Cryptocelis sinopae, Gammoudi & Bulnes & Kurt, 2022

Gammoudi, Mehrez, Bulnes, Verónica N. & Kurt, Güley, 2022, The polyclad fauna (Platyhelminthes, Rhabditophora) of the Sinop Peninsula (Black Sea, Turkey) with a description of a new species of Cryptocelis Lang, 1884 Abstract, Mediterranean Marine Science (Hong Kong, China) 22 (1), pp. 141-156 : 143-151

Cryptocelis sinopae sp. nov.

( Figs. 2–4 , 8A )

Material examined. Holotype: ESFM-PLAT/2014-2, sagittal sections of the copulatory apparatus (20 slides), 12.05.2014, station M1, 5 m deep, Sinop, Black Sea, coll. G. Kurt. Paratype: ESFM-PLAT/2014-3, one specimen (18 slides), sagittal sections of the median region containing copulatory apparatus, 12.05.2014, station M1, 5 m deep, Sinop, Black Sea, coll. G. Kurt.

Description of the holotype. Body oval-shaped and compact, 15 mm long and 6 mm wide at the broadest part of the body. The anterior margin rounded, posteriorly somewhat pointed, laterally ruffled. Dorsal surface ground color yellowish to brownish with an unpigment- ed marginal band. The dorsal color pattern consists of minute brown spots distributed mostly behind the brain region. The spots are more densely distributed in two dorsal bands parallel to the main intestine and reproductive organs. Lateral to the central bands, the spots are concentrated in small clusters, resembling rounded blotches, up to the unpigmented margin ( Fig. 2A ). Ventrally whitish. Without tentacles. Tentacular eyes present, cerebral eyes in two clusters, with frontal eyes and marginal eyes along the entire body margin. The latter more concentrated in the anterior fourth of the body, arranged in rows but scattered on the rest of the body margin ( Figs. 2F , 8A ).

At the level of the mid-longitudinal body axis, the dorsal body wall consists of an epidermal layer of cylindrical ciliated cells with basal nuclei, short and numerous spindle-shaped rhabdites, and eosinophilic and basophilic granules six times higher (29 µm) than the compact and well-developed basement membrane (4.5 µm). Inwards, there is a circular muscle layer as high as the basement membrane, followed further inwards by a longitudinal muscle layer three times higher than the basement membrane (14 µm) and an innermost well-developed circular muscle layer, almost three times higher than the longitudinal muscle layer (35 µm), blending with apparent parenchymal transversal muscle bundles ( Fig. 2C ). The ventral body wall consists of an epidermis of cylindrical cells densely ciliated, with scattered rhabdites and acidophilic granules, six times higher (28 µm) than the compact and well-developed basement membrane (5 µm). Inwards, there is a circular muscle layer slightly higher (5.4 µm) than the basement membrane, followed inwards by a longitudinal muscle layer twice as high as the basement membrane (10.2 µm) a well-developed circular muscle layer (26 µm) and an innermost longitudinal muscle layer (12 µm). Parenchymal transversal muscle bundles apparent ( Fig. 2D ).

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Necks of parenchymal eosinophilic and basophilic glands piercing the muscular wall apparent. The marginal body wall has a different arrangement. The columnar epithelium is not glandular, the cilia are longer, and nuclei are no longer basal, with scattered, small rhabdites. Basement membrane delicate and muscular body wall reduced to several longitudinal muscle fibers. The necks of two types of subepithelial glandular cells open in this area: the type with fine granular eosinophilic secretion, also distributed throughout the epidermis of the whole body, and a singular type with coarser secretion (glandular corpuscles after Bock, 1923, p. 20) ( Fig. 2F ). The body wall around the genital area is characterized by a lower columnar epithelium, shorter cilia, nuclei central, without rhabdites, and not glandular. The muscular body wall is disorganized, and the muscular layer coalesces with the muscular layers of the bulb housing the reproductive organs ( Fig. 2E ).

