei, Scombridae) in the South China Sea

Parasite biodiversity of fish in coral reefs of the South China Sea is still incompletely explored. We describe here a new species of Neohexostoma (Monogenea: Hexostomatidae) from the gill filaments of the dogtooth tuna Gymnosarda unicolor (Scombridae), collected off Yongshu Reef, South China Sea. Neohexostoma gymnosardae n. sp. is distinguished from its congeners by the following features: (i) haptor clearly marked from body proper by a strongly constricted peduncle, divided in its posterior margin into two symmetrical lobes, (ii) vagina armed with scattered small blunt spines, (iii) eggs tied by their long polar filaments, (vi) esophagus with several lateral diverticula, (v) intestinal ceca unfused and extending into the haptor. We present an analysis of the relationships of this monogenean based on partial 28S rDNA sequences. An identification key for species of Neohexostoma is provided. This is the first member of the genus Neohexostoma known to parasitize a species of Gymnosarda.


Introduction
Gymnosarda unicolor Rüppell 1836 (Perciformes: Scombridae) is an epipelagic, coral-reef associated fish endemic to the Indo-Pacific region [2]. It is the sole species of the genus Gymnosarda [2]. Very little is known about the biology and ecology of G. unicolor. It is almost exclusively solitary, associated with reef structures, and is rarely found in the open sea or in schools [6]. Coral reefs are known for their very high level of biodiversity [13]. However, parasite biodiversity in the coral reefs of the South China Sea is still incompletely explored.
The Hexostomatidae Price, 1936 [21,26,30,31]. Boeger and Kritsky [3] conducted a phylogenetic analysis of the Monogenea using morphological characters and proposed the new suborder Hexostomatinea within the Mazocraeidea to accommodate Hexostomatidae. In the South China Sea, two valid species of Hexostomatidae have been reported: H. auxisi Palombi, 1943 from Auxis thazard and N. euthynni (Meserve, 1938) Price, 1961 from Euthynnus affinis and Auxis thazard [36]. In the course of a parasitological study of monogeneans of marine fishes off the South China Sea, we collected representatives of an undescribed species of Neohexostoma on the gills of Gymnosarda unicolor. The species is described here. An identification key to Neohexostoma is also provided.

Materials and methods
Fishes Throughout May 2017, three G. unicolor were collected at Yongshu Reef (9°33 0 N, 113°E) off the South China Sea. Fish specimens were transferred to the laboratory and identified using keys [11]. Fish specimens were photographed prior to removing gills. Gills were removed from each fish and observed under the microscope for the presence of monogeneans.

Morphological methods
Gills were excised and placed in Petri dishes with sea water and examined for monogeneans with the aid of a stereomicroscope. Monogeneans were removed alive from gills using fine dissection needles. Four specimens were preserved in 70% ethanol, stained with acetic carmine, dehydrated in graded ethanol series (70%, 96% and 100%), cleared in clove oil, and mounted in Canada balsam. Two specimens were directly mounted in Berlese's fluid to study the morphology of clamps and the genital atrium. Illustrations were drawn with the help of the drawing apparatus of an Olympus BX51 microscope (Olympus Corporation, Japan), and then redrawn on a computer with Photoshop CS4.0 (Adobe, USA). Measurements were made using Olympus DP22 software. Measurements are in micrometres (lm), and indicated as the range followed by the mean and the number of measurements in parentheses.

Molecular methods
A single specimen was fixed in absolute ethanol then subjected to molecular analyses. Total genomic DNA was extracted using a TIANamp Marine Animal DNA Kit (Tiangen Biotech, China), following the manufacturer's instructions. The partial C1-D2 domain of 28S rDNA was amplified with PCR using previously published primer pairs (C1F: 5 0 -ACCCGC-TGAATTTAAGCAT-3 0 and reverse primer D2R: 5 0 -TGGT-CCGTGTTTCAAGAC-3 0 ) [10]. Each PCR amplification was performed in a 50-lL volume containing 5 lL of DNA template, 25 lL of Master Mix (dNTP, 2Â buffer, Taq polymerase), 2 lL of each primer, and 16 lL of double-distilled water, under the following conditions: initial denaturation at 95°C for 5 min; followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min; and a final elongation at 72°C for 10 min. PCR products were detected by 1% agarose gel electrophoresis and sequenced by the Sangon Biotech Company (Shanghai, China). The sequences obtained were analyzed using DNAMAN 7.0 software (Lynnon Biosoft, USA), compared to GenBank database content with BLAST, and deposited in GenBank under the accession number MN242399.

