Descriptions of acanthocephalans, Cathayacanthus spinitruncatus (Rhadinorhynchidae) male and Pararhadinorhynchus magnus n. sp. (Diplosentidae), from marine fish of Vietnam, with notes on Heterosentis holospinus (Arhythmacanthidae)

Males of Cathayacanthus spinitruncatus Amin, Heckmann & Ha, 2014 (Rhadinorhynchinae Lühe, 1912) are described for the first time from Leiognathus equulus in Hai Phong and Nha Trang and from pony fish Nuchequula flavaxilla in Quang Ninh in the Pacific waters of Vietnam. The male allotype status is designated. Males of C. spinitruncatus are smaller and have fewer and smaller proboscis hooks and trunk spines than females. The male reproductive structures are in the posterior fifth of the trunk and with 6 club-shaped cement glands gradually merging into 6 independent cement gland ducts. The proboscis receptacle is more than half as long as the trunk and with a cephalic ganglion at its anterior end. In females, the receptacle is only about one fifth the length of the trunk. Specimens described as Cathayacanthus bagarii Moravec & Sey, 1989 were shown to have been wrongly assigned to Cathayacanthus. Pararhadinorhynchus magnus n. sp. (Diplosentidae) is described from Scatophagus argus off Hai Phong in the Gulf of Tonkin. It is the third species of the genus and is readily distinguished from the Australian species by having a considerably larger trunk and male reproductive structures, and more proboscis hooks. X-ray microanalysis (EDAX) of intact and gallium-cut hooks of P. magnus showed high calcium and phosphate mainly in the central core. Specimens of Heterosentis holospinus Amin, Heckmann & Ha, 2011 (Arhythmacanthidae) are also reported from L. equulus off Quang Binh, new host and locality records.

Twenty species of acanthocephalans in 5 families were collected more recently from fishes in the Pacific and amphibians in central Vietnam, in 2016 and 2017. In the present report, we describe males of Cathayacanthus spinitruncatus Amin, Heckmann & Ha, 2014 (Rhadinorhynchidae) previously known from females only [13], a new species of the genus Pararhadinorhynchus Johnston & Edmonds, 1947 (Diplosentidae), and record Heterosentis holospinus Amin, Heckmann & Ha, 2011 in a new host and locality. For a better understanding of the chemical elements of hooks and their relationship to hook structure, intact and gallium-cut hooks of P. magnus were scanned with X-ray (EDAX) and analyzed for percent chemical elements in various parts of the hooks. This procedure has become a standard in our laboratory studies of the Acanthocephala [8,19,20].

Materials and methods
Collections Eleven males and females of C. spinitruncatus were collected from two species of fish: the common pony fish Leiognathus equulus (Forsskål) in the northern Hai Phong area (20°51 0 54 00 N,106°41 0 2 00 E) and the southern Nha Trang coast (12°15 0 N,109°11 0 E) and from the yellow-spotted pony fish Nuchequula flavaxilla Kimura, Kimura & Ikejima off northern Quang Ninh Province (21°15 0 N,107°20 0 E). Two of 10 L. equulus from Hai Phong in 2016 and 1 from Nha Trang in October, 2017, and 7 of 20 N. flavaxilla from Quang Ninh were concurrently infected with C. spinitruncatus and other acanthocephalans. Specimens of Pararhadinorhynchus magnus n. sp. were collected from the spotted scat, Scatophagus argus (Linn.) (Scatophagidae) off Hai Phong in the Gulf of Tonkin in April 2015. Additionally, specimens of Heterosentis holospinus were collected from L. equulus off the same Quang Ninh Province as noted above in May 2017.
Freshly collected acanthocephalans were extended in water until proboscides were everted, and fixed in 70% ethanol for transport to our Arizona, USA laboratory for processing and further studies. Worms were punctured with a fine needle and subsequently stained in Mayer's acid carmine, destained in 4% hydrochloric acid in 70% ethanol, dehydrated in ascending concentrations of ethanol reaching 100% (24 h each), and cleared in 100% xylene then in 50% Canada balsam and 50% xylene (24 h each). Whole worms were then mounted in Canada balsam. Measurements are in micrometers, unless otherwise noted; the range is followed by the mean values between parentheses. Width measurements represent maximum width. Trunk length does not include proboscis, neck, or bursa. Line drawing were created by using a Ken-A-Vision micro-projector (Ward's Biological Supply Co., Rochester, New York, USA) which uses cool quartz iodine 150W illumination. Color-coded objectives, and 10·, 20·, and 43· lenses were used. Images of stained whole mounted specimens were projected vertically on 300 series Bristol draft paper (Starthmore, Westfield, Massachusetts, USA), then traced and inked with India ink. Projected images were identical to the actual specimens being projected.

