Eenymeenymyzostoma nigrocorallium, Terrana, Lucas & Eeckhaut, Igor, 2017

Terrana, Lucas & Eeckhaut, Igor, 2017, Taxonomic description and 3 D modelling of a new species of myzostomid (Annelida, Myzostomida) associated with black corals from Madagascar, Zootaxa 4244 (2), pp. 277-295 : 280-290

Eenymeenymyzostoma nigrocorallium n. sp. Terrana & Eeckhaut

( Figs 1–4 )

Holotype. Great Reef of Toliara, Madagascar, 20 meters deep, 25 November 2015 Paratypes. Same data as for holotype .

Etymology. The species name is a combination of the Latin adjective ‘ nigrum’ and the noun ‘ corallium ’ referring to the antipatharian host called black coral.

Material examined. A total of 162 specimens were found on the Great Reef of Toliara in Madagascar, infesting three different antipatharian species: Stichopathes sp. (142 specimens), Cirrhipathes sp. aff. anguina (14 specimens) and Cirrhipathes sp. (6 specimens) ( Fig. 1 ). Of the 13 sampled colonies of Stichopathes sp. ( Fig. 1 A,B,C), 9 hosted myzostomids (69%) with a maximum of 39 specimens on 139 cm of coral and a maximum density of 1 individual every 0.79 cm. Single colonies of Cirrhipathes sp. aff. anguina ( Fig. 1 D) and Cirrhipathes sp. ( Fig. 1 E) were sampled (8cm and 10cm fragments respectively), and each hosted myzostomids.

Specimens “ Myzostoma sp.113” collected by Goenaga in 1977 were observed with a stereomicroscope and a scanning electron microscope. The fixative used to store the specimens did not allow any molecular analyses or histological sections.

Diagnosis. Small myzostomid found in lumen of gastrovascular tracts of black corals. Body with ovoid trunk without introvert. Five pairs of small, cone-shaped parapodia located at very margin of trunk. No marginal cirrus or parapodial cirri. Cloacal opening on margin of trunk, at sagittal midline. No visible lateral organs. Body wall with cuticle-lined columnar epidermis and underlying parenchymo-muscular layer, latter especially well developed ventrally. Digestive system with pharynx, stomach and intestine, all of equal length, and two pairs of digestive diverticula. Nervous system with compact ventral nerve chain. Simultaneous hermaphrodites. Two pairs of dorsal ovaries located in parenchyma. Female genital ducts with large sagittal uterus and two pairs of dichotomized uterine branches. Uterus and intestine merging before cloacal opening. Four pairs of dorsal testicles located in parenchyma between ovaries. Male genital ducts with two pairs of deferent ducts and two pairs of seminal vesicles opening through one pair of male gonopores located dorsally to third pair of parapodia.

Description. Host ecology and morphology. The three black coral species Stichopathes sp., Cirrhipathes sp. aff. anguina and Cirrhipathes sp. hosting E. nigrocorallium were found in the channel between the north end of the Great Reef of Toliara and the coast at depths of 15–35m ( Fig. 1 ). Currents are often strong there, often with a lot of suspended organic matter from two nearby rivers, the Fiheranana and Onilahy. The three species consist of single, unbranched stems. Stichopathes sp. is brownish and can reach up to 5m in length ( Fig. 1 C). It has a single row of polyps along the stem, which extend their tentacles at night. Both species of Cirrhipathes have several rows of polyps around the stem and reach up to 2m in length, sometimes forming wires ( Fig. 1 D,E). Cirrhipathes sp. aff. anguina is yellow ( Fig. 1 D) while Cirrhipathes sp. is dark green with orange polyps ( Fig. 1 E).

Host-parasite relations. Eenymeenymyzostoma nigrocorallium is found in the gastrovascular ducts of these black corals. In situ coral infestation by myzostomids is almost impossible to detect except in Stichopathes sp., in which the polyps are arranged along a single face of the colony ( Fig. 1 B,C). The presence of these worms can then sometimes be detected on the side opposite to the polyps, with the worms appearing as very small humps on the coral surface ( Fig. 1 B). In the two other corals, it is impossible to detect the worms by eye because of the presence of polyps around the entire stem. Random dissections of the coral fragments confirmed the presence of myzostomids. Figure 1 A shows six individuals after the removal of the upper layer of the coral soft tissue. The black skeleton (axis) of the coral is visible, covered by the thin, yellow lower layer of the soft tissue. The myzostomids sit close together. The black coral host shows no sign of any damage caused by the myzostomid presence. The skeleton spines or the different tissues are not modified at the location where the myzostomid has been found.

