Redescription and molecular analysis of Neoechinorhynchus (Neoechinorhynchus) johnii Yamaguti, 1939 (Acanthocephala, Neoechinorhynchidae) from the Pacific Ocean off Vietnam

Neoechinorhynchus (Neoechinorhynchus) johnii Yamaguti, 1939 is redescribed from Eleutheronema tetradactylum (Polynemidae), Johnius carouna (Sciaenidae), Johnius sp., and Otolithes ruber (Sciaenidae) along the north and south coasts of Vietnam. Our description completes missing and inadequate information in the original descriptions and line drawings from Johnius goma in Japan and from Pseudosciaena diacanthus in the Indian Ocean. We add new information documented by scanning electron microscopy (SEM) and photomicroscopy, and explore the wide morphological diversity attributed to host species. The redescription includes: worms cylindrical with round proboscis with prominent apical organ, and large anterior hooks distant from small middle and posterior hooks; neck longer than the proboscis, nucleated lemnisci subequal, and receptacle with large basal triangulate cephalic ganglion and attached para-receptacle structure (PRS); male reproductive system in posterior half of trunk; adult females with introvert genital vestibule; and eggs spherical or rectangular. Gallium cuts and X-ray scans of hooks show high concentrations of sulfur on edge layer aiding in forming hardened calcium phosphate apatite of that layer with calcium and phosphorus in higher concentration in central part of hook. Molecular results consistently yielded a strongly supported distinct clade for the Neoechinorhynchus species from Vietnam for both 18S gene and the ITS1-5.8S-ITS2 region of ribosomal RNA. Phylogenetic analysis demonstrated that N. johnii occupies a separate position in the trees, probably indicating an Asian origin of this species.


Introduction
Most of the recent taxonomic work on the Acanthocephala from Vietnam was reported by the Amin-Heckmann-Ha team since 2000. A number of acanthocephalan species from freshwater fish, amphibians, reptiles, birds, and mammals were previously described in Vietnam [5,[10][11][12][13][14]. Additionally, 11 species of acanthocephalans were collected from marine fish off the eastern seaboard of Vietnam in Halong Bay in 2008 and 2009. Of these, six new species of Neoechinorhynchus Stiles & Hassall 1905, one new species of Heterosentis Van Cleave, 1931, and two new species of Rhadinorhynchus Lühe 1911 were described [9,15,16]. Four other species of Echinorhynchid acanthocephalans from marine fishes in Halong Bay were also described [6], and five other new species from fishes and amphibians of eight collected host species were described [18]. Three other species of Rhadinorhynchus and one species of Gorgorhynchus were otherwise previously reported from marine fishes in Vietnam [20].
Fifteen species of acanthocephalans in five families were more recently collected from fishes in the Pacific and amphibians in central Vietnam in 2016 and 2017. The revision of the genus Neoechinorhynchus and the erection of two subgenera [2,3] complement the above reports to produce the present account redescribing N. johnii using SEM and photomicroscopy. We also report new features including results of metal analysis of hooks (energy dispersive analysis for X-ray, EDAX) and expand the host and geographical distribution of the species. We also include the molecular description of N. johnii Yamaguti, 1939 and describe its phylogenetic relationships.

Materials and methods
Collections Specimens were collected using gill nets from research vessels off shore at noted collection sites. Information on the collections of specimens of N. johnii is presented in Table 1.

Processing for microscopical studies
Freshly collected acanthocephalans were extended in water until proboscides were everted, and fixed in 70% ethanol for transport to our Institute of Parasitic Diseases (IPD) in Arizona, USA 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.
Voucher specimens were deposited in the University of Nebraska's State Museum's Harold W. Manter Laboratory (HWML) collection in Lincoln, Nebraska, USA.

