Parasite 26, 14 (2019) Ó O.M. Amin et al., published by EDP Sciences, 2019 https://doi.org/10.1051/parasite/2019015 Available online at: www.parasite-journal.org RESEARCH ARTICLE OPEN ACCESS Morphological and molecular description of Rhadinorhynchus laterospinosus Amin, Heckmann & Ha, 2011 (Acanthocephala, Rhadinorhynchidae) from marine fish off the Pacific coast of Vietnam Omar Mohamed Amin1,*, Richard Anderson Heckmann2, Sara Dallarés3, María Constenla3, and Nguyen Van Ha4 1 Institute of Parasitic Diseases, 11445 E. Via Linda 2-419, Scottsdale, AZ 85259, USA 2 Department of Biology, Brigham Young University, 1114 MLBM, Provo, UT 84602, USA 3 Department of Animal Biology, Vegetal Biology and Ecology, Universitat Autònoma de Barcelona, Cerdanyola, 08193 Barcelona, Spain 4 Department of Parasitology, Institute of Ecology and Biological Resources (IEBR), Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Received 18 January 2019, Accepted 27 February 2019, Published online 6 March 2019 Abstract – Rhadinorhynchus laterospinosus Amin, Heckmann & Ha, 2011 (Rhadinorhynchidae) was described from a single female collected from a trigger fish, Balistes sp. (Balistidae) from the northern Pacific coast of Vietnam in Halong Bay, Gulf of Tonkin. More recent collections of fishes in 2016 and 2017 revealed wider host and geographical distributions. We report this Acanthocephala from nine species of fish representing six families (including the original record from Balistes sp.) along the whole Pacific coast of Vietnam. The fish species are Alectis ciliaris (Carangidae), Auxis rochei (Scombridae), Auxis thazard (Scombridae), Leiognathus equulus (Leiognathidae), Lutjanus bitaeniatus (Lutjanidae), Megalaspis cordyla (Carangidae), Nuchequula flavaxilla (Leiognathidae), and Tylosurus sp. (Belonidae). We provide a complete description of males and females of R. laterospinosus, discuss its hook metal microanalysis using EDAX, and its micropores. Specimens of this species characteristically have lateral trunk spines bridging the anterior ring of spines with posterior field of ventral spines and a proboscis with 15–19 longitudinal alternating rows of 21–26 hooks each varying with host species. We demonstrate the effect of host species on the distribution and size of the trunk, proboscis, proboscis hooks, trunk spines, and reproductive structures. The molecular profile of this acanthocephalan, based on 18S rDNA and cox1 genes, groups with other Rhadinorhynchus species and further seems to confirm the paraphyly of the genus, which is discussed. Key words: Acanthocephala, Rhadinorhynchus laterospinosus, Host distribution, Molecular profile, EDAX, Micropores, Vietnam. Résumé – Description morphologique et moléculaire de Rhadinorhynchus laterospinosus Amin, Heckmann & Ha, 2011 (Acanthocephala, Rhadinorhynchidae) des poissons marins du large de la côte pacifique du Vietnam. Rhadinorhynchus laterospinosus Amin, Heckmann & Ha, 2011 (Rhadinorhynchidae) a été décrit d’une seule femelle prélevée chez Balistes sp. (Balistidae) sur la côte nord du Pacifique du Vietnam, dans la Baie d’Along, golfe du Tonkin. Des collections de poissons plus récentes, en 2016 et 2017, ont révélé une répartition en hôtes et en localités géographiques plus étendue. Nous rapportons cet Acanthocéphale de 9 espèces de poissons représentant 6 familles (y compris la mention originale chez Balistes sp.), le long de la côte pacifique du Vietnam. Les espèces de poissons sont Alectis ciliaris (Carangidae), Auxis rochei (Scombridae), Auxis thazard (Scombridae), Leiognathus equulus (Leiognathidae), Lutjanus bitaeniatus (Lutjanidae), Megalaspis cordyla (Carangidae), Nuchequula flavaxilla (Leiognathidae) et Tylosurus sp. (Belonidae). Nous fournissons une description complète des mâles et des femelles de R. laterospinosus, de la microanalyse des métaux des crochets à l’aide d’EDAX, et de ses micropores. Les spécimens de cette espèce ont généralement des épines latérales du tronc reliant l’anneau antérieur d’épines avec un champ postérieur d’épines ventrales et un proboscis avec 15–19 rangées longitudinales alternées de 21 à 26 crochets, variant avec l’espèce hôte. Nous démontrons l’effet des espèces hôtes sur la distribution et la taille du *Corresponding author: omaramin@aol.com This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 O.M. Amin et al.: Parasite 2019, 26, 14 tronc, du proboscis, des crochets du proboscis, des épines du tronc et des structures de reproduction. Le profil moléculaire de cet acanthocéphale, basé sur les gènes de l’ADNr 18S et de cox1, forme un groupe avec d’autres espèces de Rhadinorhynchus et semble en outre confirmer la paraphylie du genre, ce qui est discuté. Introduction were then mounted in Canada balsam. Measurements are in micrometers, unless otherwise noted; the range is followed by Most of the recent taxonomic work on the Acanthocephala the mean values between parentheses. Width measurements from Vietnam has been reported by the Amin-Heckmann-Ha represent maximum width. Trunk length does not include team since 2000. A number of acanthocephalan species from proboscis, neck, or bursa. freshwater and marine fish, amphibians, reptiles, birds, and Line drawings were created by using a Ken-A-Vision micro- mammals were previously described in Vietnam [3, 9–13, 16]. projector (Ward’s Biological Supply Co., Rochester, New Additionally, 11 species of acanthocephalans were collected York), which uses cool quartz iodine 150 W illumination with from marine fish off the eastern seaboard of Vietnam in 10, 20, and 43 objective lenses. Images of stained whole Halong Bay in 2008 and 2009. Of these, six new species of mounted specimens were projected vertically on 300 series Neoechinorhynchus Stiles & Hassall 1905, one new species Bristol draft paper (Starthmore, Westfield, Massachusetts), then of Heterosentis Van Cleave, 1931, and two new species of traced and inked with India ink. Projected images were identical Rhadinorhynchus Lühe 1911 were described [8, 14, 15]. Four to the actual specimens being projected. other species of Echinorhynchid acanthocephalans from marine Specimens were deposited in the University of Nebraska’s fishes in Halong Bay were described [4] and five other new spe- State Museum’s Harold W. Manter Laboratory (HWML) cies from fishes and amphibians of eight collected host species collection in Lincoln, Nebraska, USA. Accession numbers are were also described. Three other species of Rhadinorhynchus noted in Table 1. and one species of Gorgorhynchus were otherwise previously reported from marine fishes in Vietnam by other observers [19]. Fifteen species of acanthocephalans in five families were Scanning electron microscopy (SEM) more recently collected from fishes on the Pacific coast and amphibians in central Vietnam in 2016 and 2017. In the present About 15 specimens from four host species that had been report, we describe males and females of R. laterospinosus, fixed and stored in 70% ethanol were processed for SEM which was originally described from a single female specimen, following standard methods [36]. These included critical point from extensive collections of fishes along the Pacific coast of drying (CPD) in sample baskets and mounting on SEM sample Vietnam and provide a molecular profile of that species based mounts (stubs) using conductive double sided carbon tape. on small subunit ribosomal DNA (18S rDNA) and partial mito- Samples were coated with gold and palladium for 3 min using chondrial cytochrome c oxidase 1 (cox1) genes. Furthermore, a Polaron #3500 sputter coater (Quorum (Q150 TES) www. its phylogenetic relationships with other Rhadinorhynchus quorumtech.com) establishing an approximate thickness of and closest-related species are analyzed and discussed. 