Worm description. Individual worms are orange to dark brown and the sagittal pharynx, stomach and intestine are visible along the sagittal midline ( Fig. 1 A). The white extremities of the dichotomized digestive diverticula are observable in the trunk margin, as well as two seminal vesicles with spermatophores (not observable in the picture). When separated from their hosts, the myzostomids move very slowly by folding their trunk upward and downward. They live about three days in 5-µm filtered sea water out of contact with their host. After a few hours, they lay batches of hundreds of fertilized eggs through their cloacal opening. The eggs undergo spiral divisions during the next few hours.

Sections of myzostomids inside black corals indicate that the worms are located in the gastrovascular cavities of the polyps but also in the gastrovascular ducts that connect the polyps. Figures 3 A and 3B illustrate a section of an individual in a black coral. The axis of the host is bordered first by the coral epidermis, then by the thin mesoglea and the epithelium of the gastrovascular cavity of the polyp. The ventral side of the myzostomid faces the hosts’s axis ( Figs 3 A, B). The epithelium bordering the gastrovascular cavity does not appear affected by the presence of the worm.

The myzostomids measure 1.95–7.14 mm long and 1.73–4.36 mm wide. There are no marginal cirri and the trunk margin looks smooth under a binocular microscope, but viewed with SEM, rows of 30-µm humps are visible on the trunk margin ( Fig. 2 B,C). The presence of these humps may depend on the state of contraction of the individuals. Five pairs of extremely small parapodia of 60 µm in length are located on the trunk margins. They are very difficult to see under a binocular microscope and were fully revealed with SEM ( Figs. 2 D, E; 3C). A 12 µm long parapodial hook protrudes from each parapodial cone and has a rounded tip ( Fig. 2 E,F). When individuals are digested in bleach, the complete hooks are approximately 170 µm long, about twice as long as the acicula ( Fig. 2 F). There is a 70 µm long replacement hook at the base, and the manubrium at the tip of the acicula is rounded ( Fig. 2 F). Lateral organs are absent or not observed with SEM.

The body wall of E. nigrocorallum consists of a 4 µm thick cuticle, a columnar epidermis 44 µm high and a parenchymo-muscular layer that extends between the internal organs ( Fig. 3 C,D). Four types of cells are commonly observed in the epidermis: covering columnar cells of 44 µm in height and 5 to 10 µm wide, ciliated cells of similar shape, glandular cells and myoepithelial cells ( Fig. 3 D). The glandular cells are goblet-shaped with their nucleus located in the parenchyma and sending cellular processes out to the inner surface of the cuticula ( Fig. 3 D). The myoepithelial cells are located below the covering cells and the ciliated cells, close to the basal lamina ( Fig. 3 D). The parenchymo-muscular layer is particularly well developed on the ventral side of the body ( Fig. 3 A).

Internally, the trunk consists of a ventral part in which the main parts of the nervous system are surrounded by a hyper-developed parenchymo-muscular layer, a mid part with the digestive system and a dorsal part with the female and the male genital systems (Figs. 3,4). The excretory system has not been observed but is probably present; the analytical methods used here were not sufficient to reveal the presence of very small protonephridia. The hyper-developed ventral parenchymo-muscular layer is about one third of the 80 µm total thickness of the trunk ( Fig. 3 A). It is probably so developed because the worm’s ventral side faces the coral skeleton, which is not smooth but presents small spines; in some sections, the myzostomid’s ventral body wall fits the shape of the coral spines ( Fig. 3 B).

The nervous system principally consists of a compact ventral nerve chain that is 430 µm long and 230 µm wide in individuals about 3.3mm long ( Fig. 4 B,C,D). The compact nerve chain is composed of a central part in which the neuronal bodies lie and two lateral cords formed of the neuronal processes ( Fig. 4 C). These two lateral cords are connected anteriorly and posteriorly by two large nerve commissures and in between by small commissures. Five parapodial nerves, five side nerves and two pharyngeal nerves extend from each lateral cord ( Fig. 4 B,C,E, see also the 3D reconstruction in supplementary file). Each parapodial nerve splits in two smaller branches at the base of the respective parapodium ( Fig. 4 E and supplementary file). Small side nerves are located between the parapodial nerves and extend out to the body margins ( Fig. 4 E and supplementary file). These side nerves dichotomize into several small nerves before reaching the body margin.

The digestive system includes a tripartite pharynx, a stomach from which start two pairs of digestive diverticula and an intestine ( Fig. 4 and supplementary file). The mouth appears as a slit at the trunk margin in the sagittal plane. The pharynx, the stomach and the intestine each occupies one third of the length of the body. The pharynx is divided into three parts, each of the same length. The mid part comprises the muscular bulbus typically observed in other myzostomids ( Fig. 4 E). It is 370 µm long and 150 µm thick (in 3 mm long individuals) and consists of a muscular layer made of radial and circular muscle fibres that assist in the swallowing of particles. In the anteriormost part, the pharyngeal epithelium is not surrounded by a muscular layer, while in the posteriormost part, the pharyngeal lumen is surrounded by longitudinal muscle fibers connected to the muscular bulbus and to the stomach ( Fig. 4 E). The stomach is a pouch of 300 µm in diameter ( Fig. 4 A). Two pairs of digestive diverticula start from its lateral sides and dichotomize to finally reach the trunk margin ( Fig. 4 A and supplementary file). The digestive diverticula can barely be seen through the translucent body of the worms, and the intervening branching is obscure. The intestine is about 100 µm in diameter and fuses with the uterus at the very end of the trunk to open to the exterior by a cloacal pore (see supplementary file).

