On the Neoechinorhynchus agilis (Acanthocephala: Neoechinorhynchidae) complex, with a description of Neoechinorhynchus ponticus n. sp. from Chelon auratus in the Black Sea

We recognize four species in the Neoechinorhynchus agilis complex. We studied specimens of Neoechinorhynchus (Hebesoma) personatus Tkach, Sarabeev & Shvetsova, 2014 from Mugil cephalus in the Mediterranean Sea off Tunisia and in the Black Sea, and also specimens of Neoechinorhynchus ponticus n. sp. from Chelon auratus Risso in the Black Sea. Specimens from M. cephalus at both locations were similar. All structures of N. ponticus n. sp. were considerably smaller than those of N. personatus. Two other species of the N. agilis complex are recognized: Neoechinorhynchus agilis (Rudolphi, 1819) sensu stricto from various hosts in the Atlantic and the Mediterranean, and Neoechinorhynchus yamagutii Tkach, Sarabeev & Shvetsova, 2014 from M. cephalus and Planiliza haematocheila in the Pacific, especially the Sea of Japan. Neoechinorhynchus dimorphospinus Amin & Sey, 1996 from marine fish in the Persian Gulf and the Pacific Ocean off Vietnam may be a candidate for membership in the N. agilis complex. X-ray scans of gallium cut and intact hooks of N. personatus and N. ponticus showed differences in the mineral content of hooks with higher sulfur levels in smaller hooks and in hooks from specimens in the Black Sea compared to specimens from the Mediterranean. The relatively high genetic differences between N. ponticus n. sp. and other species of Neoechinorhynchus using a partial 18S rDNA dataset support its independent status. Neoechinorhynchus ponticus n. sp. and N. personatus have a common ancestor with species of Neoechinorhynchus collected from saltwater fish.


Introduction
Rudolphi [30] originally described the external morphology and proboscis receptacle of Neoechinorhynchus agilis (Rudolphi, 1819) Van Cleave, 1916 from 9 individuals taken from Mugil cephalus in Spezia, Italy without providing any measurements. Hamann [13] described N. agilis in detail but thought it had 6 cement glands. Van Cleave [36] described a syncytium cement gland containing 8 giant nuclei. Van Cleave [36,37] suspected that N. agilis was restricted to the Mediterranean, but this is now known not to be. For example, species presumed to be N. agilis before the establishment of the species complex concept studies by Tkach et al. [35], Sarabeev et al. [31] and ours (this paper) were reported from M. cephalus in Taiwan [32], in the Indian Ocean near Tamil Nadu [17], and in Guyana [26], as well as in Japan, Scotland, and North America. Other observers including Linton [19][20][21], Van Cleave [36,37], Meyer [23], Yamaguti [40,41], Petrochenko [24], Gaevskaya et al. [10], and Tepe and Oguz [34] reported N. agilis from at least 10 species of fish in the Black Sea and the Mediterranean, among other locations off the coasts of Scotland, North America, and Japan. This species complex appears to be widely distributed in mullets from the Atlantic and Pacific oceans ( [31,35], among others). Altogether, descriptions by the above cited authors gave an extreme range of variation in taxonomically important traits. Van Cleave [37] provided a detailed description of the variability in "N. agilis" based on the examination of Rudolphi's specimens and other materials from the Berlin Museum (Table IV of Van Cleave [37]).
None of the above authors reported on the relationship between the observed extreme variability and host species. Van Cleave [37] was unable to determine whether the "conflicting data represented individual variability within the species or resulted from inaccurate observations and erroneous identifications." Tkach et al. [35] split N. agilis into three species that showed some overlap in measurements and that were collected from M. cephalus and other mugilids from various locations from the Mediterranean to Japan. These are Neoechinorhynchus (Hebesoma) personatus Tkach, Sarabeev & Shvetsova, 2014, Neoechinorhynchus agilis sensu stricto, and Neoechinorhynchus yamagutii Tkach, Sarabeev & Shvetsova, 2014. Gargouri et al. [12] and Tepe and Oguz [34] reported "N. agilis" from C. auratus in the Mediterranean off the Tunisian coast at the Bizerte Lagoon and off the Black Sea coast of Turkey, respectively. Observations were not available to correlate their morphometric data with host species.
We also provide chemical and molecular data to explain and clarify our findings. With the use of dual beam scanning electron microscopy, it was feasible to run energy dispersive X-ray analysis (EDXA) on both intact and gallium (LMIS) cut hooks. This is usually a qualitative analysis, not quantitative, for the attachment structures [5,14,15]. This technique was applied to the study of the Neoechinorhynchus complex.
This study provides molecular profiles of N. ponticus n. sp. and N. personatus based on 18S rDNA gene. Furthermore, its phylogenetic relationships with other members of the genus Neoechinorhynchus are analyzed and discussed.