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 Nano- lab software system (FEI, Hillsboro, Oregon) and then stored Materials and methods on a USB for future reference. Samples were received under Collections low vacuum conditions using 10 kV, spot size 2, 0.7 Torr using a GSE detector. Collections of 215 specimens of R. laterospinosus from nine species of fish in six families in 2016 and 2017 along the Pacific coast of Vietnam are detailed in Table 1 along with EDXA (energy dispersive X-ray analysis) infection parameters, geographical locations and museum Standard methods were used for preparation, similar to the numbers of deposited material at the Harold W. Manter SEM procedure. Eight specimens were examined and posi- Laboratory, Nebraska State Museum, Lincoln, Nebraska. tioned with the above SEM instrument which was equipped with a Phoenix energy-dispersive X-ray analyzer (FEI, Methods Hillsboro, Oregon). X-ray spot analysis and live scan analysis were performed at 16 kV with a spot size of five and results Freshly collected acanthocephalans were extended in water were recorded on charts and stored with digital imaging until proboscides were everted and fixed in 70% ethanol for software attached to a computer. The TEAM *(Texture and transport to our Institute of Parasitic Diseases (IPD) in Arizona, Elemental Analytical Microscopy) software system (FEI, Hills- USA for processing and further studies. Worms were punctured boro, Oregon) was used. Data were stored on a USB. The data with a fine needle and subsequently stained in Mayer’s acid included weight percent and atom percent of the detected carmine, destained in 4% hydrochloric acid in 70% ethanol, elements, following correction factors, and were stored on a dehydrated in ascending concentrations of ethanol reaching USB. All figures on the USB can be viewed by contacting 100% (24 h each), and cleared in 100% xylene then in 50% the second author. The hooks were cut and scanned at two posi- Canada balsam and 50% xylene (24 h each). Whole worms tions (tip and middle) with a gallium beam (LIMS) using a dual O.M. Amin et al.: Parasite 2019, 26, 14 3 Table 1. Host and geographic distribution of Rhadinorhynchus laterospinosus in the Pacific Ocean off Vietnam. Hosts No. No. infect. Specimens Date of Location Coordinates HWML exam. (%) (mean) collection (North, South) coll. no. Alectis ciliaris (Bloch) 10 1 (10) 1 (0.1) May, 2016 Nha Trang (S) 12°150 N, 109° 139,496 (Carangidae) African 110 E pompano Auxis rochei (Lacépède) 10 7 (70) 101 (10.1) May, 2016 Nha Trang (S) 12°150 N, 109° 139,488– (Scombridae) Bullet tuna* 110 E 139,491 Auxis thazard (Lacépède) 14 11 (79) 114 (8.1) Jan, 2016, Nha Trang (S) 12°150 N, 109° 139,494, (Scombridae) Frigate tuna* Oct, 2017 110 E 139,495 Balistes sp. (Linn.); type host 2 1 (50) 1 (0.5) holo. May, 2009 Halong Bay (N) 20°510 54.500 N, 49,298 (Balistidae) Trigger fish 106°410 01.800 E Leiognathus equulus Forsskal 11 3 (27) 10 (0.9) allo. Jan, 2016 Hai Phong (N) 20°510 54.500 N, 139,486 (Leiognathidae) Common 106°410 01.800 E pony fish Nha Trang (S) 12°150 N, 109° 139,487 110 E Lutjanus bitaeniatus 3 1 (33) 1 (0.3) May, 2016 Nha Trang (S) 12°150 N, 109° 139,492 (Valenciennes) (Lutjanidae) 110 E Indonesian snapper Megalaspis cordyla (Linn.) 2 1 (50) 1 (0.5) May, 2017 Quang Binh (N) 17°300 N, 106° 139,497 (Carangidae) Torpedo scad 200 E Nuchequula flavaxilla Kimura, 20 7 (35) 10 (0.5) March, 2017 Quang Ninh (N) 21°150 N, 107° 139,493 Kimura, Ikejuma 200 E (Leiognathidae) Yellow- spotted pony fish Tylosurus sp. (Cocco) 2 2(100) 2 (1.0) ? Binh Thuan (S) 10°560 N, 108° (Belonidae) Needle fish 60 E Total 74 34 (46) 215 (2.9) * Measurements were mostly based on specimens from these two hosts. beam scanning electron microscope. The alignment of the hook Kit, and following the manufacturer’s instructions. Partial previous to cutting generated a cross section of the area. nuclear small subunit ribosomal DNA (18S rDNA) and partial fragments of mitochondrial cytochrome c oxidase 1 (cox1) gene Ion sectioning of hooks were amplified (50 lL total volume) using ExcelTaqTM SMOBIOÒ PCR Master Mix (Taiwan) containing: 5 A dual-beam SEM with a gallium (Ga) ion source (GIS) concentrated master mix, that is, a mixture of recombinant was used for the LIMS (Liquid Ion Metal Source) part of the Taq DNA polymerase, reaction buffer, MgCl2 (2 mM), dNTPs process. The gallium beam (LIMS) is a gas injection magnetron (0.2 mM), and enzyme stabilizer; 0.25 lM of each PCR primer sputtering technique whereby the rate of cutting can be regu- and 2 lL of extracted gDNA. Primer pairs and amplification lated. The hooks of six acanthocephalans were centered on conditions used were as follows. the SEM stage and cross-sectioned using a probe current Partial fragments of the 18S rDNA gene were amplified between 0.2 nA and 2.1 nA according to the rate at which using the primers 18SU467F (forward, 50 -ATCCAAGGAAGG- the area is cut. The time of cutting is based on the nature and CAGCAGGC-30 ) and 18SL1310R (reverse, 50 -CTCCACCAA- sensitivity of the tissue. Following the initial cut, the sample CTAAGAACGGC-30 ) [46] under the following thermocycling also goes through a milling process to obtain a smooth surface. conditions: initial denaturation at 94 °C for 3 min followed by The cut was then analyzed with X-ray at the tip, middle, and 40 cycles (denaturation for 30 s at 94 °C, annealing for 45 s base of hooks for chemical ions with an electron beam at 56 °C, and extension for 2 min at 72 °C), and a final extension (Tungsten) to obtain an X-ray spectrum. Results were stored step at 72 °C for 7 min. with the attached imaging software then transferred to a USB Partial fragments of the cox1 gene were amplified using the for future use. The intensity of the GIS was variable according primers LCO1490 (forward, 50 -GGTCAACAAATCATAAA- to the nature of the material being cut. GATATTGG-30 ) and HCO2198 (reverse, 50 -TAAACTT- CAGGGTGACCAAAAAATCA-30 ) [23] under the following Molecular methods thermocycling conditions: initial denaturation at 95 °C for 15 min followed by 40 cycles (denaturation for 5 min at 80 °C, Total genomic DNA was extracted from four specimens of followed by 1 min 30 s at 92 °C, annealing for 1 min at 42 °C, R. laterospinosus from Auxis rochei preserved in 70% ethanol and extension for 2 min at 72 °C), and a final extension step at using a Qiagen™ (Valencia, California, USA) DNeasyÒ Tissue 72 °C for 10 min. 4 O.M. Amin et al.: Parasite 2019, 26, 14 In every PCR run, a negative and a positive control were Rhadinorhynchus laterospinosus Amin, used to detect any potential contamination and to have a reliable Heckmann, Ha, 2011 sample to compare with, respectively. PCR amplicons were sequenced directly for both strands using the same PCR Family: Rhadinorhynchidae Travassos, 1923 primers. Genus: Rhadinorhynchus Lühe 1911 Sequences were assembled and edited using Mega v6 [47] Type host: Triggerfish Balistes sp. (Linn.) (Balistidae). and submitted to GenBank under accession numbers: Other hosts: See Table 1. MK457183 – MK457185 (18S) and MK572741–MK572744 Type locality: Halong Bay (20°510 N, 54.500 E). (cox1). Sequences were aligned using Muscle as implemented Other localities along the Pacific coast of Vietnam: See in MEGA v6 together with published sequences of Rhadi- Table 1. norhynchus and most closely-related published sequences to Specimens: HWML collection no. 49,298 (holotype members of this genus. Rotaria rotatoria (Pallas, 1776) was female) Amin et al., 2011; no. 139,486 (allotype male); nos. used as the outgroup in both the 18S (DQ089736) and cox1 139,487–139,497 of many paratypes from eight other host (EU499879) datasets. Both alignments (18S: 760 nt positions species in Table 1. of which eight were excluded prior to analysis; cox1: 537 nt positions of which 26 were excluded prior to analysis) were Description used for comparative sequence analysis. The SeaView v4 interface [27] was used to select blocks of General: With characters of the genus Rhadinorhynchus evolutionarily conserved sites. Maximum likelihood (ML) and Lühe 1911. Trunk relatively long, uniformly cylindrical, with Bayesian inference (BI) algorithms were used for phylogenetic electron dense micropores that vary in diameter and distribution tree reconstruction after determination of the best-fit model of by region (Figs. 18, 22 and 23), spinose anteriorly in two nucleotide substitution with jModelTest v2.1.4 [22] using the regions bridged with lateral spines within range of proboscis Akaike Information Criterion (AIC) and the Bayesian Informa- receptacle (Figs. 1 and 17). Counts of spines on one side of tion Criterion (BIC), respectively. For the ML algorithm, the trunk. Trunk spines and all other structures vary by position, best-fitting model selected was the GTR + G model (nst = 6, host species and worm sex (Table 2). Trunk spines (Fig. 19) lar- rates = gamma, ngammacat = 4) both for the 18S and cox1 ger in females (Fig. 2) than in males. Anterior spines in com- datasets. In the case of BI, the best-fitting model was plete circle with 1–4 dorsal, 1–4 ventral and 1–4 per circle at TVMef + G (nst = mixed, rates = gamma, ngammacat = 4) middle in males and females (Figs. 1 and 17). Posterior ventral for the 18S dataset and TrN + G (nst = 6, rates = gamma, ngam- spines 0–13 and lateral spines more numerous, 1–31. Length of macat = 4) for the cox1 dataset. ML analyses were performed in anterior trunk spines 31–73. Posterior ventral spines larger, PhyML v3.0 [30] with a non-parametric bootstrap of 100 repli- 32–95 long. Posterior lateral spines 22–82 long. Posterior trunk cates. BI analyses were carried out with MrBayes v3.2.6 [42] spines larger at middle. Proboscis long, cylindrical, straight, on the CIPRES Science Gateway v3.3 [39]. Log likelihoods gradually widening anteriorly (Figs. 1 and 10) with posterior were estimated over 10,000,000 generations using Markov sac-like membrane evaginating into receptacle (Fig. 29), and Chain Monte Carlo (MCMC) searches on two simultaneous 15–19 longitudinal alternating rows of 21–26 hooks each vary- runs of four chains, sampling trees every 1000 generations. ing with host species (Table 2). Dorsal hooks slightly shorter The first 25% of the sampled trees were discarded as “burn- and more slender that stouter and more sharply curved ventral in” and a consensus topology and nodal support estimated as hooks (Figs. 6 and 7). Hooks slightly arched (Fig. 12) with thin posterior probability values [35] were calculated from the grooved cortical layer and thick core (Figs. 13 and 14), smallest remaining trees. Pairwise genetic distance matrices were anteriorly, largest at middle, gradually smaller posteriorly calculated using the “uncorrected p-distance” model imple- except at basal circle of abruptly larger hooks (Figs. 10–12 mented in MEGA v6. and 29). Hook roots simple, markedly shorter than blades, directed posteriorly (Figs. 6 and 7). Neck prominent, slightly longer than wide posteriorly, with paired sensory pores Results (Figs. 15, 16 and 29). Proboscis receptacle double-walled, about twice as long as proboscis with cephalic ganglion near Rhadinorhynchus laterospinosus was originally described its middle. Lemnisci digitiform, equal, uniformly broad from one female specimen collected from an individual trigger- throughout, slightly shorter than receptacle (Fig. 1). Gonopore fish, Balistes sp. (Linn.) (Balistidae), from the Pacific coast at terminal in males but subterminal in females at level of poste- Halong Bay in May of 2009. It has since been found in eight rior abrupt narrowing of trunk. other species of fish in five other families along the Pacific coast Males (based on 30 adults with sperm from A. rochei and of Vietnam from the north at Hai Phong and Quang Binh to the A. thazard). Trunk 4.75–11.25 (7.04) mm long by 0.35–0.80 south at Nha Trang and Binh Thuan (Table 1). We have studied (0.56) mm wide at middle. See Table 2 for position, distribution specimens from all host species but provide measurements of and sizes of trunk spines. Proboscis 1.00–1.67 (1.30) long by specimens from the more extensive collections from two hosts, 0.17–0.23 (0.20) mm wide anteriorly. See Table 2 for measure- Auxis rochei (Lacépède) and Auxis thazard (Lacépède). ments of proboscis hooks. Neck 200–350 (277) long by 175– The description is inclusive of morphometric differences noted 250 (217) wide posteriorly. Proboscis receptacle 1.62–3.45 between specimens collected from these two-host species (2.34) mm long by 0.14–0.35 (0.23) mm wide. Lemnisci (Table 2). 1.50–2.50 (1.87) mm long by 0.11–0.23 (0.15) mm wide. Table 2. The relationship between host species and size of certain anatomical structures of measured specimens of Rhadinorhynchus laterospinosus collected off the Pacific coast of Vietnam in 2016. Worm sex Character Host species Auxis thazard (n = 12 males, 15 females) Auxis rochei (n = 18 males, 18 females) Male Trunk length (mm) 5.75–11.25 (8.09) 4.75–8.37 (6.33) Female Trunk length (mm) 7.80–26.25 (16.01) 8.00–21.25 (13.16) Male No. ant. trunk spines in row (dorsal, mid, vent.)* 1–3 (2), 2–4 (3), 1–2 (2) 1–3 (2), 2–4 (3), 1–2 (2) Female No. ant. trunk spines in row (dorsal, mid, vent.) 1–4 (2), 1–4 (3), 2–4 (3) 2–4 (3), 3–5 (4), 2–5 (3) Male No. post. trunk spines in row (vent., lateral) 1–4 (3), 5–12 (8) 0–8 (3), 2–23 (9) Female No. post. trunk spines in row (vent., lateral) 0–13 (6), 1–19 (11) 5–16 (10), 5–31 (17) Male L of ant. trunk spines (dorsal, mid, vent.) 42–62 (50), 31–42 (36), 21–63 (42) 31–73 (51), 31–52 (43), 31–62 (46) O.M. Amin et al.: Parasite 2019, 26, 14 Female L of ant. trunk spines (dorsal, mid, vent.) 52–86 (64), 42–73 (54), 51–62 (56) 48–72 (60), 31–62 (48), 46–61 (53) Male L of ventral post. trunk spines (ant., mid, post.) 40–62 (50), 42–66 (52), 20–42 (37) 32–73 (54), 51–83 (63), 40–72 (64) Female L of ventral post. trunk spines (ant., mid, post.) 42–81 (63), 62–96 (79), 42–73 (59) 52–84 (64), 61–95 (78), 51–95 (67) Male L of lateral post. trunk spines (ant., mid, post.) 22–32 (29), 31–52 (44), 31–43 (37) 41–52 (40), 40–72 (47), 30–62 (40) Female L of lateral post. trunk spines (ant., mid, post.) 31–72 (57), 63–86 (65), 42–70 (54) 41–73 (52), 62–82 (68), 41–73 (55) Female Proboscis length (mm) 1.25–1.82 (1.55) 1.45–1.90 (1.71) Proboscis hook rows 15–17 (16.4) 15–19 (17.3) Hook length Dorsal Ventral Dorsal Ventral Male Apical prob. hook L 37–40 (39)  7–10 (9) 42–50 (46)  11–12 (12) 35–47 (39)  7–11 (10) 37–50 (44)  