All the sectioned individuals are simultaneous hermaphrodites. Both genital systems are located in the upper part of the trunk ( Fig. 4 A). The female genital system consists of four ovaries, a uterus and many uterine branches (Supplementary file). It lies above the digestive system in such a way that a section through the dorsal part of a worm shows the epidermis, then the parenchymo-muscular layer with developing female germinal cells, the genital duct and finally the digestive system. Oogonia, previtellogenic oocytes and vitellogenic oocytes develop in the four ovaries, each situated in one quarter of the trunk ( Figs. 3 E,4A,4E). These germinal cells grow in the parenchyma above the uterine branches, themselves located above the digestive diverticula. Mature oocytes are found in the uterine branches and in the sagittal uterus as well ( Fig. 4 A,E). The uterus lies above the stomach and the intestine (see supplementary file). It is about 250–300 µm in diameter in sectioned individuals at its beginning and becomes narrower up to the cloacal pore.

……continued on the next page The male genital system comprises four testes, four deferent ducts and four seminal vesicles. The testes are diffuse with spermatocysts developing into the parenchyma between the branches of the ovaries ( Fig. 3 and supplementary file). Spermatocysts of different stages, including spermatogonia, spermatids and spermatozoa, are found close together. They measure from 30 to 50 µm in diameter and are conducted by short deferent ducts into four seminal vesicles. The seminal vesicles are two pairs of sacs, each being 250 µm long and 70 µm wide (in individuals of 3.3 mm in length). The vesicles of each pair flank the third pair of parapodia ( Fig. 4 D). Their extremities converge and fuse into a male gonopore, the two gonopores being located above these parapodia. The spermatophores observed inside the seminal vesicles are V-shaped structures consisting of the germinal products coming from each pair of seminal vesicles. The branches of the “V” are 200 µm long and composed of many spermatocyts of about 30 µm in diameter full of spermatozoa ( Fig. 4 D). The point of contact of the two branches includes degenerated spermiocysts similarly to the situation in M. cirriferum ’s spermatophore ( Eeckhaut & Jangoux 1991).

Observation of museum specimens. The two specimens obtained from the Smithsonian’s NMNH (catalogue number USNM 1421641) are ovoid, about 1.3–1.4 mm long and 0.8–0.9 mm wide, flattened but centrally depressed dorsally and convex ventrally ( Fig. 5 A,B). There are only four pairs of parapodia visible under scanning electron microscope on one side of the myzostomid, while on the other side two of them are less visible ( Fig. 5 A,B). The cone-shaped parapodia are located ventrally at about 100 µm of the trunk margin ( Fig. 5 A,B,C). They are also visible with a x50 stereomicroscope if they are not contracted, measuring 70–90µm in height including the hook ( Fig. 5 C). A small, round opening at one end and a slit vertical just below the edge at the other end are visible under SEM, suggesting the presence of the mouth without any introvert visible ( Fig. 5 B). No parapodial cirrus, no marginal cirrus and no lateral organ visible with a x50 stereomicroscope or a SEM. Male genital pores at the basis of the third pair of parapodia ( Fig. 5 B). The myzostomid is dorsally densely ciliated ( Fig. 5 D).

Phylogenetic position. In all analyses (MP, ML and Bayesian analysis) made either on separate genes (16S rDNA, 18S rDNA and COI) or on the three genes together, the new species always grouped with Eenymeenymyzostoma cirripedium with probabilities superior to 98% ( Figs. 6 , 7 ). With an outgroup including all 7 polychaete species, two different topologies were obtained depending on the phyogenetic inference method employed (ML, MP or Bayesian). In the Baysian tree, the Endomyzostoma clade is sister to all the other myzostomids ( Fig. 6 ). In the ML and MP trees, Endomyzostoma is in a clade together with Protomyzostomum, Asteromyzostomum , Eenymeenymyzostoma and Pulvinomyzostomum and its sister clade comprises Myzostoma, Contramyzostoma , Notopharyngoides , Hypomyzostoma and Mesomyzostoma ( Fig. 7 ). Although these five genera are always grouped together, regardless of the analysis or the outgroup, the phylogenetic relationships within the group are not clearly defined as shown by the low support values. When only Phyllodoce maculata was taken as outgroup in Bayesian analyses (see Appendix), the pattern changed and a clade consisting of Endomyzostoma, Protomyzostomum , Asteromyzostomum and Eenymeenymyzostoma (without Pulvinomyzostomum ) appeared as the sister group of the other myzostomids. With varying numbers of polychaete species in the outgroup, i.e. from 1 to 6, the 127 resulting ML analyses always showed the same pattern as the ML tree obtained with 7 species ( Fig. 7 ).

