Potamostoma shizunaiense gen. et sp. nov. (Nemertea: Hoplonemertea: Monostilifera) is described from the mouth of the River Shizunai, Hokkaido, Japan. This genus is readily distinguished from other monostiliferans by an oesophagus opening far anteriorly into the rhynchodaeum, a well developed excretory system extending the whole body length, terminals of the excretory collecting tubules situated between the body wall circular muscle layer and the dermis, and bilobed testes in males.
INTRODUCTION
Nemertean worms (phylum Nemertea), with approximately 1150 known species (Gibson, 1995), are typically marine invertebrates but several species are known to occur in brackish waters and some have been able to colonise either freshwater or terrestrial habitats. Some species of brackish-water nemerteans are known to thrive in stable, albeit reduced, salinities. An example is the hoplonemertean Cyanophthalma obscura (Schultze, 1851), which has been found in salt marshes or salt marsh pools on the Atlantic coast of North America (Norenburg, 1986), and is very common in the Gulf of Finland where salinities are below 5–6 psu (Brunberg, 1964). On the other hand, there have been only a few reports on species inhabiting in those environments, where the salinity fluctuates rapidly, like in man-grove swamps, river mouths or brackish-lakes (e.g. Gibson and Qi, 1991; Iwata, 1970; Moore and Gibson, 1981) (Table 1).
Table 1
Nemertean species recorded from estuarine or other brackish-water habitats.
During the first author's faunal study around Hokkaido, northern Japan, a hoplonemertean species was obtained at the mouth of the River Shizunai. Subsequent histological study revealed that the combination of morphological characters found in the present nemerteans excludes them from all of the described genera of Monostilifera. The present paper aims to describe the new genus and species of monostiliferous hoplonemertean inhabiting in salinity down to 2 psu.
Current classifications of nemerteans, particularly above generic level, are in need of thorough taxonomic revision (Gibson, 1985). The traditional taxonomic scheme will change in the future (Sundberg and Gibson, 1995). The new species is not assigned to family for this reason.
MATERIAL AND METHODS
Specimens of the nemerteans were collected from beneath stones on sand at the mouth of the River Shizunai, Hokkaido, Japan (42°20′N, 142°22′E) on 9 May 1996, 16 November 1997, and 24 November 1999. Sampling was carried out at ebb tide when the salinity was about 2 psu. The nemerteans were anaesthetised either in 4% MgCl2 or MS222, fixed in Bouin's solution for 24hr, embedded in 56–57°C m.p. paraffin wax and sectioned at 6, 7 or 10 μm. Most sections were stained by the Mallory trichrome method, but for one slide series haematoxylin and eosin was used. Observations on the stylet apparatus of 11 specimens were made by the squeezing method described by Kirsteuer (1967).
Type material and voucher specimens are deposited in the Zoological Institute, Hokkaido University, Japan (ZIHU).
Abbreviations used in figures.
ao = apical organ; ap = anterior pouch of intestinal caecum; br = brain; cg = cephalic gland; cm = body-wall circular muscle layer; co = cerebral sensory organ; cv = cephalic blood vessel; de = dermis; dm = dorsoventral muscle; dv = mid-dorsal vessel; ep = epidermis; ex = excretory collecting tubule; g1 = orange-staining acidophilic gland cell; g2 = purple-staining basophilic gland cell; gc = glandular component of cerebral sensory organ; ic = intestinal caecum; il = inner layer of body wall longitudinal musculature; in = intestine; ld = lateral diverticulum of intestinal caecum; lm = body-wall longitudinal muscle layer; ln = lateral nerve cord; lv = lateral blood vessel; nc = putative nurse cell; ng = neuroganglionic component of cerebral sensory organ; oc = ocellus; oe = oesophagus; ov = ovary; pi = proboscis insertion; pr = proboscis; py = pylorus; rc = rhynchocoel; rd = rhynchodaeum; sg = sub-muscular gland; st = stomach; ts = testis; vp = vascular plug.
DESCRIPTION
Potamostoma gen. nov.
Type species: Potamostoma shizunaiense sp. nov.
Etymology: The generic name (neuter) is formed by combining the Greek words potamos (= a river) and stoma (= a mouth) to indicate the habitat in which the animals were found living. The specific epithet is derived from the name of the River Shizunai by adding the Latin adjectival suffix-ense (= belonging to).