Scanning electron microscopy (SEM)
Four to six specimens that had been fixed and stored in 70% ethanol were processed for SEM following standard methods [23]. These included critical point drying (CPD) in sample baskets and mounting on SEM sample mounts (stubs) using conductive double-sided carbon tape. Samples were coated with gold and palladium for 3 min using a Polaron #3500 sputter coater (Quorum (Q150 TES) www.quorumtech.com) establishing an approximate thickness of 20 nm. Samples were placed and observed in an FEI Helios Dual Beam Nanolab 600 (FEI, Hillsboro, Oregon, USA) Scanning Electron Microscope with digital images obtained in the Nanolab software system (FEI, Hillsboro, Oregon, USA) and then stored on a USB for future reference. Samples were received under low vacuum conditions using 10 KV, spot size 2, 0.7 Torr using a GSE detector.

Energy dispersive analysis for X-ray (EDAX)
Standard methods were used for preparation similar to the SEM procedure. Specimens were examined and positioned with the above SEM instrument which was equipped with a Phoenix energy-dispersive X-ray analyzer (FEI, Hillsboro, Oregon, USA). X-ray spot analysis and live scan analysis were performed at 16 kV with a spot size of 5 and results were recorded on charts and stored with digital imaging software attached to a computer. The TEAM *(Texture and Elemental Analytical Microscopy) software system (FEI, Hillsboro, Oregon, USA) was used. Data were stored on a USB. The data included weight percent and atom percent of the detected elements following correction factors, and were stored on a USB. All figures on the USB can be viewed by contacting the second author. The hooks were cut and scanned at two positions (Tip and Middle) with a gallium beam (LIMS) using a dual beam scanning electron microscope. The alignment of the hook prior to cutting generated a cross-section of the area.

Ion sectioning of hooks
A dual-beam SEM with a gallium (Ga) ion source (GIS) is used for the LIMS (Liquid Ion Metal Source) part of the process. The gallium beam (LIMS) is a gas injection magnetron sputtering technique whereby the rate of cutting can be regulated. The hooks of the acanthocephalans were centered on the SEM stage and cross-sectioned using a probe current between 0.2 nA and 2.1 nA according to the rate at which the area is cut. The time of cutting is based on the nature and sensitivity of the tissue. Following the initial cut, the sample also goes through a milling process to obtain a smooth surface. The cut was then analyzed with X-ray at the tip, middle, and base of hooks for chemical ions with an electron beam (Tungsten) to obtain an X-ray spectrum. Results were stored with the attached imaging software then transferred to a USB for future use. The intensity of the GIS was variable according to the nature of the material being cut.
Type specimens were deposited in the University of Nebraska's State Museum's Harold W. Manter Laboratory (HWML) collection in Lincoln, Nebraska, USA.

Results
Four of the 11 specimens of C. spinitruncatus collected were males. Female specimens in the same collection were identical to those described by Amin et al. [13] thus confirming the diagnosis of males. The 4 male and 7 female specimens were processed, studied and measured. Four males and five females of P. magnus n. sp. were collected from S. argus in April 2015. One male and five gravid females of H. holospinus were also found in 2 specimens of L. equulus.
Type specimen: HWML collection no. 139405 (allotype male), no. 139406 (paratype male). Apical and subapical hooks relatively small becoming largest in anterior half of proboscis and gradually smaller and more crowded posteriorly. Basal hooks not specialized but slightly larger than smallest pre-basal hooks. Length and width (at base) of dorsal and ventral hooks nos. 1, 6, 12, 16, 20, 24, 28, 32, 36, and basal hooks in 1 male: Dorsal hooks: 27 (4), 30 (5), 35 (7), 32 (7), 30 (6), 27 (5), 27 (5), 22 (3), 17 (3)   The pattern of dorso-ventral differentiation of hooks along the length of the proboscis is the same in both sexes. The proportion of lemnisci length to receptacle length was also comparable. However, in females, the receptacle was considerably shorter occupying about one fifth (23%) of trunk space (Fig. 23 of Amin et al. [13]) while it was over one half (57%) in males (Fig. 1). The description of the female reproductive system in Amin et al. [13], however, did not include two large paravaginal muscular lobes close to the body wall (Fig. 5, arrows). General: With characters of the genus Pararhadinorhynchus (Rhadinorhynchidae). Trunk unarmed, long, uniformly cylindrical (Fig. 6) with prominent micropores of variable pore size and distribution in various parts of trunk and neck (Figs. 19, 20). Body wall with reticular lacunar system anastomoses with 2 main lacunar channels and many scattered nucleated cells (Fig. 7). Shared structures larger in females than in males. Proboscis long and cylindrical, widest posteriorly (Figs. 7, 15, 16), with many uniform hooks in 14-16 rows each with 23-27 hooks. Hooks largest subapically gradually decreasing in length posteriorly reaching smallest size basally (Figs. 17, 18). Hooks dorso-ventrally differentiated being more robust and more sharply curved with thicker roots ventrally, but slightly longer with more slender blades and roots, and less sharply curved dorsally. All hooks rooted. Hook roots simple, directed posteriorly, about as long as hooks anteriorly and at middle, becoming progressively more slender and shorter than blades posteriorly until reaching near vestigial state basally, but always evident (Fig. 8). Neck prominent, conical, widest posteriorly (Fig. 15). Proboscis receptacle double walled but outer wall incomplete posteriorly, widest anteriorly, about twice as long as proboscis, with large drop-shaped cephalic ganglion at its base and a nucleated pouch at its posterior ventral end (Figs. 7, 11, 12). Lemnisci equal, digitiform, plump, with definite clear cortical layer (Fig. 11), and of variable length; usually markedly longer than receptacle but occasionally as long as receptacle when considerably heavier (Figs. 7, 12).
Male (based on 4 mature adults with sperm; 1 male monorchid): See Table 1 for measurements and counts. Testes ellipsoidal, postequatorial, usually contiguous. Two long, tubular cement glands enlarged posteriorly and non-contiguous with posterior testis anteriorly (Fig. 6). Two sperm ducts visible in space between anterior cement gland and posterior testis measuring 0.31-1.5 (0.81) mm long by 0.04-0.08 (0.07) mm wide. No such space in trunk of monorchid male crowded with large testis measuring 3.12 long by 0.75 wide. Cement gland ducts prominent, relatively wider posteriorly, surrounding Sarfftigen's pouch and common sperm duct (Fig. 6). Bursa plump, rounded, with very few rays and no evident special features or sensory structures, but with occasional typical body wall nucleated cells (Fig. 14). Posterior end of trunk occasionally with deep cleft marking completely invaginated bursa (Fig. 21).