Collections
Five hundred and twenty two individuals of 5 mugilid species of fish in the Ichkeul Lagoon off Northern Tunisia (37°10 0 00 00 N, 9°40 0 00 00 E) were sampled between March 2014 and September 2017 by gill net fishing. Fish were examined for helminths under a stereomicroscope shortly after capture. The specimens of acanthocephalans collected were first placed in distilled water at 4°C until the proboscis was everted. Tunisian specimens of N. personatus, among other acanthocephalan species, from M. cephalus from this collection were deposited in the Harold W. Manter Laboratory (HWML) parasitology collection no. 102004. In the Black Sea at Zalizny Port (46°7 0 N, 32°17 0 E), individuals of M. cephalus and of Chelon auratus were examined in June, 2015. Specimens of N. personatus from M. cephalus were deposited in the HWML collections no. 102006 and those of N. ponticus from C. auratus in the HWML collection no. 102005.

Methods for microscopical 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 (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.
Microscope images were created using 10X and 40X objective lenses of a BH2 light Olympus microscope (Olympus Optical Co., Osachi-shibamiya, Okaya, Nagano, Japan) attached to an AmScope 1000 video camera (United Scope LLC, dba AmScope, Irvine, CA, USA), linked to an ASUS labtop equipped with an HDMI high definition multimedia interface system (Taiwan-USA, Fremont, CA, USA). Images from the microscope were transferred from the labtop to a USB and stored for subsequent processing on a computer.

Scanning electron microscopy (SEM)
Samples of parasites that had been fixed and stored in 70% ethanol were processed following standard methods [18] which included critical point drying (CPD) in sample baskets and mounted 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, OR, USA) Scanning Electron Microscope with digital images obtained in the Nanolab software system (FEI) and then transferred to a USB for future reference. Images were taken at various magnifications. Samples were received under low vacuum conditions using a 10 kV, spot size 2 0.7 Torr using a GSE detector.
X-ray microanalysis, energy dispersive X-ray analysis 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). 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) was used. 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 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 was 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 for chemical ions with an electron beam (Tungsten) to obtain an X-ray spectrum. The intensity of the GIS was variable due to the nature of the material being cut.

DNA extraction and PCR amplification
Total genomic DNA was extracted from 3 adult worms of N. ponticus n. sp. and 4 of N. personatus using a Qiagen DNeasy blood and tissue kit (Qiagen Inc., Valencia, CA, USA), according to the manufacturer's instructions. Partial nuclear small subunit ribosomal DNA (18S rDNA) was amplified by PCR reactions in 30 lL volumes containing 2Â red PCR premix (Ampliqon, Odense, Denmark), 20 pmol of each primer, and 3 lL of isolated DNA. Forward primer (5 0 -AGAT-TAAGCCATGCATGCGTAAG -3 0 ) and reverse primer (5 0 -ACCCACCGAATCAAGAAAGAG -3 0 ) were used for the amplification of the partial fragment of the nuclear 18S rDNA gene [7]. PCR conditions were: 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 61°C for 30 s, 72°C for 60 s, and 72°C for 7 min as a final extension. Finally, the PCR products were electrophoresed on a 1.5% agarose gel and visualized with a UV transilluminator (Vilber Lourmat, Collégien, France).