Diagnosis: Monostiliferous hoplonemerteans with rhynchocoel reaching to posterior end of body; rhynchocoel musculature in two separate layers; proboscis anterior chamber with three muscle layers (outer and inner circular, middle longitudinal); body-wall musculature with diagonal layer, longitudinal layer anteriorly divided by posterior extensions of cephalic glands; pre-cerebral septum absent; cephalic retractor muscles derived from outer portion of longitudinal musculature; oesophagus opening into rhynchodaeum just behind proboscis pore; intestinal caecum with two anterior pouches and lateral diverticula; blood system very well developed, with single vascular plug; cerebral ganglia with neither inner neurilemma nor neurochord cells; lateral nerve cords without accessory nerves but with myofibrillae; peripheral nervous system well developed; single apical organ present, cephalic glands well developed, extending behind brain, sub-muscular glands present; cerebral organs close in front of brain; excretory system well developed, extending throughout body length, collecting tubule terminals situated between body wall circular muscle layer and dermis; dioecious, testes bilobed.
Potamostoma shizunaiense sp. nov.
Material examined: A total of 32 living individuals were collected, of which 11 were examined in vivo by the squeezing method described by Kirsteuer (1967): 4 specimens, 9 May 1996; 24 specimens, 16 November 1997; 4 specimens, 24 November 1999. Holotype, immature female, complete series of transverse sections, 7 μm, 81 slides (ZIHU-2037); paratypes, immature male, transverse sections of anterior body region, 6 μm, 39 slides, (ZIHU-1930) and immature female, complete series of transverse sections, 6 μm, 84 slides (ZIHU-2040). Nine sectioned voucher specimens are also deposited under ZIHU-1931, -1932, -1933, -2038, -2039, -2041, -2042, -2043, -2044.
Type locality: Mouth of the River Shizunai, Hokkaido, Japan (42°20′N, 142°22′E), under stones on a sandy bottom. Salinity at ebb tide 2 psu.
External features: Body slender, up to 6 cm long and 1.2 mm wide (Fig. 1). Head oval or truncate triangular shape. Eyes arranged in four groups, each containing 2-22 black to dark green pigment-cup ocelli (Fig. 2). White pigment spots on mid-dorsal surface of snout. Two pairs of cephalic furrows present; posterior furrows encircle the body, forming caudally directed V-shape on dorsal surface (Fig. 2). Body gradually tapered posteriorly, ending in bluntly rounded tail.
Fig. 1
Potamostoma shizunaiense gen. et sp. nov. Photograph of a living specimen, viewed dorsally. Scale bar = 5 mm.
Fig. 2
Potamostoma shizunaiense gen. et sp. nov. Enlargement of the head, viewed dorsally, to show the arrangement of the eyes and cephalic furrows. The position of the brain lobes and anterior portions of the lateral nerve cords is also indicated. Scale bar = 1 mm.
General body colour yellowish-green to olive, rhynchocoel appearing as yellowish-ochre median dorsal line extending almost to posterior tip. Ovaries in ripening females visible as deep greenish bands arranged pseudometamerically in posterior half of body. Lateral nerve cords also visible through lateral body margins as dark orange lines fringed with whitish dots.
Body wall, musculature and parenchyma: Epidermis ciliated, with maximum thickness of about 38 μm in anterior body regions generally thinner posteriorly and in places only 15–20 μm tall (Fig. 3). Dermis consistently about 5 μm thick throughout body (Fig. 3).
Fig. 3
Potamostoma shizunaiense gen. et sp. nov. Camera lucida drawing of a transverse section to show the organization of the body in the foregut region. Scale bar = 400 μm.
Body wall muscles consisted primarily of outer circular and inner longitudinal layers; circular layer 3–7 μm thick and frequently much thinner than dermis, longitudinal layer varies in thickness from 20–55 μm depending upon local contraction (Fig. 3). In cerebral region longitudinal layer divided into outer and inner zones by posterior extensions of cephalic glands (Fig. 4A). Outer portion, not contributing to proboscis insertion, extended to head tip. Most of inner longitudinal muscle fibres terminated in proboscis insertion, but a few extended anteriorly surrounding rhynchodaeum (Fig. 4B). Cephalic retractor muscles derived from outer portion of longitudinal layer. Thin diagonal muscle layer (Fig. 4C), extending between the two main body wall muscle coats, not enclosed by connective tissue. Dorsoventral muscles moderately well developed in foregut and intestinal regions.