Trees and distances
Sequences of 10 species belonging to 6 families available in GenBank, and one sequence of Neohexostoma gymnosardae n. sp. generated in this study were included in the phylogenetic analyses (Table 1). Evolutionary analyses were conducted in MEGA 7.0. [18]. The trees were inferred using the maximum likelihood (ML) method and the neighbor-joining method, with a sequence of Polystoma gallieni Price, 1938 as the outgroup [8,28]. For the ML tree, the best model, estimated by MEGA7, was the general time-reversible model with discrete gamma distribution (GTR + G). The tree was re-sampled with 1000 bootstrap replicates to evaluate the reliability of the groups. Kimura 2-parameter distances between sequences were estimated with MEGA7.0 [16].

Molecular analyses
28S rDNA sequence data with 881 bp for N. gymnosardae n. sp. was generated, and the BLAST result indicated a 92.42% identity with 98% coverage for H. thynni (Delaroche, 1811) Rafinesque, 1815 (EF653383), and less than 85% identity with 100% coverage for other monogeneans in GenBank. For trees and genetic distances, there was a total of 837 positions in the final dataset, including 388 conserved sites, 449 variable sites, and 301 parsimony informative sites. The species most closely related to N. gymnosardae n. sp. was H. thynni, with Kimura two-parameter distance of 8.2% ( Table 2). The neighbour-joining and maximum likelihood methods led to identical tree topologies and thus only the ML tree is shown (Fig. 1) Etymology: The species is named after its host.

Differential diagnosis
Monogeneans found on the gills of G. unicolor are allocated to Neohexostoma by the following morphological features: body elongate, widest in ovarian region; haptor with four pairs of clamps arranged as two more or less vertical rows, decreasing in size anteroposteriorly; vitellarium not extending posteriorly beyond the distal portion of the testes [26]. Apart from the above, the new species can also be distinguished from its congeners by its body shape, intestine, and clamp disposition. Neohexostoma gymnosardae n. sp. is characterized by a well visible peduncle: of the seven species of Neohexostoma mentioned above, only N. thunninae and N. mochimae have a constriction at the beginning of the haptor, separating the latter from the rest of the body [24,25,35]. The new species most closely resembles N. thunninae by its body shape and the possession of a peduncle. Neohexostoma gymnosardae n. sp. differs from N. thunninae by having an esophagus with lateral diverticula, ceca extending more posteriorly into the haptor and the post-atrial intestines bifurcating. Moreover, in N. gymnosardae n. sp. the anchors are placed between clamps of the most posterior pair, whereas they are borne on a short lappet in N. thunninae [24,25].
Neohexostoma gymnosardae n. sp can be distinguished from N. kawakawa, N. mochimae and N. euthynni by having testes tightly packed into a post-ovarian pile [20,34,35]. Furthermore, in N. gymnosardae n. sp., intestinal bifurcation is post-atrial and the unfused intestinal ceca extend into the haptor; in N. kawakawa and N. euthynni, the intestinal bifurcation is pre-atrial and ceca are united posteriorly [20,34]. Neohexostoma gymnosardae n. sp. differs from N. pricei by the extension of intestinal ceca into the haptor (up to a shorter distance in N. pricei) and the shape of lateral sclerites of clamps (straight in N. pricei vs. double-pronging in N. gymnosardae n. sp.) [17]. Neohexostoma gymnosardae n. sp. can be further distinguished from N. extensicaudum and N. robustum by body shape and clamp disposition. Both N. extensicaudum and N. robustum, like most of Neohexostoma spp., have an elongated waist-like constriction in the testicular region, which is absent in the new species [7,26]. Moreover, clamps of the right row in N. extensicaudum lie very close to N. euthynni (Meserve, 1938) N. extensicaudum (Dawes, 1940) N. robustum Price, 1961 N. pricei (Koratha, 1955 The width of the body given for N. extensicaudum is that of the third region. The data for N. euthynni in square brackets are from Millemann (1956) [21]. * Diameter. ** Ratio large clamp/body length. *** Ratio small clamp/body length. A., Atlantic Ocean. M., Mediterranean Sea. P., Pacific Ocean.
those of the left row so that the posterior end of the body is much more attenuated than is the case in other species [7]; and in N. robustum, clamps are arranged in oblique transverse rows on a roughly triangular haptor [26]. The comparison of morphometrics of Neohexostoma gymnosardae n. sp. and Neohexostoma spp. is presented in Table 3. Neohexostoma gymnosardae n. sp. most closely resembles its congeners by having clamps decreasing in size anteroposteriorly. It differs from N. euthynni, N. kawakawa, and N. mochimae by the number of testes, host and locality for the latest species. In addition, N. gymnosardae n. sp. can be distinguished from N. kawakawa, N. euthynni and N. extensicaudum by having a longer polar filament. We note that body length in N. gymnosardae n. sp. is greater than in N. euthynni, N. pricei, N. mochimae, and N. kawakawa. However, such a measurement is generally not a reliable characteristic for species differentiation [4]. We present herein a key to species of Neohexostoma as follows, which is modified from Millemann [21] and Zambrano [35].

