Rhadinorhynchus hiansi Soota and Bhattacharya, 1981

Amin, Omar M., Heckmann, Richard A., Dallarés, Sara, Constenla, María & Ha, Nguyen Van, 2020, Morphological And Molecular Description Of Rhadinorhynchus Hiansi Soota And Bhattacharya, 1981 (Acanthocephala: Rhadinorhynchidae) From Marine Fish Off The Pacific Coast Of Vietnam, Journal of Parasitology 106 (1), pp. 56-70 : 58-65

Rhadinorhynchus hiansi Soota and Bhattacharya, 1981 ( Figs. 1–31 )

The present report represents a dramatic extension of the host and geographical distribution of a rhadinorhynchid acanthocephalan, R. hiansi , reported only once from 2 specimens collected from a flat needle fish, A. hians (Belonida) , off the southwestern coast of India almost 40 yr ago and provides a full morphological and molecular description of that species for the first time. Rhadinorhynchus hiansi was originally described from 2 male specimens collected from the flat needlefish Ablennes hians Valenciennes (Belonidae) off Trivandrum, Kerala, on the southwestern coast of India. Of the 129 specimens of R. hiansi from 9 of 10 striped bonito, S. orientalis off the southern Pacific coast of Vietnam at Nha Tran, we processed and whole mounted 32 specimens (19 males and 13 females) for microscopic examination, 15 specimens for SEM studies, 8 specimens for metal analysis of hooks, and 4 specimens for molecular studies. The remaining specimens are in ethanol in the Omar M. Amin collection .

General: With characters of the genus Rhadinorhynchus Lühe 1911 . Trunk relatively long, uniformly cylindrical, spinose anteriorly in 2 regions within range of proboscis receptacle ( Figs. 1, 2 , 19 ). Trunk and all shared structures markedly larger in females than in males with rare males almost reaching length of longest females. Cuticular surface flat with many electron-dense micropores ( Fig. 22 ). Trunk spines with prominent dense central core ( Figs. 4 , 20, 21 ) (counted on 1 side of trunk) larger and usually more numerous in females than in males. Anterior trunk spines in near complete circles, fewer dorsally, not exceeding 5 vertically or horizontally. Posterior trunk spines reaching 9 ventrally and 11 laterally, larger at middle ( Table I ). Posterior lateral trunk spines smaller than posterior ventral spines and closer to ventral side of worms. Proboscis long, cylindrical, straight, titling ventrad ( Figs. 1, 2, 5 , 12 ), gradually widening and rounded anteriorly, with 21–24 longitudinal alternating rows ( Fig. 14 ) of 36–48 hooks each ( Figs. 5 , 12 ), varying with worm sex. Hooks with thin cortical layer and thick solid core ( Fig. 19 ), directed posteriorly with external striations (6, 7, 15, 16). Dorsal hooks slightly longer and slenderer than stouter ventral hooks especially in anterior half of proboscis ( Figs. 6, 7 ). Apical hooks smallest ( Fig. 14 ), third and forth hooks largest and thickest. Subsequent hooks progressively smaller posteriorly and then increasing in size nearing basal hooks. Basal crown hooks longest ( Fig. 17 ) but slender and larger ventrally than dorsally (Table II; Figs. 6, 7 ). Hook roots simple, relatively shorter than blades, directed posteriorly except few larger posterior 5 or 6 hooks having small roots with short anterior manubria (Table II; Figs. 6, 7 ). Neck prominent, longer dorsally than wide posteriorly with paired sensory pores ( Fig. 18 ). Proboscis receptacle about twice as long as proboscis, double-walled, about twice as long as proboscis with cephalic ganglion near its middle. Lemnisci digitiform, equal, uniformly broad throughout, usually slightly shorter than receptacle ( Figs. 1, 2 ) but occasionally longer. Gonopore terminal in males and subterminal in females to various degrees ( Figs. 8, 10 , 23 ).