Scanning electron microscopy (SEM)
Specimens that had been fixed and stored in 70% ethanol were processed for SEM following standard methods [29]. 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) https://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) Scanning Electron Microscope, with digital images obtained in the Nanolab software system (FEI, Hillsboro, Oregon), and then transferred to 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.
X-ray microanalysis using 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). X-ray spot analysis and live scan analysis were performed at 16 kV with a spot size of five 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) was used. Data were stored on a USB for future analysis. The data included weight percent and atom percent of the detected elements following correction factors.

Ion sectioning of hooks
A dual-beam SEM with a gallium (Ga) ion source (GIS) was used for the liquid ion metal source (LIMS) part of the process. 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 was cut. The time of cutting was based on the nature and sensitivity of the tissue. Following the initial cut, the sample also went 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.

Molecular methods
Genomic DNA of the worms preserved in 95% ethanol was extracted individually using a DNeasy™ Blood and Tissue kit (Qiagen, Germany), according to the manufacturer's instructions. Isolated DNA was amplified by PCR using the primer pairs: Worm A (5 0 -GCGAATGGCTCATTAAATCAG-3 0 ), 270R (5 0 -CCGTCAATTCCTTTAAGT-3 0 ) [30]  Alignment and analysis of sequences obtained during the study were carried out using BioEdit, version 7.2.5 [26]. Sequences of 18S and the ITS1+5.8S+ITS2 region from closely related acanthocephalans were determined by BLASTn search and downloaded for further analysis.
Multiple sequences alignment was performed using Clus-talW as implemented in MEGA v. 6.0 [36] together with other species of acanthocephalans. Phylogenetic analyses were conducted using MEGA v. 6.0 [36] with 1000 bootstrap replicates for prior testing of reliability and Bayesian inference (BI) analyses in Topali 2.5 [31]. For nucleotide alignment of 18S and the ITS1+5.8S+ITS2 region, a phylogenetic tree was constructed using the maximum likelihood (ML) method in MEGA v. 6.0. Pairwise distance analyses were carried out using the Maximum Composite Likelihood model [36]. For Bayesian inference (BI) analyses, the substitution models were tested by the Bayesian Information Criterion and GTR+G+I was chosen. Posterior probabilities were estimated over 1,000,000 generations via five independent runs of four simultaneous MCMCMC chains with every 100th tree saved. The "burn in" was set to 25%. Representative DNA sequences: The 18S and ITS1-5.8S-ITS2 region of rDNA sequences of N. johnii were deposited in GenBank under the accession numbers MK260005 and MK260007 (for the 18S gene), and MK260006 and MK260008 (for the ITS region).

Morphological description
General. Neoechinorhynchidae. With characters of the genus Neoechinorhynchus and the subgenus Neoechinorhynchus as described by Amin [2]. With prominent sexual dimorphism in size of shared structures. Trunk elongate, cylindrical and slender, slightly but decidedly wider anteriorly. Body wall with osmiophilic micropores throughout (Figs. 6 and 7), often with paired cuticular folds and occasionally with dorsal hump (Fig. 13). Giant hypodermal nuclei not readily observed. Proboscis rounded but slightly wider than long (Fig. 1); with prominent nucleated apical organ. Anterior hooks long with shorter posteriorly directed spoon shaped roots, at apical end of proboscis (Figs. 2 and 14). Middle and posterior hooks small, almost equal, with short stout roots, close together and distant from anterior hooks. Middle hooks, near posterior end of proboscis; posterior hooks slightly smaller, at the attenuated posterior end of proboscis (Fig. 3). Hooks with thin cortical layer and solid but vacuolated core (Figs. 4 and 5). Neck prominent, longer than proboscis, wider at base (Figs. 1 and 15). Proboscis receptacle more than twice as long as proboscis, with ovoid cephalic ganglion at its base and para-receptacle structure (PRS) at least on one side (Fig. 15, arrow). Lemnisci long, slightly sub-equal, with two prominent ovoid giant nuclei each. Gonopore terminal in males and subterminal in females (Figs. 17 and 18).
Males (based on 11 adult specimens from 4 species of fish). See Tables 2 and 3 for measurements. Reproductive system in posterior half of trunk with two long tubular contiguous testes filling width of body cavity. Posterior testis longer than anterior testis. Syncytial cement gland distant from posterior testis; with many linearly arranged large spherical nuclei. Cement reservoir prominent, bulky, elongate, at posterior end of cement gland followed posteriorly by large triangular sperm vesicle, broadest anteriorly, overlapping Saefftigen's pouch. Bursa without prominent ornamentation or marked sensory structures (Fig. 9).     Females (based on 32 adults with eggs and ovarian balls from 4 species of fish). See Tables 2 and 3 for measurements. Reproductive system with prominent vaginal sphincter, bulb and tube, thick-walled fusiform uterus, few large uterine glands of selector apparatus, and large conical uterine bell attached to body wall ventrally. Adults with introverted genital vestibule lined with invaginated body wall (Figs. 17 and 18) but occasionally adults and immatures with terminal or subterminal digitiform prolongation of posterior trunk tip with terminal gonopore (Figs. 11 and 19). Eggs either spherical and thick walled showing no prolongation of fertilization membrane or more rectangular with soft corners and two cone-shaped poles (Figs. 12 and 20).