Sequencing and phylogenetic analysis
All PCR products were sequenced on an ABI 3730 automatic sequencer (Applied Biosystems, Foster City, CA, USA) in both directions using the same PCR primers. The obtained sequences were edited and analyzed using Chromas v.2.01 (Technelysium Pty Ltd., Brisbane, Queensland, Australia) and BioEdit software v.7.0.9. The sequences were compared with available data in GenBank using the BLAST algorithm (http:// www.ncbi.nlm.nih.gov/). The nucleotide sequences of the 18S rDNA gene from this study were submitted to the GenBank database (Accession Numbers: MT020789 -MT020791 for N. ponticus n. sp. isolates and MT020792 -MT020795 for N. personatus isolates).
A phylogenetic tree was constructed using the maximumlikelihood algorithm and Tamura-3-parameter model in MEGA7 software (http://www.megasoftware.net/). A bootstrap value with 1000 replications was also implemented to evaluate the reliability of the tree topologies. Furthermore, genetic distances were calculated with the maximum composite likelihood model using MEGA7 software. The sequences used for the phylogenetic analysis are listed in Table 1.  The following redescription is based on the microscopic examination of 26 specimens (9 males, 17 females) from M. cephalus in the Black Sea and 16 specimens (8 males, 8 females) off the Tunisian coast, and on 6 more specimens of each collection examined by SEM. Additional specimens were used for molecular analysis. The redescription addresses mostly qualitative features that apply to the two comparable populations of N. personatus studied in these two geographical locations, presenting a number of features not noted in the original description. The specimens from the Black Sea and the Mediterranean were collected in the same month, June, 2014 and 2015 (in the Black Sea). Morphological and morphometric data are presented in Table 2. Biochemical, EDXA and molecular analysis are addressed separately, below.
Male (see Table 2 for measurements): Reproductive system in posterior two thirds of trunk. Testes large, equal, oblong, contiguous, slightly anterior to mid-trunk, with male reproductive structures extending into bursa. Cement gland longer than either testes, usually with 8-18 giant nuclei; mature adults with fewer nuclei. Cement reservoir just posterior to cement gland, less than half size of cement gland. Common sperm duct prominent. Saefftigen's pouch prominent, overlapping common sperm duct and often obscured by it (Fig. 1). Bursa rounded, usually longer than wide, with no apparent sensory structures (Fig. 21). Gonopore terminal.
Female (see Table 2 for measurements). Reproductive system about 10% length of trunk (Fig. 3). Vagina not especially muscular. Uterus and uterine bell of moderate length with prominent ligament strand connecting anterior end of uterus at uterine glands with posterior body wall near the vagina dorsally (Fig. 6). Gonopore near terminal on ventral side. Eggs fusiform, elongate, with bluntly pointed ends and with polar prolongation of fertilization membrane (Figs. 5, 22).