Males (based on 19 adults with sperm from S. orientalis ): Trunk 4.75–11.25 (7.04) mm long by 0.45–0.87 (0.58) mm wide at middle. See Table I for position, distribution, and size of trunk spines. Proboscis 1.62–2.20 (1.88) long by 0.19–0.28 (0.23) mm wide anteriorly. See Table II for measurements of proboscis hooks and roots. Neck 312–520 (418) long dorsally by 250–302 (279) wide posteriorly. Proboscis receptacle 3.50–4.87 (3.86) mm long by 0.22–0.32 (0.26) mm wide. Lemnisci 2.30–4.37 (3.24) mm long by 0.06–0.21 (0.13) mm wide. Reproductive system in posterior-most third of trunk in contiguous structures with genitalia opening into bursa ( Figs. 1, 3 ). Testes ovoid; anterior testis 0.57–1.25 (0.77) mm long by 0.21–0.50 (0.33) mm wide, larger than posterior testis 0.42–1.12 (0.63) mm long by 0.21–0.55 (0.33) wide. Cement glands 4, rod-shaped, in 2 contiguous pairs, longer posteriorly ( Fig. 3 ). Anterior glands 0.36–1.04 (0.87) mm long by 0.34–0.56 (0.21) wide; posterior glands 0.94–1.92 (1.50) mm long by 0.16–0.21 (0.18) mm wide. Individual cement gland ducts surround prominent fusiform Saefftigen’s pouch, 312–832 (489) mm long by 146–281 (193) mm wide, anteriorly and joining its posterior duct at thick-walled bursa ( Figs. 1, 3 ). Bursa thickwalled with many sensory papillae in inner rings and in outer clusters ( Figs. 26–28 ), 520 by 364 in 1 specimen.

Females (based on 13 mature females with eggs and ovarian balls from S. orientalis ): Trunk 10.50–24.75 (17.83) mm long by 0.42– 0.90 (0.61) mm wide at middle. See Table I for position, distribution and size of trunk spines. Proboscis 2.10–2.37 (2.30) mm long by 0.22–0.37 (0.27) mm wide anteriorly. See Table II for measurements of proboscis hooks and roots. Neck 416–520 (467) long dorsally by 250–322 (293) long by 109–300 (233) wide posteriorly. Neck 200–375 (315) long by 109–300 (233) wide posteriorly. Proboscis receptacle 3.00–5.75 (4.65) mm long by 0.20–0.32 (0.27) mm wide. Lemnisci 2.57–5.07 (3.74) mm long by 0.11–0.21 (0.13) wide. Reproductive system 2.60–6.12 (4.23) mm long (30% of trunk length: 25% in shortest worm and 29% in longest worm) ( Fig. 8 ), with well-defined subterminal vagina ( Figs. 10 , 23 ), very long uterus, small and elongated uterine bell with few cells ( Figs. 8, 9 ). Eggs elliptic with thick coarse outer shell, without polar prolongation of fertilization membrane ( Figs. 11 , 24 ), 37–62 (50) long by 15–23 (20) wide.

Taxonomic summary

Type host: Flat needlefish Ablennes hians Valenciennes (Belonidae) .

Other hosts: Striped bonito Sarda orientalis Temminck and Schlegel (Scombridae) ; this paper.

Type locality: Trivandrum , Kerala, India (08°29 ′ 15 ′′ N, 76°57 ′ 9 ′′ E) .

Other localities: Southern Pacific coast of Vietnam at Nha Trang (12°15 ′ N, 109°11 ′ E). Allotype female from Vietnam: HWML collection no. 139894 .

Specimens: HWML collection no. 139895 (voucher males and females). GenBank accession nos: Rhhi18S1-S3: MN203133 - MN203135 ; RhhiCOI1-3: MN203136 - MN203138 . Z.S.I.Reg. No. WN 360/1 (holotype male and paratype male on same slide from Albennes hians at Trivandrum ) (Soota and Bhattacharya, 1981).

Remarks

The collection of a large number of specimens from S. orientalis along the Pacific coast of Vietnam provided an opportunity to fully describe males and females of R. hiansi for the first time. The description of 2 males by Soota and Bhattacharya (1981) did not include complete text and/or illustrations information on the proboscis receptacle, trunk spines, neck, lemnisci, or cement glands and made no reference to hook roots or Saefftigen’s pouch. The range of measurements was limited by the number of specimens that they examined (2). Irrespective, it appears that their specimens had smaller trunks (8.8–10.00 mm long) and somewhat longer proboscides (2.2–2.25 mm long). Their specimens had shorter lemnisci (2.5–2.6 mm long), anterior testis (470–490 long), and posterior testis (440– 460 long). Soota and Bhattacharya (1981) also indicated the length of the thinner dorsal and the thicker ventral hooks as 33– 66 and 33–55 long, respectively. Our specimens were, however, readily recognizable as R. hiansi and are fully described and illustrated.