Figures

FIGURE 1. Eenymeenymyzostoma nigrocorallium n. sp. and its antipatharian hosts. (A) View of a cluster of six reddish living specimens revealed by removing overlying coral tissue; myzostomids situated within the coral tissue are often invisible. (B) In places where there are no coral polyps, the presence of myzostomids can sometimes be detected as a slight swelling of the coral surface (red arrow). (C – E) Antipatharian species recorded herein as hosts of E. nigrocorallium: (C) Stichopathes sp.; (D) Cirrhipathes sp. aff. anguina; (E) Cirrhipathes sp. Scales: A, B: 1 mm; C, D, E, F, G: 1 cm / square.

FIGURE 2. Eenymeenymyzostoma nigrocorallium n. sp. (A) SEM photo, dorsal view. (B) SEM photo, ventral view of partially folded specimen. (C) Detail of humps along margin in (B); three sets of about 10 humps each are present on each side. (D, E) Very small parapodia (not always visible), each with curved hook protruding from tip. (F) Light micrograph of parapodium digested in bleach to show entire hook apparatus. Scales: A, B: 1 mm; C: 100 µm; D, E: 10 µm; F: 50 µm. Abbreviations: ac, acicula; h, humps; ho, hook; m, mouth; ma, manubrium; mr, lateral margin; p, parapodium; pc, parapodial cone, ph, parapodial hook; rh, replacement hook.

FIGURE 3. Eenymeenymyzostoma nigrocorallium n. sp., internal structure. (A) Transverse section through a black coral infested by a myzostomid lying in the lumen of a digestive duct of the coral host. (B) Detail of the host epithelial structure under the myzostomid. (C) Parapodium fully retracted into epithelial folds, thus invisible externally. (D, E, F) Hermaphroditic gonads extending out to the body margins, with spermatozoids ready to fertilize oocytes inside the gonadal ducts. Scales: A, B: 200 µm; C: 60 µm; D: 100 µm; E, F: 10 µm. Abbreviations: ac, acicula; c, cuticle; d, dorsal side of myzostomid; e, epidermis; ec, ectoderma; ee, external environment, en: endoderma; gv, granular vesicles; lu, lumen of digestive tract; m, myzostomid; mc, myoepithelial cells; me, mesoglea; o, oocyte; s, coral skeleton; sg, spermatogonia; ss, skeletal spine; v, ventral side of myzostomid.

FIGURE 4. Eenymeenymyzostoma nigrocorallium n. sp., internal structure. (A, E) Upper (A) and lower (E) sections of body showing entire digestive tract, reproductive apparatus and nervous system. (B, C) Details of nervous system. (E) Two seminal vesicles associated with third parapodium, both containing spermatophores. Scales: A, E: 200 µm; B: 100 µm; C, D: 50 µm. Abbreviations: c, chaeta; ca, digestive caeca; fg, female gametes; i, intestine; mg, male gametes; nf, nervous fibers; ns, nervous system; p, parapodia; pe, perikarya; ph, pharynx; pn, parapodial nerve; s, spermatophore; sn, side nerves; st, stomach; sv, seminal vesicles; u, uterus; ub, uterine branches; uh, uterine horns, branches.

FIGURE 5. Undescribed specimen collected by Goenaga in 1977 on the inside of the black coral Stichopathes sp. (A) Dorsal view showing barely any structure except the parapodia. (B) Dorsal view under SEM showing the myzostomid ventrally convex. (C) View of a parapodium. (D) Dense cilia found on the dorsal side of the myzostomid. Scales: A: 500 µm; B: 100 µm; C, D: 10 µm. Abbreviations: ci, cilia; dc, dorsal cilia; ds, dorsal side; gp, male genital pore; m, mouth; mr, lateral margin; p, parapodia; pc, parapodial cone; ph, parapodial hook; vs, ventral side.

FIGURE 6. Phylogenetic position of Eenymeenymyzsotoma nigrocorallium n. sp. in a tree computed with Bayesian analysis using 16 S- 18 S-CO 1 alignment. Numbers above branches indicate posterior probabilities; a star means a posterior probability superior to 95. Red arrow points to the position of the new species.

FIGURE 7. Phylogenetic position of Eenymeenymyzsotoma nigrocorallium n. sp. in a tree obtained using the maximum likelihood model (MetaPIGA). Numbers above branches indicate posterior probabilities; a star means a posterior probability superior to 95. Red arrow points to the position of the new species.