Remarks
Morphologically, our specimens of N. personatus from Tunisia and the Black see were almost identical to those described by Tkach et al. [35]. The extent of the length of the lemnisci reaching the anterior testis, the shape and organization of the proboscis, hooks, and reproductive systems, and the number of giant sub-cuticular and cement gland nuclei were practically identical to those in the original description and to their Figures 12-18 (page 298). Our specimens, however, had slightly larger trunk, testes, cement glands and eggs, and slightly smaller hooks. References to the anterior trunk muscle ring, the ligament strand connecting the anterior end of uterus at the uterine glands with the posterior body wall, the micropores, and the neck's sensory pores and pebblelike texture of its epidermis were missing from the original description. Characters used by Tkach et al. [35] to distinguish N. personatus from species of Neoechinorhynchus     Fig. 29 showing detail of the micropore channels.
other than those in the N. agilis complex apply to the present discussion. The eggs in our specimens were slightly larger (35-50 Â 10-15) compared to theirs (30-34 Â 9-11). Egg size difference may be related to degree of development; their Figure 14 (mislabeled) shows 2 apparently underdeveloped eggs.
EDXA results for N. personatus Results for energy dispersive X-ray analysis are shown in Tables 3-6 (Table 3). The hardening elements (Ca, P) are more plentiful in the large anterior hooks. Table 3 also shows a difference in mineral content for the large and small hooks with small hooks showing higher sulfur and lower calcium levels (Figs. 37, 38 and Tables 3, 4). All elements were highest at the tip of hooks compared to the middle and bottom cuts (Table 5), and calcium was especially high at the base of hooks ( Table 6). The hardening elements (Ca, P) are more plentiful in the large anterior hooks.
Neoechinorhynchus ponticus n. sp. (Figs. 2, 4, 8 The following description is based on the microscopic examination of 19 specimens of acanthocephalans (9 males, 10 females) from Chelon auratus in the Black Sea and on 6 more specimens examined by SEM. The supply of hosts limited the number of specimens available for this study. The description addresses mostly qualitative features that apply to specimens from this one host species in the Black Sea. Morphological and morphometric data are presented in Table 2. Biochemical, EDXA and molecular analysis are addressed separately, below.

Description
General: With characters of the genus Neoechinorhynchus as designated by Amin [1] (Neoechinorhynchidae). Mediumsized sexually mature adults. Shared structures larger in females than in males. Trunk somewhat enlarged anteriorly, gradually tapering at both ends (Figs. 2, 4). Body wall with electron dense micropores ducting into subcutaneous layer with multiple  branching. Neck prominent with paired sensory pores (Fig. 31) and elevated pebble-like protrusions (Fig. 35). Body wall with prominent reticular lacunar system characteristically manifesting as heavily stained and lightly stained bands alternating at regular intervals, and with 5-7 (usually 6) dorsal and 1-3 (usually 2) giant hypodermal nuclei (Figs. 2, 4). Muscular ring at anterior trunk variable but often prominent (Fig. 8). Proboscis cylindrical, truncated and widest in anterior half, relatively but consistently wider than long, with prominent apical organ having 2 conspicuous nuclei at its distal end. Apical organ (Fig. 32) may reach or surpass level of posterior hooks, with 2 prominent giant nuclei (Fig. 8). All hooks with longitudinal serrations (Figs. 33, 34), rooted, most robust anteriorly, smallest posteriorly. Hook roots prominent but markedly shorter than blades, with anteriorly directed manubria. Manubria least prominent in largest anterior hooks (Fig. 10). Proboscis receptacle single-walled, slightly longer than 4 times as long as proboscis with cerebral ganglion at its base. Lemnisci of moderate size, digitiform, subequal, with 2 large oval giant nuclei each at widest part, falling far short of anterior testis and somewhat shorter in females (Figs. 2, 4).
Male (see Table 2 for measurements): Reproductive system in posterior half of trunk. Testes equal, equatorial to post equatorial, oblong, contiguous with male reproductive structures extending into bursa. Cement gland larger than either testes, usually with about 8 giant nuclei in adults but with up to 18 nuclei in juveniles. Cement reservoir just posterior to cement gland and less than half its size. Common sperm duct prominent. Saefftigen's pouch prominent, about as long as cement gland, overlapping common sperm duct and often obscured by it (Fig. 2). Bursa usually longer than wide.
Female (see Table 2 for measurements). Reproductive system about 10% length of trunk, with prominent strand connecting anterior end of uterus at uterine glands with posterior body wall near vagina dorsally. Vagina not especially muscular. Uterus and uterine bell of moderate length, with terminal to near terminal gonopore (Fig. 36). Eggs not observed; only ovarian balls present.  (Table 7). These are figures from the base of hooks. Intact hooks showed the same pattern ( Table 4). The base of the large anterior hooks appears to exhibit the highest level of calcium, as with N. personatus (Fig. 6).