Energy Dispersive X-ray Analysis

The results of large and small gallium (Ga) cut hooks and trunk spines are given in Tables III–V and represented by Figures 29– 31 . The elements necessary for the mineralization and hardening of the hooks and spines especially calcium and phosphorus are present with sulfur, especially highest in small hooks and trunk spines.

Molecular analyses

Three partial 18S rDNA (742–764 nt) and 3 COI (619–628 nt) sequences were generated from 4 adult specimens of R. hiansi . Two 18S rDNA sequences were identical (and thus only one of them was thus included in the corresponding phylogenetic tree), and, with respect to the third one, intraspecific divergence was 0.003% (2 nt difference). Intraspecific sequence divergence for the COI gene ranged between 0.002 –0.024 % (1–12 nt difference).

Table VI provides data for the sequences retrieved from GenBank and used in the phylogenetic analyses based on the 2 alignments. Since ML and BI algorithms produced trees with identical topology for the 2 sequenced genes, only the BI tree is shown for 18S ( Fig. 32 ) and the ML tree for the COI gene ( Fig. 33 ).

In the phylogenetic tree based on 18S rDNA, R. hiansi formed a highly supported clade with the congeneric species Rhadinorhynchus pristis (Rudolphi, 1802) and Rhadinorhynchus laterospinosus Amin, Heckmann and Ha, 2011 , and also with Gymnorhadinorhynchus mariserpentis Steinauer, Garcia-Vedrenne, Weinstein and Kuris, 2019 (0.000 –0.003 %, 0–2 nt difference from newly generated sequences in all cases). Less related to R. hiansi was a sequence belonging to an unknown species of Rhadinorhynchus (0.013%, 10 nt difference) that grouped, with low support, with Gymnorhadinorhynchus decapteri Braicovich, Lanfranchi, Farber, Marvaldi, Luque and Timi, 2014 (0.039 and 0.040%, 29 and 30 nt difference from newly generated sequences).

According to phylogenetic analyses based on the COI gene, the 3 newly generated sequences for R. hiansi were sisters to a group including sequences of R. laterospinosus (0.130 –0.136 %, 66–69 nt difference) as well as representatives of Bolbosoma and the species Neorhadinorhynchus nudus (Harada, 1938) Yamaguti, 1939 (0.130 –0.149 %, 66–76 nt difference from newly generated sequences). Separately, it remained a clade including a sequence assigned to the same unknown species of Rhadinorhynchus present in the 18S phylogeny (0.234%, 119 nt difference from newly generated sequences) and which grouped with G. decapteri and Transvena annulospinosa Pichelin and Cribb, 2001 (0.250 –0.312 %, 127–159 nt difference from newly generated sequences).

Figures

Figure 29. Energy dispersive X-ray spectrum of the base center of a gallium cut large anterior hook of a Rhadinorhynchus hiansi specimen showing high levels of calcium and phosphorus. The x-ray data are the elemental analysis of the hook base (see boldfaced figures in Table III). Insert: SEM of a cross and lateral longitudinal gallium cut hook.

Figure 30. Energy dispersive X-ray spectrum of the tip of a gallium cut small base hook of a Rhadinorhynchus hiansi specimen showing high levels of sulfur. The x-ray data are the elemental analysis of the hook tip (see boldfaced figures in Table IV). Insert: SEM of a cross and lateral longitudinal gallium cut hook.

Figure 31. Energy dispersive X-ray spectrum of a gallium cut trunk spine of a Rhadinorhynchus hiansi specimen showing high levels of sulfur. The x-ray data are the elemental analysis of the center of the spine; see boldfaced numbers in Table V. Insert: SEM of a lateral longitudinal cut spine.

Figure 32. Bayesian inference (BI) phylogram reconstructed using newly generated 18S rDNA sequences for Rhadinorhynchus hiansi and retrieved sequences from GenBank for the closest-related sequences. Outgroup: Rotaria rotatoria. Nodal support from maximum likelihood (ML) and BI analyses are indicated as ML/BI. Bootstrap values lower than 70 and posterior probability values lower than 0.9 are omitted. The scale bar indicates the expected number of substitutions per site.