Fig. 4
Potamostoma shizunaiense gen. et sp. nov. A. Transverse section through the brain to show the divided body-wall longitudinal muscle layer. Arrowheads indicate the inner portion of the longitudinal musculature. B. Transverse section through the proboscis insertion. C. Tangential section through the body wall musculature to show the non-fasciated diagonal fibres, indicated by arrowheads. D. Sagittal section through the anterior tip of the head. E. Higher magnification of the boxed area in D to show the oesophagus opening (indicated by the arrowhead) from the ventral wall of the rhynchodaeum just behind the proboscis pore. F. Transverse section near the tip of the head at the point where the oesophagus is about to branch off from the rhynchodaeum. Scale bars: A, B, D, F = 100 μm; C, E = 50 μm.
Parenchymatous connective tissues well developed throughout body length, most obvious below rhynchocoel just behind brain.
Proboscis apparatus: Proboscis pore, opening ventrally just behind head tip, leading into short ciliated chamber (Fig. 4D, E). Rhynchodaeum with delicate and unciliated epithelium, 10 μm in average thickness (Fig. 4E, F). Immediately in front of proboscis insertion, rhynchodaeum surrounded by sphincter-like layer of circular muscles.
Rhynchocoel reaching almost to posterior tip of body, with wall containing separate inner longitudinal and outer circular muscle layers (Fig. 3). Each layer mostly 2–5 μm in thickness, but circular layer in posterior portion of rhynchocoel thickened to about 15 μm.
Proboscis slightly shorter than body, white in colour when everted. Larger anterior portion, 30–47% of body diameter when retracted, consisted of papillate epithelium, 60–80 μm or more tall, containing acidophilic gland cells, distinct connective tissue basement layer, weak layer of outer circular muscles, inner longitudinal muscle stratum 15 μm in maximum thickness, a delicate inner circular muscle layer and a flattened inner lining. Proboscis nerves running in longitudinal muscle layer (Fig. 5A); of 12 sectioned specimens one individual possessed 11 proboscis nerves, one had 12 nerves, and the remainder 13.
Fig. 5
Potamostoma shizunaiense gen. et sp. nov. A. Transverse section through the anterior portion of the proboscis to show the 13 proboscis nerves, indicated by arrowheads. B. Photomicrograph of a typical central stylet and basis. C. Transverse section through the posterior region of the stomach. D. Transverse section through the pyloric region of the gut. E. Transverse section to show the single vascular plug. Foregut nerves are indicated by the arrowheads. Scale bars: A, B, E = 100 μm; C, D = 200 μm.
Central stylet smooth (Fig. 5B), variable in length, 82–110 μm long (mean 93 μm); basis 98–158 μm long (mean 128 μm), 32–54 μm wide (mean 45 μm) (in vivo observation; n=11). Stylet/basis ratio 0.62–0.88 (mean 0.73). Number of accessory stylet pouches 2–4 (mostly 3), each containing 2–4 accessory stylets.
Posterior proboscis chamber about 25–35% of the body diameter, with wall containing basophilic glandular epithelium not forming papillae, outer longitudinal muscle layer about 5 μm thick, thin and indistinct inner circular muscle layer and delicate lining layer. No nerves were distinguished in posterior portion of proboscis.
Alimentary canal: Oesophagus moderately thick-walled, opening at ventral wall of rhynchodaeum just behind proboscis pore (Fig. 4D, E). Oesophageal epithelium neither ciliated nor glandular. After passing below ventral cerebral commissure, oesophagus expanded leading to stomach; transitional region between oesophagus and stomach contained violet-staining basophilic gland cells with granular cytoplasm.
Stomach epithelium (Fig. 3, 5C), reaching to 70 μm or more in height, dominated by basophilic glands but also contained a few acidophils. Degree of epithelial folding varied among individuals. Stomach gradually narrowed posteri-orly, with both epithelial height and gland cell number decreasing posteriorly, leading into narrow pyloric canal (Fig. 5D) opening into dorsal wall of intestine.
Intestinal caecum with a pair of anterior pouches and several pairs of deep lateral diverticula (Fig. 5D, 6): anterior pouches extended forward to middle portion of stomach, not reaching brain. Intestine with distally branched deep lateral diverticula along whole length. Anus at posterior end of body.
Fig. 6
Potamostoma shizunaiense gen. et sp. nov. Schematic diagram to show the arrangement of the anterior pouches and lateral diverticula of the intestinal caecum, drawn from living material.
Blood system: Blood vascular system well developed. Pair of cephalic vessels joinning anteriorly by loop running dor-sally over front of rhynchodaeum (Fig. 4B, D). Posteriorly, vessels entering cerebral ring. Mid-dorsal vessel arises from one of the lateral vessels. Behind brain three longitudinal vessels, one mid-dorsal and two lateral, extend through remaining body length without connection until meeting posteriorly by supra-intestinal loop. Mid-dorsal vessel, soon after origin, entering ventral rhynchocoel wall forming single large vascular plug up to 55 μm wide and 30 μm tall (Fig. 5E). Blood vessels with distinct walls throughout, up to 60 μm wide anteriorly; gradually narrower in posterior intestinal region.