Remarks
Tkach et al. [35] and Sarabeev et al. [31] satisfactorily differentiated 3 species in the N. agilis complex: N. agilis sensu stricto, N. personatus, and N. yamagutii from each other and from related species of Neoechinorhynchus Stiles and Hassall, 1905 outside of the N. agilis complex. The morphological features of our specimens from M. cephalus off the Tunisian Mediterranean and in the Ukrainian Black Sea are consistent with those of N. personatus, and so are the molecular profiles. Specimens of N. ponticus n. sp. from C. auratus in the Black Sea are clearly morphologically distinguished from those of N. personatus by the markedly smaller size of their trunk, proboscis, hooks, receptacle, lemnisci, testes and other male reproductive system structures ( Table 2). In addition, the shorter lemnisci in N. ponticus occupy a considerably smaller trunk space distant from the anterior testis (Fig. 2) compared to usually overlapping with it in N. personatus (Fig. 1).
In N. agilis, the lemnisci are also distant from the testes, as in N. ponticus, but the roots of its anterior and middle hooks are much shorter in N. agilis with diminished manubria. In N. ponticus n. sp., the proboscis and proboscis receptacle are   markedly longer while the hooks and testes are considerably smaller than in N. agilis; see Table 2 of Tkach et al. [35] and Table 2 (this paper). Specimens of N. ponticus lack the terminal papillae posterior to the gonopore characteristic of N. agilis. The description of N. yamagutii by Tkach et al. [35] was heavily based on the description of N. agilis by Yamaguti [40] from M. cephalus in the Inland Sea and the Pacific coast of Mie Prefecture. Specimens of N. yamagutii, like those of N. personatus, have long unequal lemnisci reaching and overlapping the anterior testis (our Fig. 1 and Fig. 24 of Tkach et al. [35]) but they are larger worms with a considerably longer trunk (females reaching 25 mm) than N. ponticus and, unlike N. ponticus (Fig. 4), with decidedly subterminal female gonopore (Fig. 20 of Tkach et al. [35]). In N. yamagutii, the middle hooks are about half as long as the anterior hooks and barely longer than the posterior hooks. In N. ponticus n. sp., the transition in hook size from anterior is more gradual (our Table 2, and Table 4 of Tkach et al. [35]). In N. ponticus, the receptacle is larger but the testes and cement glands are considerably smaller than in N. yamagutii. In the description of N. yamagutii by Tkach et al. [35] references to Saefftigen's pouch (1.00 long in Yamaguti [40]), or to the bursa (1.4 mm long in Yamaguti [40]) were missing.

Molecular analysis
All N. ponticus n. sp. and N. personatus specimens successfully showed amplification of about 1300 bp for the partial 18S rDNA gene. The 18S rDNA dataset (1047 nt) included 25 sequences for different species of the genus Neoechinorhynchus, 3 N. ponticus n. sp. and 4 N. personatus sequences obtained in this study. Multiple alignment indicated that intra-species homology within N. ponticus n. sp. was 100%. Furthermore, pairwise distance illustrated the presence of an intra-species distance rate of 0-0.19% within N. personatus specimens obtained in the current study, and 0.18-0.67% compared to those with the sequences available in GenBank. Inter-generic differences based on partial 18S rDNA sequence between N. ponticus n. sp. with N. personatus, N. agilis, N. yamagutii, N. dimorphospinus, N. buttnerae, N. crassus, N. pseudemydis, N. cylindratus, N. beringianus, N. saginata and N. simansularis as other members of the genus Neoechinorhynchus were 3.24-4.49%, 3.26-3.69%, 3.84%, 3.49%, 5.06%, 5.45%, 14.91%, 4.55%, 4.02%, 5.12% and 4.02%, respectively. The sequence divergence based on the partial sequence of 18S rDNA between N. personatus with N. ponticus n. sp., N. agilis, N. yamagutii, N. dimorphospinus, N. buttnerae, N. crassus, N. pseudemydis, N. cylindratus, Figure 37. Energy dispersive X-Ray spectrum of the large anterior hook of a Neoechinorhynchus personatus specimen from Mugil cephalus in the Mediterranean showing high levels of phosphorus and calcium and low levels of sulfur. The X-ray data are the elemental analysis of the whole hook (see bolded figures in Table 3). Insert: SEM of a deep Gallium cut at the base of a hook. Figure 38. Energy dispersive X-ray spectrum of the small posterior hook of a Neoechinorhynchus personatus specimen from Mugil cephalus in the Mediterranean showing markedly lower levels of phosphorus and calcium, and considerably lower levels of sulfur than anterior hooks (Fig. 37). The X-ray data are the elemental analysis of the whole hook (see bolded figures in Table 3 (Fig. 40).