Relationships to host species
The measurements provided for specimens of N. johnii collected from four species of fish (Table 2) demonstrate considerable variability in the size of certain structures that appear to be associated with host species (Table 3). Most noticeably, the longest worms from E. tetradactylum (females reaching 120.0 mm in length) had the shortest anterior proboscis hooks in males and females averaging 64-83 long compared to anterior hooks of worms from J. carouna and O. ruber that exceeded 100 in length. Size of the proboscis, proboscis receptacle, and testes was similarly smaller in worms obtained from E. tetradactylum compared with worms from J. carouna and O. ruber.

Remarks
We have provided the morphological description of 11 male and 32 female specimens of N. johnii from 20 and 40 specimens obtained from four new species of fish collected along the Pacific coast of Vietnam (Table 1). The previous three descriptions were incomplete and inadequate. Yamaguti [40] originally described the species from four females with two line drawings (his Figs. 37 and 38) collected from Johnius goma (Tanaka) in the East China Sea. Bilqees [21] described one 2.8 mm long male of uncertain identity with ovate anterior testis and four females with line drawings (her Figs. 9-16) from Pseudosciaena diacanthus Lacepède off the Karachi coast of Pakistan. Gupta and Jain [25] described only six males from D. diacanthus also in the Indian Ocean off Panaji and Bombay, India, even though they collected 51 males and 35 females but provided line drawings of both males and females (their Fig. 2a-h). Bilqees [21] reported unusually large middle proboscis hooks, two or three times as large as the posterior hooks, which is a serious departure from the original description of the species where middle and posterior hooks have the same size. Gupta and Jain [25] mentioned egg size in females that were not described. The size of most structures of the single diminutive male from Pakistan  (Fig. 21). [21] could not be objectively compared to that of corresponding structures from the six males from India [25] collected from the same host species, P. diacanthus, to examine the influence of host species. Comparative measurements in Table 2 show that our specimens from four host species in Vietnam collectively had the largest size of trunk, proboscis, proboscis hooks, neck, and testes, and more cement gland nuclei. When those measurements were broken down by host species (Table 3), the relationship of host species to the size of taxonomically important structures such as trunk, anterior proboscis hooks, proboscis and testes became quite apparent. We consider the line drawings of the above authors sufficient to give a sense of the morphology of N. johnii and we provide additional insights and new information of various anatomical structures of this species using photo-microscopy (Figs. [13][14][15][16][17][18][19][20] and SEM (Figs. 1-12) of our specimens collected off the Pacific coast of Vietnam (Table 1). Our Vietnamese hosts are of Indo-West Pacific distribution extending between India and east Africa to the west and the Gulf of Thailand, southern China, and the West Pacific to the east [32,35]. Our findings provide the first complete description of this species and show considerable variations between our Vietnamese specimens and those reported by other authors (Table 2). Our findings also demonstrate the relationships between size of certain structures and host species (Table 3). Table 4 and Figure 21 show the results of the hook chemical element analysis. Chemical elements observed were typical for acanthocephalan hooks with high sulfur content on the outer edge of the hook and increased amounts of calcium and phosphorus in the center areas. Both cross-section cuts and longitudinal cuts were analyzed.