Figure 33. Maximum likelihood (ML) phylogram reconstructed using newly generated cox1 sequences for Rhadinorhynchus hiansi and retrieved sequences from GenBank for the closest-related sequences. Outgroup: Rotaria rotatoria. Nodal support from maximum likelihood (ML) and BI analyses are indicated as ML/BI. Bootstrap values lower than 70 and posterior probability values lower than 0.9 are omitted. The scale bar indicates the expected number of substitutions per site.

Figures 1–11. Line drawings of whole mounted specimens of Rhadinorhynchus hiansi from Sarda orientalis collected off the southern Pacific coast of Vietnam at Nha Trang. (1) A young male specimen. (2) A young female specimen. (3) Detail of the reproductive system of the male specimen in Figure 1. (4) A posterior trunk spine. (5) The proboscis of a male specimen. (6, 7) Profiles of selected dorsal (6) and ventral (7) proboscis hooks from Figure 5 showing 5 types of hooks. Hook no. 1 (apical), no. 3 (largest), no. 15 (smaller mid-proboscis hook), no. 40 (larger hooks near posterior end), basal crown (longest). (8) Reproductive system of an 11 mm long female showing long uterus and the attachment of the simple uterine bell to the ventral wall of the trunk. (9) A larger magnification of the uterine bell. (10) Larger magnification of the vagina. (11) An egg.

Figures 12–16. SEM of specimens from Sarda orientalis collected off the southern Pacific coast of Vietnam at Nha Trang. (12) A fully extended proboscis of a male specimen. (13) An apical view of the proboscis of specimen in Figure 12 showing the smaller apical hooks and the arrangement of hook rows. (14) A gallium cut hook showing the thin cortical layer and the dense prominent core. (15) A view of hooks at mid-proboscis showing their shape, external texture, and orientation. (16) A higher magnification of a hook showing detail of its external striations. All hooks are striated.

Figures 17–22. SEM of specimens from Sarda orientalis collected off the southern Pacific coast of Vietnam at Nha Trang. (17) Posterior proboscis hooks are longest and in a perfect ring. (18) Sensory pore just posterior to basal hooks. (19) Anterior trunk of a male specimen showing anterior rings of spines and posterior ventral and lateral spines characteristic of this species. (20) A higher magnification of a trunk spine. (21) A gallium cut spine showing a middle dense core and a think spongy outer layer under the cuticle. (22) An example of micropores in the middle of trunk.

Figures 23–28. SEM of specimens from Sarda orientalis collected off the southern Pacific coast of Vietnam at Nha Trang. (23) Rounded posterior end of a female specimen showing the sub-ventral position of gonopore appearing as a lateral slit. (24) Eggs with rounded poles and prominent coarse surface. (25) The posterior end of a male specimen showing the terminal position of an invaginated bursa. (26) A lateral view of terminal bursa. (27) A face view of the thick bursa showing the distribution of sensory structures in an outer ring of clusters and inner rings of single units. (28) Higher magnification of a sensory cluster from the outer ring showing its organization and the elevated dome-shaped center of each unit. The organization of sensory structures of the bursa is species specific and is a useful diagnostic tool for species recognition.

Tables

Table I. Size and distribution of trunk spines of 19 male and 13 female specimens of Rhadinorhychus hiansi from Sarda orientalis in Vietnam.

Anterior trunk spines		Posterior trunk spines
Dorsal	Ventral	Across	Ventral	Lateral
Males
0–3 (1.1)* 31–62 (47.3)*	0–4 (2.5) 31–62 (47.3)	2–5 (3.0) 31–62 (47.3)	2–7 (5.0) 42–73 (61.5)	0–12 (5.0) 35–52 (43.8)
Females
0–4 (1.6) 52–73 (62.3)	1–4 (2.6) 52–73 (62.3)	0–5 (3.5) 52–73 (62.3)	5–9 (6.7) 62–84 (71.1)	0–11 (4.3) 52n73 (64.4)

* Range of number of spines on 1 side of worms (mean); range and mean of spine length in micrometers.