Discussion
Morphometrics None of the earlier descriptions of this variable acanthocephalan, N. agilis, accounted alone for its extreme morphometric variability. Host species and geography were not considered in the accounts given by Hamann [13], Linton [19][20][21], Van Cleave [36,37], Meyer [23], Yamaguti [40,41], Petrochenko [24], Gaevskaya et al. [10], Tepe & Oguz [34], and Tkach et al. [35], among others who described "N. agilis" from at least 10 species of hosts. Altogether, descriptions by the above cited authors gave an extreme range of variation in taxonomically important traits including trunk length of 7.00-45.00 mm, anterior, middle and posterior hook length of 70-150, 39-87, 20-71 respectively, proboscis receptacle 178-700 Â 89-240, and testes 394-1820 Â 97-570. Van Cleave [37] provided the most detailed description of the variability in N. agilis based on the examination of Rudolphi's specimens from the Berlin Museum and the reports of the Dujardin, Stossich, Hamann, Condorelli, Porta, Monticelli, and Parona collections from Toulouse, Trieste, Rome, and Genoa (Table IV of Van Cleave [37]). Van Cleave [37] "found characterizations so diverse that it was difficult to determine whether the conflicting data represented individual variability within the species or resulted from inaccurate observations and erroneous identifications." The species complex None of the above accounts considered the possibility of dealing with a species complex. Tkach et al. [35] split N. agilis into three species (the nominal N. agilis and two new species) that overlapped in measurements of such important taxonomic traits as hook size and were collected from M. cephalus and other mugilids from various locations from the Mediterranean to Japan. Tkach et al. [35] collected one species, N. personatus, from M. cephalus, C. auratus and Planiliza haematocheila in the Mediterranean and in the Black Sea. Our specimens from M. cephalus in both locations proved to be N. personatus but those from C. auratus in the Black Sea turned out to be a different species, N. ponticus n. sp. The case for correlating taxonomic decisions with morphometric data, individual host species and geography was not clearly made by Tkach et al. [35]. These authors listed their N. agilis only from thick lipped grey mullet, Chelon labrosus Risso, leaving the type host "undefined" because of difficulties of ascertaining the host species from which Rudolphi's [30] nine specimens were taken in Spezia, Italy (Type locality) as well as from the Bay of Biscay (Atlantic), and the Gulf of Santa Pola, Júcar Estuary (Mediterranean). It is worth noting that specimens of N. agilis  Table 7). Insert: SEM of anterior hooks.   identified by Linton [19] in the Atlantic off Wood's Hole, Massachusetts, USA from the common eel, Anguilla rostrata Lesueur (Anguillidae), and the dusky shark, Carcharhinus obscurus Lesueur (Carcharhinidae), and in Fulton Market, New York from the white perch, Roccus americanus Gmelin (Moronidae) were correctly identified. Linton [19] did not provide a description, measurements or measurement bars of his whole male specimen (Fig. 70) that showed compatible proportions and disposition of the internal organs, including short lemnisci distant from the anterior testis characteristic of that species. This 130-year-old record alone expands the range of host and geographical distribution of N. agilis. In our study, morphological differences noted in specimens from M. cephalus and C. auratus in the same location in the Black Sea (Table 2) correlate well with the designation of two different species of acanthocephalans supported by molecular data, N. personatus and N. ponticus n. sp., respectively. Measurements of key taxonomic characteristics of males and females of "N. agilis", e.g. trunk, proboscis, hooks, testes, and eggs, from C. auratus on the Black Sea coasts of Turkey by Tepe and Oğuz ([34]; their Table 2) are very similar to those of N. ponticus n. sp. from the same host and body of water. We, therefore, assign the specimens reported by Tepe and Oğuz [34] to N. ponticus n. sp.