Molecular description
In this study, we compared DNA sequences of N. johnii species from Vietnam with sequences of the closely related species of the same genus, Neoechinorhynchus, and other acanthocephalans retrieved from GenBank (Tables 5 and 6). For the analysis, we choose the 18S gene as it is highly conserved and better for an analysis of the upper level phylogeny, while  Table 4). Insert: SEM of a lateral longitudinal gallium cut hook. The X-ray data are the elemental analysis of the hook base. the ITS1-5.8S-ITS2 region is additionally beneficial to differentiate between species. DNA fragments of the 18S and ITS1-5.8S-ITS2 datasets were sequenced for individuals of N. johnii. Our sequences from two isolates from two different hosts of N. johnii for 18S and the ITS1+5.8S+ITS2 region show no detectable intraspecific sequence variability among the individuals sampled. Isolates from all host species were not possible because of limited sample sizes. The genetic divergence estimated among the species of Neoechinorhynchus used for phylogenetic analysis (Table 5 and 6) ranged from 0.06 to 0.28% for 18S and from 0.15 to 0.24% for the ITS1-5.8S-ITS2 region, respectively. For the 18S gene, both ML and BI analyses recovered the newly generated sequence for N. johnii from Vietnam and formed a strongly supported individual sister clade (100/1.00). The isolate of N. johnii obtained demonstrated the association with Neoechinorhynchus salmonis Ching, 1984 (KF156878) from Lake Chistoe, Russia. Both the species show 92.86% identity along with other species of Neoechinorhynchus from a clade belonging to Mexico, China, Iran, Russia and the USA (Fig. 22). For the ITS1+5.8S+ITS2 sequence dataset, the phylogenetic analyses resulting from both ML and BI analyses were highly congruent. Newly generated sequences for N. johnii clustered together with the sequences of N. roseum Salgado-Maldonado, 1978 (FJ388981) from Mexico were strongly associated (98/1.00%) with other species of Neoechinorhynchus from Mexico (Fig. 23). When the data set consisted of 18S and the ITS1-5.8S-ITS2 region used to generate the tree, the tree showed paraphyly of the genus Neoechinorhynchus that can be studied more congruently after addition of other congeneric species data worldwide.
In spite of the increasing number of described species of acanthocephalans from Vietnam, no molecular data are available for species of the genus Neoechinorhynchus to date. Molecular sampling of members of the genus Neoechinorhynchus is still lacking. The 18S and ITS1-5.8-ITS2 datasets included in our study are the only representative sequences for N. johnii to date.

Discussion
We provide a complete description of N. johnii for the first time from four new species of fish off the Vietnam Pacific coast. Our material fills certain serious gaps in knowledge of the species, which was based on previous incomplete and inadequate descriptions. This acanthocephalan species is clearly of Indo-Pacific distribution and is found in hosts that naturally inhabit waters off the Japanese islands, India and Pakistan. Its distribution in the Pacific waters of Vietnam extended from Tien Yen in the north to Vung Tau, Nha Trang, and Bac Lieu in the south (Table 1). It will not be surprising to find this acanthocephalan from the same host species elsewhere within their endemic range in the Indo-Pacific region.

Variability
Much of the reported variability in the size of taxonomically important structures such as the trunk, proboscis hooks, proboscis, testes, etc. has been attributed to host species. Such relationships have previously been reported in other species of acanthocephalans including Echinorhynchus salmonis Müller, 1784 in Lake Michigan where male and female specimens from bloater, Coregonus hoyi (Gill) (Salmonidae) achieved not only larger size but also different body form (broad anteriorly) compared to the slender specimens from rainbow smelt, Osmerus mordax (Mitchell) (Osmeridae) [4]. The larger and heavier worms from bloater almost invariably showed higher regression coefficients (adjusted coefficient of determination) compared to Table 6. Acanthocephala species, origin and GenBank accession numbers used for phylogenetic analysis based on the ITS1-5.8S-ITS2 gene cluster. Sequences marked with an asterisk were obtained in this study. Na = not available.