11–12 (11) Male Subapical prob. hook L  W 45–55 (51)  10–15 (12) 52–62 (58)  12–17 (15) 42–60 (53)  10–12 (12) 47–63 (55)  12–15 (13) Male Mid prob. hook L  W 55–60 (58)  12–14 (13) 60–67 (65)  14–18 (16) 52–62 (59)  12 60–77 (67)  13–18 (15) Male Post. prob. hook L  W 35–37 (36)  8–11 (10) 37–47 (42)  11–13 (12) 27–42 (35)  7–10 (8) 35–50 (41)  9–12 (11) Male Basal prob. hook L  W 50–60 (53)  11–15 (13) 56–72 (62)  13–17 (15) 47–55 (51)  10–14 (11) 55–72 (63)  12–17 (14) Female Apical prob. hook L  W 50–60 (54)  10–12 (11) 52–65 (59)  11–15 (13) 45–52 (48)  10–15 (12) 45–60 (53)  12–15 (14) Female Subapical prob. hook L  W 60–65 (62)  13–17 (15) 63–70 (66)  15–17 (17) 62–72 (65)  12–20 (15) 60–70 (66)  14–20 (16) Female Mid prob. hook L  W 70–77 (73)  13–17 (15) 73–80 (77)  20–22 (21) 70–77 (73)  12–15 (14) 75–78 (76)  18–20 (19) Female Post. prob. hook L  W 40–45 (42)  10–11 (10) 42–50 (48)  10–15 (12) 32–45 (40)  10–12 (11) 50–57 (54)  10–15 (13) Female Basal prob. hook L  W 65–72 (68)  10–15 (12) 72–82 (75)  15–17 (16) 57–82 (68)  12–17 (14) 75–87 (79)  12–18 (16) Male Prob. Recept. L  W (mm) 2.08–3.45 (2.45)  0.14–0.35 (0.25) 1.62–2.62 (2.28)  0.15–0.30 (0.21) Male Anterior testis (mm) 0.69–1.62 (1.19)  0.31–0.50 (0.40) 0.59–1.75 (0.98)  0.22–0.52 (0.35) Male Posterior testis (mm) 0.52–1.25 (0.99)  0.25–0.57 (0.42) 0.47–1.50 (0.84)  0.22–0.47 (0.33) Male Ant. cement glands (mm) 0.78–1.04 (0.95)  0.17–0.32 (0.24) 0.31–1.09 (0.64)  0.10–0.27 (0.18) Male Post. cement glands (mm) 0.78–1.25 (1.05)  0.17–0.26 (0.21) 0.36–1.09 (0.62)  0.14–0.26 (0.16) Male Saefftigen’s pouch (mm) 0.83–1.27 (1.08)  0.18–0.27 (0.22) 0.42–1.00 (0.78)  0.15–0.26 (0.20) * All observations of trunk spines are made on one side of the trunk. 5 6 O.M. Amin et al.: Parasite 2019, 26, 14 Figures 1–9. Line drawings of whole mounted specimens of Rhadinorhynchus laterospinosus from Auxis rochei and Auxis thazard in the Pacific Ocean off Vietnam. (1) A paratype male showing the anteriorly enlarged proboscis, the distribution of trunk spines within the range of the long receptacle, and the posterior distribution of the reproductive system. (2) A posterior ventral trunk spine of a female specimen. (3) Detailed male reproductive system. Note the large tubular giant nuclei of the cement glands and the posterior extension of the cement gland ducts surrounding Saefftigen’s pouch anteriorly. (4) A ripe egg. (5) Detail of the uterine bell of the female specimen shown in Figure 9. Note the inner paired rod-like structures. (6, 7) Dorsal (Fig. 6) and ventral (Fig. 7) hooks at the mid proboscis of a female specimen. Note differences in the thickness, length, and curvature of dorsal vs. ventral hooks. (8) Detail of the vagina from Figure 9. Note the inner muscular plug lining of the posterior tip of the trunk. (9) A complete female reproductive system characterized by the long and wide uterus. O.M. Amin et al.: Parasite 2019, 26, 14 7 Figures 10–15. SEM of specimens of Rhadinorhynchus laterospinosus from Auxis rochei and Auxis thazard in the Pacific Ocean off Vietnam. (10) The proboscis of a female specimen. (11) The apical end of the proboscis in Figure 10 showing the smaller apical hooks, the organization of hook rows and no external evidence of an apical organ. (12) A typical example of hook shape and orientation from the midsection of a proboscis. (13) A magnified view of a hook showing its surface serrations. (14) A broken hook demonstrating its thick core and thin cortical layer. (15) Posterior end of a proboscis showing the larger hooks in the posterior circle and a sensory pit. 8 O.M. Amin et al.: Parasite 2019, 26, 14 Reproductive system in posterior half of trunk in contiguous posterior cement glands, and Saefftigen’s pouch, but relatively structures with genitalia opening into bursa. Testes ovoid; ante- fewer and smaller trunk spines than specimens from A. rochei rior testis 0.59–1.75 (1.05) mm long by 0.22–0.52 (0.36) mm (Table 2). wide, larger than posterior testis 0.47–1.50 (0.89) mm long by 0.22–0.57 (0.36) wide. Cement glands four, rod-shaped, in two contiguous pairs, each with one tubular giant nucleus Energy dispersive X-ray analysis (EDXA) (Figs. 1, 3 and 30). Anterior glands 0.31–1.09 (0.73) mm long We report the X-ray scans and metal composition of large and by 0.14–0.32 (0.20) wide; posterior glands 0.36–1.25 small proboscis hooks (Figs. 34, 35 and Tables 3, 4) and trunk (0.74) mm long by 0.11–0.26 (0.17) mm wide. Individual spines (Table 5 and Fig. 36) of R. laterospinosus that were cut cement gland ducts surround prominent fusiform Saefftigen’s with a gallium beam (LMIS) and viewed with a dual beam pouch, 0.42–1.27 (0.89) mm long by 0.15–0.26 (0.21) mm scanning electron microscope with X-ray capabilities (EDXA). wide, anteriorly (Fig. 3) and joining its genital terminalia There are variable levels of calcium, phosphorus, and sulfur (Figs. 26 and 31) at thick-walled bursa (Figs. 24 and 25). Bursa depending on the type of hook and whether readings are made with many elaborate sensory papillae (Fig. 27) at center and in at the base, core, tip or edge of hooks. Other common ele- outer rings (Figs. 25 and 33), 208–775 (569) long by 416–831 ments of living organisms (carbon and oxygen) and elements (625) wide. used for specimen preparation (gallium, palladium, gold) are Females (based on 32 mature females with eggs and ovar- not included in the analysis. In large hooks, the calcium and ian balls from A. rochei and A. thazard). Trunk 7.80–21.25 phosphorus levels were highest at the center of the hook base (14.41) mm long by 0.35–1.00 (0.59) mm wide at middle. (Table 3 and Fig. 34). In small hooks, calcium and phosphorus See Table 2 for position, distribution and sizes of trunk spines. were highest at hook tips (Table 4 and Fig. 35). Sulfur was high Proboscis 1.25–1.90 (1.64) mm long by 0.17–0.30 (0.24) mm in both spine tip and base compared to calcium and phosphorus wide anteriorly. See Table 2 for measurements of proboscis (Table 5, Fig. 36). hooks. Neck 200–375 (315) long by 109–300 (315) long by 109–300 (233) wide posteriorly. Neck 200–375 (315) long by 109–300 (233) wide posteriorly. Proboscis receptacle Molecular results 2.24–3.95 (3.14) mm long by 0.17–0.37 (0.25) mm wide. Lemnisci 2.29–3.64 (2.83) mm long by 0.14–0.27 (0.19) wide. Three partial 18S rDNA (741–767 nt) and four cox1 Posterior end bluntly pointed with subterminal laterally slit (606–622 nt) sequences were generated from four adult lipless gonopore (Figs. 20). Reproductive system 2.18–5.50 specimens (two males and two females) of R. laterospinosus. (3.67) mm long; 25% of trunk length (Fig. 9) with well- While 18S rDNA sequences were identical (only the longest developed vagina (Figs. 8 and 28), very long and broad uterus, one was thus included in the corresponding phylogenetic trees), small and elongated uterine bell with unequal walls and two intraspecific sequence divergence for cox1 ranged between central rod-shaped elongate tubes (Figs. 5 and 32) extending 0.008 and 0.018% (5–11 nt difference). into body cavity, and no uterine bell glands. Eggs fusiform with Table 6 provides data for the sequences retrieved from prominent polar prolongation of fertilization membrane (Figs. 4 GenBank and used in the phylogenetic analyses based on the and 21), 57–68 (63) long by 12–18 (15) wide. two alignments. While both ML and BI algorithms produced trees with identical topology for the 18S gene (Fig. 37), a Remarks slightly different topology was observed for the cox1 gene (Figs. 38 and 39). The present report represents an expansion of our under- Phylogenetic analyses based on 18S rDNA gene demon- standing of R. laterospinosus since its description from only strated the strong association between R. laterospinosus and one female in 2011 [8] from a trigger fish, Balistes sp. from other representatives of the genus (R. pristis) with maximum the northern Pacific coast of Vietnam at Cat Ba Island, Halong support (0.000–0.013%, 0–10 nt difference), but also with a Bay, Gulf of Tonkin. The single female had a proboscis with sequence classified as Gymnorhadinorhynchus sp., which 18 longitudinal rows of 24 hooks each, and eight ventral and showed no differences with the present sequence (0%, 0 nt). 