Intraspecific morphological variations
Morphometric variations in specimens of N. personatus from the same host species, M. cephalus, in the Mediterranean and from the Black Sea (Table 2), would point to intraspecific geographical variations. Van Cleave [37] made a vague reference to "geographical varieties" that was not supported by hard data. The observed variations are comparable to the more dramatic case of Mediorhynchus papillosus Van Cleave, 1916 (Gigantorhynchidae) featured by Amin and Dailey [2]. These authors studied key taxonomic characteristics in various geographical populations of M. papillosus which has a wide range of distribution in at least 73 species of birds outside of North and South America in Asia from Taiwan to the east into China, many of the former Soviet Republics, and to Eastern Europe to the west. Amin and Dailey [2] demonstrated a distinct geographically-based variability, especially in the size of proboscis and its armature, neck, receptacle, and testes, that appeared related to geographical restrictions, intermediate and definitive host specificity and distribution, and host feeding behavior. We recognize that many fish hosts of species of Neoechinorhynchus are migratory and that their feeding grounds and specimen collecting localities may not always be the same. However, this situation is also comparable to that of migratory bird hosts of M. papillosus.

Electron dense micropores
Micropores are present throughout the epidermal surface of the trunk of species of Neoechinorhynchus, like those reported in other species of the Acanthocephala, are associated with internal crypts, and vary in diameter and distribution in different trunk regions corresponding with differential absorption of nutrients. We have documented this phenomenon in 16 species of acanthocephalans [16] and a few more since. The functional aspects of micropores in a few other acanthocephalan species including Rhadinorhynchus ornatus Van Cleave, 1918, Polymorphus minutus (Goeze, 1782) Lühe, 1911, Moniliformis moniliformis (Bremser, 1811) Travassos (1915), Macracanthorhynchus hirudinaceus (Pallas, 1781) Travassos (1916,1917), and Sclerocollum rubrimaris Schmidt and Paperna, 1978 were reviewed earlier by Amin et al. [3]. We demonstrated the tunneling from the cuticular surface into the internal crypts by TEM (Figs. 26, 29, 30). Wright and Lumsden [39] and Byram and Fisher [8] 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] 26,29,30). We cannot explain the nature or functionality of the pebble-shaped texture of the neck in specimens of these two acanthocephalan species from either M. cephalus or C. auratus. We can, however, use this unique feature as a diagnostic tool for these two species. We do not know if the necks of other species of the N. agilis complex are similar. We do know that other species of Neoechinorhynchus that we have studied did not possess this feature.