Species
Host Females of the same developmental stage recovered during the same period were found to have attained larger sizes in certain hosts than in others, with the largest females being found in Lepomis macrochirus Rafinesque. The size of the trunk in males was also found to follow the same pattern. Similarly, testes also attained a larger size in males recovered from Catostomus commersonii Lacépède (Catostomidae) than in males from Semotilus atromaculatus (Mitchill) (Cyprinidae). Amin [1] stated that these size variations "result from differential growth rates of these worms in the various host intestinal environments (and) are probably mediated by certain host specific factors." The para-receptacle structure (PRS) The PRS inserts anteriorly in the body wall near the neck and posteriorly at the posterior end of the receptacle. The presence of the PRS in eoacanthocephalans with a weak single proboscis receptacle wall was first demonstrated in Neoechinorhynchus (N.) qatarensis Amin, Saoud, Alkuwari, 2002 [19] and has since been reported in other species of Neoechinorhynchus and Acanthogyrus (Acanthosentis) Verma and Datta, 1929 reviewed in part in Amin et al. [15] and reported here for the first time in various species of Neoechinorhynchus from marine fishes off the east coast of Vietnam. In the description of the PRS, Amin et al. [7,19] proposed that it may regulate the hydrostatic pressure in the receptacle to facilitate the retraction and eversion of the proboscis.

Electron dense micropores
Micropores are present throughout the epidermal surface of the trunk of reported species of Neoechinorhynchus, like those reported in other species of the Acanthocephala, and are associated with internal crypts and vary in diameter and distribution in different trunk regions, corresponding to differential absorption of nutrients. We have documented this phenomenon in 16 species of acanthocephalans [27]. The functional aspects of micropores in a few other acanthocephalan species including Rhadinorhynchus ornatus Van Cleave, 1918, Polymorphus minutus (Goeze, 1782) Lühe, 1911, Moniliformis (Bremser, 1811) Travassos (1915), Macracanthorhynchus hirudinaceus (Pallas, 1781) Travassos (1916,1917), and Sclerocollum rubrimaris Schmidt and Paperna, 1978 were reviewed earlier [8]. We demonstrated the tunneling from the cuticular surface into the internal crypts by TEM. Wright and Lumsden [39] and Byram and Fisher [22] reported that the peripheral canals of the micropores are continuous with canalicular crypts. These crypts appear to "constitute a huge increase in external surface area implicated in nutrient uptake." Whitfield [38] estimated a 44-fold increase at a surface density of 15 invaginations per 1 lm 2 of Moniliformis moniliformis (Bremser, 1811) Travassos, 1915 tegumental surface. The micropores and the peripheral canal connections to the canaliculi of the inner layer of the tegument of Corynosoma strumosum (Rudolphi, 1802) Lühe, 1904 from the Caspian seal Pusa caspica (Gmelin) in the Caspian Sea were demonstrated by transmission electron micrographs [17].

EDAX of hooks
Most acanthocephalan hooks have three major elements: calcium, phosphorus, and sulfur [15,16], among others, and small amounts of magnesium plus common protoplasm elements (C, N, O, H). The arched area at the base of the anterior hook of N. johnii exhibited high levels of calcium and phosphorus (Table 4, Fig. 21) very similar to the mammalian tooth with its layers. The X-ray scans of the gallium cut hooks help explain the morphological nature of N. johnii and identify its unique "personality." The uniqueness of the metal analysis as expressed by X-ray scans appear to be species-specific and can be regarded as finger prints of key diagnostic value that are just as important as molecular analysis.