18 lateral spines in the posterior field of trunk spines connecting Sequences belonging to Gymnorhadinorhynchus decapteri anteriorly with the anterior field of trunk spines. The collec- Braicovich, Lanfranchi, Farber, Marvaldi, Luque and Timi, tion of over 200 specimens from eight additional hosts along 2014 and Transvena annulospinosa Pichelin and Cribb, 2001 the Pacific coast of Vietnam provided an opportunity to were also included in this clade (0.024–0.039%, 18–29 nt describe males, lemnisci, the female reproductive system, and difference from newly generated sequences). Two sequences eggs for the first time, and to clarify the dorso-ventral belonging to R. pristis (Rudolphi, 1802) and R. lintoni Cable differentiation of proboscis hooks that were inaccurately and Linderoth, 1963 (0.184–0.185%, 138–139 nt difference declared as “similar in shape and size, and in their posteriorly from newly generated ones) remained on a separate clade and directed angle of projection from proboscis” [8] with the were strongly associated with members of Pomphorhynchus. availability of more specimens for study. The new description According to phylogenetic analyses based on the cox1 gene, made it possible to examine the relationship between host the four newly generated sequences for the R. laterospinosus species and the expression of certain morphometric parameters. grouped, with low support, with a clade formed by representa- Specimens from A. thazard had larger size of trunk, some tives of Bolbosoma and the species Neorhadinorhynchus nudus proboscis hooks, proboscis receptacle, testes, anterior and (Harada, 1938) Yamaguti, 1939 (0.076–0.141%, 39–72 nt O.M. Amin et al.: Parasite 2019, 26, 14 9 Figures 16–21. SEM of specimens of Rhadinorhynchus laterospinosus from Auxis rochei and Auxis thazard in the Pacific Ocean off Vietnam. (16) A larger magnification of the neck sensory pit showing no rim outline. (17) The anterior end of two specimens showing the posterior zone of ventral and lateral spines. (18) Micropores at the anterior part of the trunk. (19) A high magnification of a trunk spine. (20) The bluntly pointed posterior end of a female specimen showing the sub-ventral gonopore. (21) A small cluster of eggs. 10 O.M. Amin et al.: Parasite 2019, 26, 14 Figures 22–27. SEM of specimens of Rhadinorhynchus laterospinosus from Auxis rochei and Auxis thazard in the Pacific Ocean off Vietnam. (22, 23) Microspores from the middle and posterior parts of the trunk, respectively. Note the different density and diameter of the pores, also compared with Figure 18 related to differential absorption rates. (24) A lateral view of the bursa. (25) A ventrolateral view of a bursa showing its thick muscular margin and the organization of the outer circle and the central cluster of sensory papillae. (26) A high magnification of the center of the bursa showing the terminal genitalia surrounded by close circles of sensory papillae. This organization is species-specific. (27) A higher magnification of one sensory papilla made up of small units embedded in elliptic depression. O.M. Amin et al.: Parasite 2019, 26, 14 11 Figures 28–33. Microscopical images of some internal structures as seen in their natural state not readily demonstrable in line drawings of specimens of Rhadinorhynchus laterospinosus from Auxis rochei and Auxis thazard in the Pacific Ocean off Vietnam. (28) A sub-ventral vagina at the constriction of posterior end of trunk of a female. (29) The posterior loop of a thin sac (arrow) emerging from the insertion of the proboscis receptacle at the base of the proboscis. (30) The four tubular cement glands with their long nuclei just anterior to Saefftigen’s pouch. (31) The penis emerging from the bursa of one specimen. (32) Uterine bell in one female. Note the unequal sides of the bell. (33) The posterior end of one male showing the bursa with rings of sensory papillae (arrow). 12 O.M. Amin et al.: Parasite 2019, 26, 14 Figure 34. Energy Dispersive X-ray spectrum of the base center of a large hook of a Rhadinorhynchus laterospinosus specimen showing high levels of calcium and phosphorus (see Table 3). Insert: SEM of a lateral and cross gallium cut hook. difference), and remained apart from a third clade which size of trunk, proboscis, longest proboscis hooks, receptacle, included the only available published sequence on this gene testes, lemnisci, and eggs. The taxonomic implications of this for Rhadinorhynchus (0.238–0.242%, 122–124 nt difference variability were discussed (Amin and Redlin, 1980). Earlier, from newly generated sequences). Amin [1] demonstrated a similar relationship for Acantho- cephalus dirus (Van Cleave, 1931) Van Cleave and Townsend, 1936 in Wisconsin fishes. Females of the same developmental Discussion stage recovered during the same period were found to have Morphometric comparisons attained larger sizes in certain hosts than in others with the largest females being found in Lepomis macrochirus The observed relationship between host species and size Rafinesque. The size of the trunk in males was also found to and even shape of acanthocephalans observed in this study follow the same pattern. Similarly, testes also attained a larger (Table 2) has been previously demonstrated for other acantho- size in males recovered from Catostomus commersonii cephalans including Echinorhynchus salmonis Müller, 1784 Lacépède (Catostomidae) than in males from Semotilus whose variability in the size of taxonomically important struc- atromaculatus (Mitchill) (Cyprinidae). Amin [1] stated that tures such as the trunk, proboscis hooks, proboscis, testes, etc. these size variations “result from differential growth rates of has been attributed to host species. Such relationships have these worms in the various host intestinal environments (and) been reported in Lake Michigan where male and female spec- are probably mediated by certain host specific factors.” imens from bloater, Coregonus hoyi (Gill) (Salmonidae) achieved not only larger size but also different body form Distribution (broad anteriorly) compared to the slender specimens from rainbow smelt, Osmerus mordax (Mitchell) (Osmeridae) [17]. Amin [2] and Amin et al. [8] recognized 38 valid species The larger and heavier worms from bloater invariably showed of Rhadinorhynchus and invalidated 30 others. Only five a higher regression coefficient (adjusted coefficient of determi- more species of Rhadinorhynchus were described since, four nation) compared to those from smelt in all characters including from marine fishes off Australia [43] and Rhadinorhynchus O.M. Amin et al.: Parasite 2019, 26, 14 13 Figure 35. Energy Dispersive X-ray spectrum of the tip of a small hook of a Rhadinorhynchus laterospinosus specimen showing high levels of calcium and phosphorus but less calcium than large hooks (see Table 4). Insert: SEM of posterior hooks and hook tips in cross gallium cuts. Table 3. X-ray scans for chemical elements of a Gallium cut (LMIS) large hook of Rhadinorhynchus laterospinosus. Elements* Hook tip edge Hook tip center Mid hook edge Mid hook center Hook base edge Hook base center Magnesium (Mg) 0.00 0.00 0.15 0.59 0.33 0.42 Phosphorus (P) 0.96 0.87 3.27 9.78 7.21 14.87 Sulfur (S) 11.96 15.39 16.59 8.96 12.61 0.00 Calcium (Ca) 2.02 2.04 6.64 22.11 13.90 45.30 * Common protoplasmic elements (C, N, O) as well as processing and coating elements (Pd, Au, Ga) are not included. List in cut%. Table 4. X-ray scans for chemical elements of a Gallium cut small Table 5. X-ray scans for chemical elements of a Gallium cut spine hook at the base of the proboscis of Rhadinorhynchus laterospinosus. of Rhadinorhynchus laterospinosus. Elements* Hook Hook Hook base Elements* Spine tip Spine base tip edge tip center edge Magnesium (Mg) 2.07 0.49 Magnesium (Mg) 0.01 1.23 0.02 Phosphorus (P) 4.46 3.67 Phosphorus (P) 1.61 14.22 5.00 Sulfur (S) 18.23 11.64 Sulfur (S) 17.88 1.18 17.65 Calcium (Ca) 4.63 3.48 Calcium (Ca) 2.35 30.57 9.82 * Common protoplasmic elements (C, N, O) as well as process- * Common protoplasmic elements (C, N, O) as well as processing and ing and coating elements (Pd, Au, Ga) are not included. List coating elements (Pd, Au, Ga) are not included. List in cut%. in cut%. 14 O.M. Amin et al.: Parasite 2019, 26, 14 Figure 36. Energy Dispersive X-ray spectrum of the tip of a trunk spine of a Rhadinorhynchus laterospinosus specimen showing high levels of sulfur (see Table 5). Insert: SEM of a spine in lateral gallium cut. oligospinosus Amin and Heckmann, 2017 off the Peruvian 10. Rhadinorhynchus laterospinosus Amin, Heckmann, Ha, Pacific coast. The 43 valid species of Rhadinorhynchus include 2011 from Halong Bay, Vietnam. 20 species from the Pacific Ocean, especially off Australia, 11. Rhadinorhynchus oligospinosus n. sp. from Port of Japan, and Vietnam. These species are: Chicama, La Libertad, Peru. 12. Rhadinorhynchus ornatus Van Cleave, 1918 from the Atlantic coast of the USA, Japan, and the Pacific Ocean 1. Rhadinorhynchus bicircumspinus Hooper, 1983 from off South America. New South Wales, Australia. 13. Rhadinorhynchus pichelinae Smales, 2014 from Point 2. Rhadinorhynchus biformis Smales, 2014 from Heron Peron, Western Australia. Island, Australia. 14. Rhadinorhynchus polydactyli Smales, 2014 from 3. Rhadinorhynchus carangis Yamaguti, 1939 from the Moreton Bay, Queensland, Australia. Seto Inland Sea, Japan. 15. Rhadinorhynchus polynemi Gupta and Lata, 1967 from 4. Rhadinorhynchus chongmingnensis Huang, Zheng, India and north-east Australia. Deng, Fan et Ni, 1988 from Chongming, China. 16. Rhadinorhynchus pomatomi Smales, 2014 from New 5. Rhadinorhynchus cololabis Laurs et McCauley, 1964 Brighton, New South Wales, Australia. from Oregon, USA. 6. Rhadinorhynchus decapteri Parukhin et Kovalenko, 1976 17. Rhadinorhynchus selkirki Van Cleave, 1920 from Juan from Hawaii. Fernandez Island, Chili. 7. Rhadinorhynchus ditrematis Yamaguti, 1939 from the 18. Rhadinorhynchus seriolae (Yamaguti, 1963) Golvan, Seto Inland Sea, Japan. 1969 from Japan and Australia. 8. Rhadinorhynchus dorsoventrospinosus Amin, 19. Rhadinorhynchus trachuri Harada, 1935 from a Tokyo Heckmann, Ha 2011 from Halong Bay, Vietnam. market, Japan. 9. Rhadinorhynchus johnstoni Golvan, 1969 from South 20. Rhadinorhynchus zhukovi Golvan, 1969 from the Kuril Australia. Islands, Japan–Russia. Table 6. Data for the sequences belonging to Paleacanthocephala retrieved from GenBank and included in the phylogenetic analyses. Order (Family) Species GenBank ID Location Reference 18S cox1 Echinorhynchida Rhadinorhynchus pristis KR349117 Minho and Mondego rivers Bao et al. [20] (Rhadinorhynchidae) (Rudolphi, 1802) (Western Iberian Peninsula) JQ061133 Atlantic Ocean (Vigo, Spain) Gregori et al. [29] JX014226 Indian Ocean Verweyen et al. [48] (Java, Indonesia) Rhadinorhynchus lintoni JX014224 Pacific Ocean (Hawaii, USA) Verweyen et al. [48] O.M. Amin et al.: Parasite 2019, 26, 14 Cable and Linderoth, 1963 Rhadinorhynchus sp. AY062433 Unknown García-Varela et al. [24] Rhadinorhynchus sp. DQ089712 Unknown García-Varela and Nadler [26] Echinorhynchida Neorhadinorhynchus nudus MG757445 Pacific Ocean – South China Sea Li et al. [38] (Cavisomidae) (Harada, 1938) Yamaguti, 1939 (Shanwei, China) Echinorhynchida Gymnorhadinorhynchus decapteri KJ590123 KJ590125 Atlantic Ocean (Cabo Frío, Brazil) Braicovich et al. [21] (Gymnorhadinorhynchidae) Braicovich, Lanfranchi, Farber, Marvaldi, Luque and Timi, 2014 Gymnorhadinorhynchus sp. MK014866 Pacific Ocean (Japan) Steinauer et al. [45] Echinorhynchida Transvena annulospinosa AY830153 Unknown García-Varela and (Transvenidae) Pichelin and Cribb, 2001 Nadler [25] DQ089711 Unknown García-Varela and Nadler [26] Echinorhynchida Pomphorhynchus laevis JX014223 Atlantic Ocean – Baltic Sea Verweyen et al. [48] (Pomphorhynchidae) (Zoega in Müller, 1776) AY423346 Ouche river (Dijon, France) Perrot-Minot [41] Pomphorhynchus tereticollis AY423347 Ouche river (Dijon, France) Perrot-Minot [41] (Rudolphi, 1809) Pomphorhynchus zhoushanensis KY490051 Pacific Ocean – South China Sea Li et al. [37] Li, Chen, Amin and Yang, 2017 (Zhoushan Islands, China) Polymorphida Bolbosoma balaenae (Gmelin, 1790) JQ040303 Atlantic Ocean (Vigo, Spain) Gregori et al. [28] (Polymorphidae) Bolbosoma sp. KX098556 Atlantic Ocean – Gulf of Mexico Andres et al. [18] 15 16 O.M. Amin et al.: Parasite 2019, 26, 14 Figure 37. Bayesian inference (BI) phylogram reconstructed using a newly generated 18S rDNA sequence for Rhadinorhynchus laterospinosus and retrieved sequences from GenBank for Rhadinorhynchus and the closest-related sequences to members of this genus. Outgroup: Rotaria rotatoria. Nodal support from maximum likelihood (ML) and Bayesian inference (BI) analyses are indicated as ML/BI. Bootstrap values lower than 70 and posterior probability values lower than 0.95 are omitted. The scale-bar indicates the expected number of substitutions per site. Morphological comparisons comparable to the inner core of mammalian teeth [6]. Sulfur levelsshowed a higher differential concentration at the edge Morphologically, Rhadinorhynchus stunkardi Gupta et than the middle of cut hooks (Tables 3 and 4). This element Fatma, 1987 from India is the only other species of Rhadi- is part of the prominent outer layer of most acanthocephalan norhynchus that has lateral trunk spines connecting the anterior hooks and is a major contributor to the hardening process of and posterior fields of trunk spines like R. laterospinosus. this attachment structure. There is a difference in the distribu- Rhadinorhynchus stunkardi, however, has only 3–4 posterior tion of calcium ions in the smaller hooks in relation to large trunk spines on the ventral side, only 8–10 proboscis hook rows hooks, this level being highest in the core and base of large each with 24–26 small hooks that reach a maximum length of hooks (45/30%) but highest at the tip of small hooks only 46, considerably larger eggs, 120–150  25–28, and a (30.57%) (Tables 3 and 4). A similar EDAX study of the pro- terminal