Digestive system. The mouth opens near the center of the body, and the pharyngeal pocket is located in the second third of the body, connected to the intestine at almost the same level as the mouth.

Reproductive system. Testis arranged ventrally between intestinal branches. Male copulatory apparatus immediately behind the pharyngeal pocket directed backward and enclosed in a well-developed muscular bulb. The outermost fibers of the muscular bulb intermingle with the longitudinal muscle layer of the body wall. The vasa deferentia also anchored to the body wall. The paired vasa deferentia filled with sperm, first run anteriad before turning to the middle body line and joining into a median vas deferens ( Fig. 3A ). The latter runs anteriad up to the level of the prostatic vesicle. After leaving the vas deferens, the ejaculatory duct develops a strong circular muscle layer between the ciliated inner epithelium and the external longitudinal muscle layer. The ejaculatory duct turns dorsal and, after piercing the muscular bulb from ventral, projects into the prostatic vesicle ( Fig. 3B ).

Anterior to the prostatic vesicle, there is a strong layer of transversal muscles, anchored dorsally and ventrally to the body’s muscular wall. Prostatic vesicle rounded; its muscular wall consists of a few muscular bundles mixed with the muscular bulb. The inner glandular lining is deeply ridged and secretory, filling the lumen of the vesicle anteriorly, while distally the ridges become shorter and the lumen of the vesicle becomes apparent. Distally, the inner epithelium becomes cuboidal and ciliated before opening to the ejaculatory duct of the cirrus. Without extra vesicular glands. Cirrus long, unarmed, set coiled and somehow twisted in the muscular bulb. The cirrus muscular wall consists of a compact layer of circular muscle fibers intermingling with glandular cells, which run along the entire tortuous path up to the male atrium ( Fig. 3C ). The cirrus is enclosed in a muscular bulb. This bulb consists of an inner, looser muscular matrix and an outermost layer of circular muscles, both devoid of glandular secretions. The muscular fibers coating the muscular bulb blend dorsally and ventrally with the body’s muscular layers, anchoring the male copulatory system to the body wall. Ejaculatory duct coated with a cuboidal,

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ciliated, folded epithelium ( Figs. 3 C-D), alternating with secretory patches. In the last third of its length, the ejaculatory duct widens. At this point, the folds of the duct´s wall become deeper. Shortly before its opening, the ejaculatory duct turns into a narrow tube ( Fig. 3C ). The cirrus opens to the male atrium in the center of shallow conical penis papillae. The male atrium is narrow, covered with a tall and heavily ciliated epithelium, except dorsally where it widens laterally, forming two pouches in the roof of the atrium. An anterodorsal intrabulbar musculo-glandular conical fold projects into each atrium´s pouch ( Figs. 3E ,

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E´, and F). The glandular folds consist of scatter muscular fibers, immersed in a mass of glandular cells, covered with a squamous epithelium without cilia. The male atrium opens to the exterior through a narrow canal via the male pore ( Figs. 3C , 5 ).

Ovaries dorsal, immersed in the parenchyma. The uteri run slightly ventral to the ovaries, filled with eggs ( Fig. 4D ). The oviducts enter separated from the ventral side to a distended portion of the vagina interna ( Fig. 4A ). From this point, the female canal continues anteriorly. At the level of the male gonopore, the female canal makes

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a long posteroventral turn and then runs posteriorly up to the level of the female gonopore. Distally the vagina turns ventrally, opening to the ample female atrium and gonopore. The female gonopore is located 3 mm behind the male pore. From the vagina interna up to the female atrium, the female canal develops deep folds in its wall ( Figs 4 A-C). Oviducts slender, with cuboidal inner epithelium with short cilia. The vagina interna lined with high columnar epithelium and long cilia. The rest of the female canal and female atrium is covered by a columnar

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ciliated glandular epithelium. The female canal is immersed in a dense mass of cement glands ( Figs 4A–C ). Posteriorly, the cement glands mass extends not far from the level of the female gonopore and is limited by an external muscular layer. The outermost fibers of this layer blend dorsally and ventrally with the muscular body wall.