Discussion
This study describes a new species of Neohexostoma, N. gymnosardae n. sp., obtained from Gymnosarda unicolor, caught off Yongshu Reef, South China Sea, Northern Pacific. We note that we previously named this species Leptohexostoma gymnosarda n. gen. n. sp. in an abstract of a symposium held in China [19]. According to Article 9 for "What does not constitute published work" in the International Code of Zoological Nomenclature (ICZN), abstracts of articles, papers, posters, texts of lectures, and similar material when issued primarily to participants at meetings, symposia, colloquia or congresses does not constitute published work within the meaning of the Code [12]. The name, Leptohexostoma gymnosarda, proposed in the abstract is a nomen nudum.
Millemann synonymized H. macracanthum with H. euthynni and noted that all species of Hexostoma could be separated into two different morphological groups [9,21]. In species assigned to the first group, clamps are arranged in straight transverse rows. Clamps of representatives of the remaining species are disposed in two longitudinal rows. Price [26] established Neohexostoma to accommodate three species transferred from Hexostoma, along with the newly described N. robustum. He also provided the main differences between the new genus and Hexostoma. Subsequently, Yamaguti [33] and Unnithan [30] amended the diagnoses of Hexostomatidae and that of Neohexostoma. All the previously mentioned studies valued the haptor and body shape as a generic feature. Our new species shares the "classic" morphological characteristics with species of Neohexostoma as stated in the differential diagnosis [29,33].
The waist-like constriction in the testicular region, characteristic of Neohexostoma [26,33], is not observed in the new species. Actually, this waist-like constriction was not mentioned in the original description of N. thunninae [24,25]. Some species in Hexostoma, such as Hexostoma dissimile (Yamaguti, 1937) Sproston, 1946, Hexostoma sibi Yamaguti, 1968 and Hexostoma grossum (Goto, 1894) Sproston, 1946, also have a slightly constricted waist in the testicular region or in front of the haptor [22,29,33,34]. Hence, we suggest that the soft parts of monogeneans should be treated with caution when defining a new taxon, as these features may be associated with the in situ position of the worm on the host. Although measurements of some body parts are conventionally used for the systematics of Monogenea, some measurements (e.g. size of body, size of clamps) vary widely within this new species and overlap with other species (Table 3), making determination to species level difficult. The sclerotized structures such as clamp sclerites are considered important taxonomical features for species identification. Generally, the middle skeletal piece in Neohexostoma spp. is X-shaped with a base plate [2]. However, clamp sclerites are actually complicated three-dimensional structures, resulting in different shapes observed from different sides (Fig. 2G vs. Fig. 3G).
Also noteworthy is the genital system. Based on previous descriptions and figures, although some authors failed to determine the number of testes in their descriptions, the number of testes in Neohexostoma is usually less than 50 and testes are arranged in two or three alternating rows [9,27,34,35]. Abundant testes (185-246 in our new species) are usually seen in some species of Hexostoma, such as H. grossum, H. sibi, and H. dissimile [14,29,33,34]. Moreover, the terminal portion of the vagina in members of the Hexostomatidae is typically represented by two symmetrical pads with densely conical spines forming two serrated edges, which is dissimilar to that in our species. Another point of interest is that the eggs of new species are joined in a chain by their long filaments. Monogeneans shows a remarkable diversity in the shape and size of eggs, as well as their appendages. Although this type of egg assembly is also observed in other monogeneans (e.g. Squalonchocotyle catenulate Guberlet, 1933) [15], it is unique in Hexostomatidae. Unfortunately, as a single sequence of H. thynni is available, we cannot further comment on the phylogenetic relationships within Neohexostoma.