gonopore [31]. boscis hooks of Echinorhynchus baeri Kostylew, 1928 showed that large hooks have higher calcium, phosphorus, and sulfur Energy dispersive X-ray analysis (EDXA) than miniature rootless hooks [6]. Comparable patterns for the numerous trunk spine gallium cuts (Table 5) demonstrate The results of the X-ray analysis (Tables 3–5 and the rigid nature of the spine which is explained by the X-ray Figs. 34–36) of gallium cut hooks and spines of R. laterospino- scans (Fig. 36). There is a reasonably high level of phosphorus, sus show that the large hooks in the mid-proboscis and the calcium and especially sulfur at the tip (18.23%) and base small posterior hooks had a high level of sulfur at the tip edge, (11.64%) of the spine, which have mineralized to form the rigid which is consistent with the base of the hooks. This element support. The X-ray scans of the gallium cut hooks and spines along with calcium and phosphorus aid in the mineralization help explain the morphological nature of R. laterospinosus and hardening of the outer layer of hooks, similar to the enamel and identify its unique “personality” [44]. The uniqueness of layer of the mammalian tooth (calcium phosphate apatite) [32]. the metal analysis as expressed by X-ray scans appears to be The base center of large hooks shows increased levels of species-specific and can be regarded as a fingerprint of key calcium (45.30%) and phosphate ions (14.87) (Table 3), diagnostic value that is just as important as molecular analysis. O.M. Amin et al.: Parasite 2019, 26, 14 17 Figure 38. Maximum likelihood (ML) phylogram reconstructed using four newly generated cox1 sequences for Rhadinorhynchus laterospinosus and retrieved sequences from GenBank for Rhadinorhynchus and the closest-related sequences to members of this genus. Outgroup: Rotaria rotatoria. Bootstrap values lower than 70 are omitted. The scale-bar indicates the expected number of substitutions per site. Figure 39. Bayesian inference (BI) phylogram reconstructed using four newly generated cox1 sequences for Rhadinorhynchus laterospinosus and retrieved sequences from GenBank for Rhadinorhynchus and the closest-related sequences to members of this genus. Outgroup: Rotaria rotatoria. Posterior probability values lower than 0.95 are omitted. The scale-bar indicates the expected number of substitutions per site. 18 O.M. Amin et al.: Parasite 2019, 26, 14 This was well demonstrated in the study of Rhadinorhynchus clade has recently been classified into the genus Gymnorhadi- oligospinosus Amin and Heckmann, 2017 from mackerels in norhynchus [45]. The null difference between this sequence the Pacific Ocean off Peru [5], among others. and the newly generated one for R. laterospinosus points to a need for reclassification of this Gymnorhadinorhynchus sp. specimen most probably into the genus Rhadinorhynchus. Micropores The outcome of the phylogenetic analysis based on the cox1 The presence of micropores on various trunk regions of gene is less complete than the 18S rDNA-based one due to the specimens of R. laterospinosus (Figs. 18, 22 and 23) suggests near absence of cox1 gene sequences for Rhadinorhynchus in differential nutrient absorption related to the diameter and dis- GenBank. Even so, it shows conflictive relationships for tribution of micropores as appears to be the case in practically members of this genus, with present sequences forming a sub- all acanthocephalans. We have documented this phenomenon clade within a group including Bolbosoma members and in 16 species of acanthocephalans [33] and a few more since. N. nudus, apart from the group formed by Rhadinorhynchus The functional aspects of micropores in a few other acantho- sp., T. annulospinosa, and G. decapteri. Although the goal of cephalan species including Rhadinorhynchus ornatus Van the present study is not to discuss the higher level classification Cleave, 1918, Polymorphus minutus (Goeze, 1782) Lühe, of Paleacanthocephala, the inclusion of the echinorhynchid 1911, Moniliformis moniliformis (Bremser, 1811) Travassos N. nudus within the Polymorphidae (i.e. Bolbosoma) further (1915), Macracanthorhynchus hirudinaceus (Pallas, 1781) demonstrates the extent of these inconsistencies at the supra- Travassos (1916, 1917), and Sclerocollum rubrimaris Schmidt familiar level. In fact, the paraphyly within the palaeacantho- and Paperna, 1978 were reviewed earlier [7]. The micropore cephalan at the family level is well established [25, 34, 40, canals appear to be continuous with canalicular crypts that con- 48], which highlights the existing problems with their taxonomic stitute a huge increase in external surface area implicated in arrangement and points to the need for a reclassification based nutrient uptake [7]. on better morphological, ecological and molecular characteriza- tion of their members. To summarize, following the 18S rDNA-based analysis, a Molecular analysis single clade including all the valid species of the genus Rhadinorhynchus described up until now is recognized. To date, genetic data have been provided for only three However, Rhadinorhynchus relationships in phylogenetic anal- species of Rhadinorhynchus: R. laterospinosus (present results), ysis based on cox1 sequences are not so clear, mostly due to the R. pristis, and R. lintoni (see Table 6 for references). The scar- lack of published sequences of this gene so far. Conflicting city of molecular profiles described for this genus poses relationships with other genera (i.e. Gymnorhadinorhynchus, difficulties for correctly determining relationships among its Transvena, Bolbosoma and Neorhadinorhynchus) are apparent members and with other genera, and adds importance to the in both phylogenies, underlining the importance of elucidating new molecular data presented herein. relationships within the Paleacanthocephala in future studies. The lack of congruence between taxonomy and evolution- ary history within Rhadinorhynchus observed in the 18S rDNA- and cox1-derived phylogenies has been noted previ- ously by other authors based on morphological [40] and genetic Conflict of interest markers (18S and 28S rDNA and cox1 genes) [21, 29]. Indeed, The authors declare that they have no conflict of interest. while the sequences provided for R. pristis and R. lintoni [48] form a strongly supported clade with members of the genus Acknowledgements. This project was supported by the Pomphorhynchus in the 18S rDNA-derived phylogram, the rest Department of Biology, Brigham Young University (BYU), Provo, of the available Rhadinorhynchus sequences (including newly Utah, the Vietnam National Program No. 47 under Grant code generated ones) form a clearly separate group that also includes VAST.DA47.12/16-19, and by an Institutional Grant from the Para- sequences from T. annulospinosa and G. decapteri. This pattern sitology Center, Inc. (PCI), Scottsdale, Arizona. We thank Naomi was highlighted previously [21, 29]. While Gregori et al. [29] Mortensen, Bean Museum (BYU) for expert help in the preparation questioned the genetic identification of the specimens character- and organization of plates and figures and Michael Standing, Elec- ized by Verweyen et al. [48], Braicovich et al. [21] attributed tron Optics Laboratory (BYU), for his technical help and expertise. this pattern to incorrect assignment to Rhadinorhynchus by García-Varela et al. [24]. In fact, in their revision of the genus, Amin et