Energy dispersive X-ray analysis
Our studies of acanthocephalan worms have usually involved X-ray scans (EDXA) of gallium cut hooks and other worm structures [14,15,33]. Both large and small hooks were evaluated for chemical ions, with sulfur (S), calcium (Ca) and phosphorus (P) being the prominent elements. Sulfur as expected, was high especially at the outer edge of large hooks of specimens from the Black Sea. Calcium and phosphorus are major ions at the base and middle of the hooks (Table 3). There was a difference in chemical content for large and small hooks. Large hooks play a major role in host tissue attachment, which may account for the difference. The anterior and posterior hooks of our N. personatus from M. cephalus in the Mediterranean and Black Sea had comparable biochemical profiles, especially for phosphorous. Variable levels of sulfur and calcium in hooks between the Mediterranean and the Black Sea specimens (Table 3) may be attributable to different levels of accumulation of metals in the two bodies of water. It should be noted, however, that extreme differences may occur in different parts of the same hook of acanthocephalan species found in the same host at a single location, or between anterior vs. posterior hooks on the same proboscis. The levels of sulfur were markedly higher in N. ponticus from the Black Sea compared to N. personatus from the Mediterranean which may also be attributable to the rich resources of sulfur in the Black Sea [9]. For instance, in Cavisoma magnum (Southwell, 1927) Van Cleave, 1931 from Mugil cephalus in the Arabian Sea, unusually high levels of sulfur in hook tips (43.51 wt%) and edges (27.46 wt%) were found. The center and base of hooks of the same worms had negligible sulfur levels and contained mostly phosphorus and calcium, the two other essential elements for hook structure [5]. Like fingerprints, the EDXA appear to be species-specific and has significant diagnostic value in acanthocephalan systematics [6]. For example, Moniliformis cryptosaudi Amin, Heckmann, Sharifdini, and Albayati, 2019 was erected based primarily on its EDXA pattern [6]. Our methodology for the detection of the chemical profile of hooks in the Acanthocephala has also been used in other parasitic groups including the Monogenea [27,29] and Cestoda [28]. We also provide chemical and molecular data to explain and clarify our findings.

Molecular analysis
In the last few decades, a PCR-sequencing technique was applied for assessment of taxonomic identification, diversity and phylogenetic relationships among acanthocephalan species [11]. Inter-generic differences are noted between N. ponticus n. sp. and N. personatus with other members of the genus Neoechinorhynchus based on partial 18S rDNA being 3.24-14.91% and 2.18-14.70%, respectively. The relatively high genetic differences between N. ponticus n. sp. and other species of the Neoechinorhynchus supports the morphological observation indicating it is an independent species. Sarabeev et al. [31] showed that N. agilis, N. personatus and N. yamagutii are three independent species using the same phylogenetic analysis of partial sequences of the 18S rRNA gene. Our data, consistent with results of other studies [22,31], indicated genetic divergences among these three species were relatively high, at the range between 2.18% and 6.60%. The phylogenetic analysis of the partial 18S rDNA gene illustrated that our sequence of N. personatus clustered with N. personatus isolates from Ukraine. Genetic differences between N. agilis and N. personatus were found to be low. Moreover, the tree also showed they are two closely related species. Our sequences of N. ponticus n. sp. were placed separately from other species of Neoechinorhynchus in clade I with strong support. The position of N. ponticus n. sp. along with other species of Neoechinorhynchus from saltwater fishes including N. dimorphospinus, N. yamagutii, N. agilis and N. personatus in the tree, indicated that all these species share a common ancestor. This would make N. dimorphospinus a candidate for membership in the N. agilis complex (see Fig. 40) especially that specimens of N. dimorphospinus are morphologically comparable to the 4 species of the N. agilis complex. It is also grouped with them in one major clade with 93% similarity with the Tunisian N. personatus. However, N. dimorphospinus is distinguishable morphologically by having the 2 lateral proboscis hooks in the anterior ring being considerably longer than the other 4 hooks in the same ring. It has been found in 6 species in 4 families of marine fish in the Persian Gulf and in the Pacific Ocean off Vietnam. In species of Neoechinorhynchus, high level of variation in the cox1 gene would provide better resolution of the relationships within closely related taxa [25]. Due to the lack of sufficient sequences of cox1 sequences of N. agilis complex in GenBank for comparison, we did not perform molecular analysis of these specimens with this gene. Pinacho-Pinacho et al. showed that the cox1 gene is an informative molecular marker compared to the ITS and 28S rDNA genes for detection of cryptic species complexes in Neoechinorhynchus in Middle America [25]. However, their analysis is for the species inhabiting fresh and brackish water fish across Middle America, a different zone from our study areas. Providing additional genetic markers as well as obtaining more specimens of these species from different geographical locations and hosts would be useful to better understand their phylogenetic relationships.