X-ray scans (EDAX)
The results of the X-ray scans using EDAX are listed in Table 2 and Figure 22. A definite layering was visible with high sulphur content in the outside layer.

Remarks
Johnston & Edmonds [22] characterized their new genus Pararhadinorhynchus as being Rhadinorhynchus-like by having 2 long tubular cement glands, a long proboscis with numerus hooks, and a double walled proboscis receptacle. In Pararhadinorhynchus, however, the cephalic ganglion is  normally at the base of the receptacle, the trunk is aspinose, and basal proboscis hooks are not enlarged or projecting at a right angle to the proboscis. These features are well represented in P. mugilis described from the flathead gray Mugil cephalus Linn. (Mugilidae) in South Australia [22] and Pararhadinorhynchus coorongensis Edmonds 1973 also collected from South Australia from the yellow-eyed mullet Aldrichetta forsteri (Cuvier & Valenciennes) (Mugilidae) as well as those of P. magnus n. sp. from Vietnam. Additionally, all three species usually have contiguous testes and cement glands distant from posterior testis especially in P. mugilis, similar eggs, and similar proboscis hook blades and shape of trunk, lemnisci and reproductive structures. The lemnisci in P. mugilis were about as long as the receptacle but about twice as long in P. coorongensis. In P. magnus, they were variable but often markedly longer than the receptacle; they were shorter when more plump.
Pararhadinorhynchus coorongensis primarily differs from P. mugilis and P. magnus by having a considerably smaller proboscis with markedly fewer hooks and smaller eggs ( Table 1). The marked dorso-ventral differentiation of proboscis hooks noted in our specimens was not reported in either one of the two Australian species. However, we suspect that this was simply overlooked. Line drawings of hooks of P. mugilis (Fig. 11 of Johnston & Edmonds, 1947 [22]) appear to represent dorsal hooks, while those of P. coorongensis (Fig. 2 of Edmonds, 1973 [15]) represent ventral hooks, especially the anterior and middle hooks. Posterior hooks in the same figures lack roots, which is also probably an oversight because of their minute size. In our specimens from the spotted scat S. argus from a different family (Scatophagidae) in Vietnam, the male reproductive system is markedly larger than in the two Australian species. In addition, the female gonopore in P. magnus is decidedly subterminal behind a prominent round posterior tip but terminal in the two Australian species.

About Heterosentis holospinus
The 1 male and 5 gravid females were practically identical to those described by Amin et al. [12]. The rooted apical and larger subapical proboscis hooks, the 3-4 posterior unrooted spine-like hooks, the unspined anterior trunk cone,     the receptacle shape and plump longer lemnisci, and trunk shape and size were almost identical. The male reproductive structures, especially the shape of the thick sperm ducts, the rounded bursa, and the shape and position of the testes and cement glands were very similar. The female reproductive system especially the complex vagina, the prominent and widening uterus, the uterine bell with many cells, and the eggs were also very similar. However, the nucleated pouch at the posterior end of the receptacle was not consistently prominent and the trunk spines were faint and less readily visible.

Discussion
Golvan [18] relegated Rhadinorhynchus exilis Van Cleave, 1928 from the crucian carp, Carassius carassius (Lin.) in China to his new genus Cathayacanthus based on the absence of large specialized basal proboscis hooks as is typical in Rhadinorhynchus. The proboscis of Golvan's [18] new genus characteristically exhibited dorso-ventral differentiation of hooks, the cephalic ganglion was positioned at the anterior end of the receptacle, and the trunk was spinose. Trunk spines were only anterior in C. exilis but covered the whole trunk in C. spinitruncatus. Both species were described from females only. The present description of males provides for the first time a description of males of any member of the genus with its characteristic reproductive system.  Table 2. Insert: SEM of cross gallium cut hook.