al. [8] classified R. pristis and R. lintoni from Atlantic References and Mediterranean waters as invalid species, which supports the view by Gregori et al. [29] given that specimens collected by 1. Amin OM. 1975. Variability in Acanthocephalus parksidei Verweyen et al. [48] were from Pacific waters. This solves Amin, 1974 (Acanthocephala: Echinorhynchidae). Journal of Parasitology, 61, 307–317. the paraphyly “problem” observed in these previous phyloge- 2. Amin OM. 2013. Classification of the Acanthocephala. Folia nies and in the ones presented herein based on the 18S rDNA Parasitologica, 60, 273–305. gene. Because previously described 18S rDNA Rhadi- 3. Amin OM, Ha NV. 2008. On a new acanthocephalan family and norhynchus sequences [20, 29] and present results group with new order, from birds in Vietnam. Journal of Parasitology, 94, those provided by García-Varela et al. [24], the suggestion by 1305–1310. Braicovich et al. [21] of a misidentification by the latter author 4. Amin OM, Ha NV. 2011. On four species of echinorhynchid could be ruled out. Another specimen belonging to the same acanthocephalans from marine fish in Halong Bay, Vietnam, O.M. Amin et al.: Parasite 2019, 26, 14 19 including the description of three new species and a key to from the northern Gulf of Mexico. Gulf and Caribbean species of Gorgorhynchus. Parasitology Research, 109, 841–847. Research, 27, 11–19. 5. Amin OM, Heckmann RA. 2017. Rhadinorhynchus oligospi- 19. Arthur JR, Te BQ. 2006. Check list of parasites of fishes of nosus n. sp. (Acanthocephala, Rhadinorhynchidae) from mack- Vietnam. FAO Fisher Tech Paper 369/2. p. 123. erels in the Pacific Ocean off Peru and related rhadinorhynchids 20. Bao M, Roura A, Mota M, Nachón DJ, Antunes C, Cobo F, in the Pacific, with notes on metal analysis. Parasite, 24, 19. MacKenzie K, Pascual S. 2015. Macroparasites of allis shad 6. Amin OM, Heckmann RA, Evans RP, Tepe Y. 2016. A (Alosa alosa) and twaite shad (Alosa fallax) of the Western description of Echinorhynchus baeri Kostylew, 1928 (Acantho- Iberian Peninsula Rivers: ecological, phylogenetic and zoonotic cephala: Echinorhynchidae) from Salmo trutta in Turkey, with insights. Parasitology Research, 114, 3721–3739. notes on synonymy, geographical origins, geological history, 21. Braicovich PE, Lanfranchi AL, Farber MD, Marvaldi AE, molecular profile and X-ray microanalysis. Parasite, 23, 56. Luque JL, Timi JT. 2014. Genetic and morphological evidence 7. Amin OM, Heckmann RA, Radwan NA, Mantuano JS, Alcivar reveals the existence of a new family, genus and species of MAZ. 2009. Redescription of Rhadinorhynchus ornatus (Acan- Echinorhynchida (Acanthocephala). Folia Parasitologica, 61, thocephala: Rhadinorhynchidae) from skipjack tuna, Katsu- 377–384. wonus pelamis, collected in the Pacific Ocean off South 22. Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest America, with special reference to new morphological features. 2: more models, new heuristics and parallel computing. Nature Journal of Parasitology, 95, 656–664. Methods, 9, 772. 8. Amin OM, Heckmann RA, Ha NV. 2011a. Description of two 23. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. new species of Rhadinorhynchus (Acanthocephala: Rhadi- DNA primers for amplification of mitochondrial cytochrome c norhynchidae) from marine fish in Halong Bay, Vietnam, with oxidase subunit I from diverse metazoan invertebrates. a key to species. Acta Parasitologica, 56, 67–77. Molecular Marine Biology and Biotechnology, 3, 294–299. 9. Amin OM, Heckmann RA, Ha NV, Luc PV, Doanh PN. 2000. 24. García-Varela M, Cummings MP, Pérez-Ponce de León G, Revision of the genus Pallisentis (Acanthocephala: Quadrigyri- Gardner SL, Laclette JP. 2002. Phylogenetic analysis based on dae) with the erection of three new subgenera, the description of 18S ribosomal RNA gene sequences supports the existence of Pallisentis (Brevitritospinus) vietnamensis subgen. et sp. n., a class Polyacanthocephala (Acanthocephala). Molecular Phylo- key to species of Pallisentis, and the description of a new genetics and Evolution, 23, 288–292. quadrigyrid genus, Pararaosentis gen n. Comparative 25. García-Varela M, Nadler SA. 2005. Phylogenetic relationships Parasitology, 67, 40–50. of Palaeacanthocephala (Acanthocephala) inferred from SSU 10. Amin OM, Heckmann RA, Ha NV. 2004. On the immature and LSU rDNA gene sequences. Journal of Parasitology, 91, stages of Pallisentis (Pallisentis) celatus (Acanthocephala: 1401–1409. Quadrugyridae) from occasional fish hosts in Vietnam. Raffles 26. García-Varela M, Nadler SA. 2006. Phylogenetic relationships Bulletin of Zoology, 52, 593–598. among Syndermata inferred from nuclear and mitochondrial 11. Amin OM, Ha NV, Heckmann RA. 2008. New and already gene sequences. Molecular Phylogenetics and Evolution, 40, known acanthocephalans from amphibians and reptiles in 61–72. Vietnam, with keys to species of Pseudoacanthocephalus 27. Gouy M, Guindon S, Gascuel O. 2010. SeaView version 4: a Petrochenko, 1956 (Echinorhynchidae) and Sphaerechi- multiplatform graphical user interface for sequence align- norhynchus Johnston and Deland, 1929 (Plagiorhynchidae). ment and phylogenetic tree building. Molecular Biology and Journal of Parasitology, 94, 181–189. Evolution, 27, 221–224. 12. Amin OM, Ha NV, Heckmann RA. 2008. New and already 28. Gregori M, Aznar FJ, Abollo E, Roura A, González AF, known acanthocephalans mostly from mammals in Vietnam, Pascual S. 2012. Nyctiphanes couchii as intermediate host for with descriptions of two new genera and species of Archiacan- the acanthocephalan Bolbosoma balaenae in temperate waters thocephala. Journal of Parasitology, 94, 194–201. of the NE Atlantic. Diseases of Aquatic Organisms, 99, 37–47. 13. Amin OM, Ha NV, Heckmann RA. 2008. Four new species of 29. Gregori M, Aznar FJ, Abollo E, Roura A, González AF, acanthocephalans from birds in Vietnam. Comparative Para- Pascual S. 2013. Nyctiphanes Couchii an intermediate host for sitology, 75, 200–214. Rhadinorhynchus sp. (Acanthocephala, Echinorhynchidae) from 14. Amin OM, Ha NV, Ngo HD. 2011. First report of Neoechi- NW Iberian Peninsula waters. Diseases of Aquatic Organisms, norhynchus (Acanthocephala: Neoechinorhynchidae) from marine 105, 9–20. fish (Belonidae, Clupeidae, Megalopidae, Mugilidae, Sciaenidae) 30. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, in Vietnamese waters, with the description of six new species with Gascuel O. 2010. 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Molecular phylogeny and contrasting levels of host manipulation between forms of of the Acanthocephala (Class Palaeacanthocephala) with a Pomphorhynchus laevis (Acanthocephala). International Journal Paraphyletic assemblage of the orders Polymorphida and for Parasitology, 34, 45–54. Echinorhynchida. PLoS One, 6, e28285. 42. Ronquist F, Teslenko M, van derMark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. Cite this article as: Amin OM, Heckmann RA, Dallarés S, Constenla M & Ha NV. 2019. Morphological and molecular description of Rhadinorhynchus laterospinosus Amin, Heckmann & Ha, 2011 (Acanthocephala, Rhadinorhynchidae) from marine fish off the Pacific coast of Vietnam. Parasite 26, 14. An international open-access, peer-reviewed, online journal publishing high quality papers on all aspects of human and animal parasitology Reviews, articles and short notes may be submitted. 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