Nervous system: Brain moderately large, with dorsal and ventral lobes of similar size (Fig. 4A). Dorsal and ventral commissures 20–25 μm and 45–50 μm thick, respectively. Thin outer neurilemma investing cerebral ganglia, but without inner neurilemma. Lateral nerve cords, lacking accessory nerves and neurochords, meeting posteriorly by supra-intestinal commissure.
Peripheral nervous system rather well developed. Ante-riorly, several cephalic nerves extending from front of both dorsal and ventral brain lobes, innervating proboscis and ocelli mostly. Pair of stout and prominent nerves emerging laterally from dorsal ganglia, leading to neuroganglionic tissues of cerebral sensory organs. Pair of foregut nerves, originating from ventral cerebral ganglia, running posteriorly in parenchyma above oesophagus and stomach (Fig. 5E). Apical organ and cephalic glands: Single apical organ, comprising of ciliated pit 35–55 μm in diameter and lined by neutrophilic vacuolate epithelium of 10–25 μm thick, opening at tip of head (Fig. 4D). Cephalic glands discharging to exterior via apical organ.
Cephalic glands comparatively large, irregularly shaped and vacuolated in appearance, occupying much of cephalic space (Fig. 7A), and extending back behind brain. As well as typical vacuolate basophilic lobules, cephalic glands also contain two other distinct gland types; yellow- to orange-staining acidophils and purple-staining basophils with Mallory's trichrome (Fig. 7A).
Fig. 7
Potamostoma shizunaiense gen. et sp. nov. A. Transverse section through the pre-cerebral region. B. Transverse section through the stomach region to show the sub-muscular glands, with the aperture of one of the glands indicated by the arrowhead. C. Transverse section to show the appearance of a cerebral sensory organ. D–G. Serial transverse sections through the stomach region to show the excretory collecting tubules, indicated by black arrowheads. White arrowheads in E–G indicate another collecting tubule leading to its terminal region situated between the dermis and body wall circular muscle layer. H. Enlargement of the boxed area shown in F. White arrowheads indicate an excretory tubule leading to its terminal region which is surrounded by small cells (indicated by black arrowhead). I. Longitudinal section to show a putative terminal region of an excretory collecting tubule, indicated by arrowhead. The asterisk indicates the rhynchocoel circular muscle layer. J. Transverse section through the posterior stomach region to show one of the nephridiopores, indicated by the arrowhead. K. Transverse section through the posterior brain region to show one of the sinuses, indicated by the arrowhead, containing unidentified cells, situated in the parenchyma. L. Transverse section through the intestinal region to show the dark pigment granules in immature ovaries (indicated by arrowheads). M. Transverse section through the intestinal region of an immature male with evidence of early spermatogenesis to show the bilobed appearance of a testis. N. Transverse section through the intestinal region of an immature female to show the appearance of a developing ovary. Scale bars: A, J, N = 100 μm; B–G = 50 μm; H = 15 μm; I, K = 20 μm; L, M = 200 μm.
Sub-muscular glands: Sub-muscular glands (Fig. 7B) scattered amongst fibres of body-wall longitudinal muscle layer in foregut region on all sides of body. Each gland opening to exterior by own pore.
Sensory organs: 10–70 ocelli, arranged into two groups on each side of head (Fig. 2), 30–40 μm in diameter, with dark green to black pigment cup (Fig. 7C). Anterior groups situated near head tip, posterior groups at about level of proboscis insertion.
Cerebral sensory organs (Fig. 7C) comparatively large and situated close below anterolateral margins of cerebral ganglia, opening to exterior via distinct ventrolateral ciliated cerebral canals of about 45 μm in diameter, leading inwards from anterior pair of cephalic furrows. Canals surrounded posteriorly by neuroganglionic tissues, flanked above and below by cap-like glandular portions. Cerebral organs elliptical in cross-section, 60 μm wide and 200 μm tall. Excretory system: Excretory system very well developed and extending throughout the length of body, including head. Terminal regions of excretory system embedded between dermis and body-wall circular muscle layer (Fig. 7D-H). Terminals surrounded by small but unidentified cells; flame cells not found. Thick-walled (10–15 μm) collecting tubules, 35–45 μm or more in diameter, highly convoluted and ramify around rhynchocoel, lateral nerve cords and alimentary canal (Fig. 3). Nuclei of cells forming collecting tubules located close to tubule lumen. Final region of each collecting tubule thick-walled and surrounded by unidentified cells similar to cells around terminal (Fig. 7I). Numerous nephridiopores opening laterally (Fig. 7J). In holotype 17 and 15 nephridiopores opening on right and left side of body, respectively. Each nephridiopore leading to distinct collecting tubules and terminals.
Numerous sinuses containing highly vacuolated cells with small and dense nuclei (Fig. 7K) found in parenchyma; each sinus isolated, not opening into either blood vessels or excretory tubules.
Reproductive system: Dioecious. Gonads arranged pseudo-metamerically, alternating with lateral intestinal diverticula in immature. Gonads in individuals collected in November identifiable to sex; early spermatogenesis or developing ova seen in individuals collected in May. Sexually matured individuals exhibiting colour dimorphism; testes khaki in colour, whereas ovaries deep green. In immature females ovaries filled with dark green pigment granules (Fig. 7L). Testes approaching maturity are dorsoventrally bilobed (Fig. 7M), each lobe about 100 μm by 400 μm in dimension. Developing ovaries up to about 300 μm by 600 μm; each ovary containing at least 30 eggs. Putative nurse cells situated along inner wall of each ovary; some eggs connected to nurse cells by short stalk (Fig. 7N).
Systematic remarks: Comparatively few hoplonemertean genera that have two separate muscle layers in their rhynchocoel wall have been reported in which the oesophagus, as in the present specimens, emerges from the rhynchodaeum far in front of the brain lobes (Table 2). The present new genus closely resembles Arctonemertes Friedrich, 1957, but can be distinguished from the latter by the anteriorly divided body-wall longitudinal musculature and the extensive development of the excretory system. None of the genera listed in Table 2 possesses the anteriorly divided body-wall longitudinal muscle layer, as in the present species; this feature, as well as the position of the terminal portions of the excretory collecting tubules between the dermis and body wall circular muscle layer, together with other characters listed in Table 2, exclude the present species from any of these genera.
Table 2
Monostiliferous hoplonemertean genera with a bilayered rhynchocoel wall and an oesophagus emerging from the rhynchodaeum close behind the proboscis pore. Data compiled from Friedrich (1940, 1955, 1957), Gibson (1973), Sánchez and Moretto (1988), Senz (1993) and the present study.
DISCUSSION
Nemerteans are thought to have their origin in marine environment. In the majority of marine taxa, the excretory system is restricted in the foregut region of the body. Contrary, in all the terrestrial and freshwater species, as well as in some brackish-water forms, the excretory system is significantly developed throughout the body, regardless of their higher taxonomic ranks, such as class-level. This indicates the presence of an extensively developed excretory system alone cannot always be used as evidence of close common ancestry; Iwata (1968, 1970) suggested that the extensive development of the excretory system in brackish-water nemerteans, compared with the much more restricted systems found in marine taxa, has an osmoregulatory function.
Stenohaline marine invertebrates are at best barely able to tolerate the rapid salinity changes characteristic of estuarine conditions (Nybakken, 2001). They are thus generally restricted to salinities of 25 psu or more; most marine nemertean species probably belong in this category and are likely to be found penetrating only into the lower reaches of estuaries. On the other hand, the brackish waters, such as the Baltic and Black Seas where freshwater input exceeds evaporation, are often characterised by much more stable conditions; invertebrates living in such regions are not subject to the rapid salinity fluctuations associated with estuaries. The Amphiporus and Tetrastemma species, which were reported from Sea of Okhotsk by Korotkevich (1977), are not found in salinities less than 18 psu (Table 1) and their excretory systems are restricted to the foregut region of the body. On the other hand, those euryhaline species, including Amniclineus zhujangensis Gibson and Qi, 1991, Hinumanemertes kikuchii Iwata, 1970, Sacconemertella lutulenta Iwata, 1970, Sacconemertopsis belogurovi Chernyshev, 1991, S. olivefera Iwata, 1970, and the present species, which live in salinities fluctuating down to 1–6 psu, possess excretory systems extending throughout most of the body. These findings suggest the critical salinity, where nemerteans have to develop their excretory system throughout the body for survive, would likely to be between 6–18 psu.
Acknowledgments
We are grateful to Dr Y. Takashima for bringing to HK's notice the existence of the present specimens, found whilst he was collecting oligochaetes from the mouth of the River Shizunai for a taxonomic investigation. We would also like to thank Dr Janet Moore for criticising and commenting on an earlier version of this paper.