Whitish, spherical, ruffled structures were recognized on the dorsal surface of one specimen. They were identified as spermatophores ( Fig. 2B ). Histological sections also revealed sperm cells in several areas of the body’s

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parenchyma ( Fig. 4F ) and around the uterine ducts ( Fig. 4E ), probably as a consequence of the indirect transfer of sperm (dermal impregnation).

Etymology. The species name is a specific term alluding to the type locality’s ancient name, Sinop, which is taken from the name of the goddess Sinope from the

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Greek mythology.

Remarks. The oval body, without tentacles, with cerebral, nuchal, and marginal eyespots, male genital apparatus enclosed in a massive muscular bulb, lack of a seminal vesicle, with an intrabulbar prostatic vesicle with ridged glandular inner lining, and unarmed cirrus surrounded by

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Continued

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an intrabulbar secretory tissue, without Lang´s vesicle, agree with the characteristics of the species gathered in the genus Cryptocelis, Lang 1884 . The presence of the paired lateral musculo-glandular folds in the roof of the male atrium represents an apomorphic character, which distinguishes Cryptocelis sinopae sp. nov. from its congeners ( Table 2).

The presence of additional secretory structures associated with the male copulatory organs was described for Cryptocelis glandulata Jacubowa, 1909 and Cryptocelis lilianae Marcus and Marcus, 1968 . The annex glandular organ of C. glandulata is oblong in shape with a slightly pointed anterior tip, opening to the distal ejaculatory duct, and filled with fine-grained basophilic star-shaped secretions organized in somewhat longitudinal rows. The muscular wall consists of a thin layer of longitudinal muscular fibers, underlined by a few small oval-shaped glands opening to the organ’s lumen ( Jacubowa, 1909, p. 16). Although Jacubowa was not able to find the connection to the ejaculatory duct, she believed the annex glandular organ opened to the distal part of the ejaculatory duct and hypothesized the secretions were related due to the formation of spermatophores. Marcus & Marcus (1968) observed that the distal portion of the ejaculatory duct of Cryptocelis lilianae forms a conspicuous dorsal diverticulum. They describe this diverticulum as an inner muscular wall and an outer glandular epithelium with sunken glands and nuclei partly immersed in the muscular layer ( Marcus & Marcus 1968, p. 14). The lack of a well-defined muscular wall lined with a glandular epithelium led us to consider that these accessory secretory structures associated with the male systems of Cryptocelis sinopae , C. glandulata , and C. lilianae are not adenodactyls like that of Adenodactyloplana uruguayensis Bulnes, Faubel & Ponce de Leon, 2003 , which also belongs to the family Cryptocelididae . Additionally, the paired musculo-glandular conical folds of C. sinopae , open to the male atrium, while the accessory secretory structures of C. glandulata and C. lilianae are connected to the ejaculatory duct.

Lang (1884) created the genus Cryptocelis based on the description of Leptoplana alba . In his description, he stated that the copulatory system of Cryptocelis resembles the copulatory organ of the genus Planocera (p. 248-249): an intrabulbar prostatic vesicle that opens to a cirrus and the male atrium. But while Planocera cirri are armed, meaning the inner lining of the ejaculatory duct secretes scleroproteins in the form of hooks and teeth, in Cryptocelis the ejaculatory duct is lined with an epithelium secreting viscous substances, probably acid mucins, since they are dyed red in basophilic stains. The extreme development of the muscular wall and the predominance of secretory epithelia lining the male system led Bock to consider that the muscular bulb housing the male copulatory organ of Cryptocelis ijimae was a prostatic vesicle, even when he discovered the secretions of the proximal bulbous component were different from those of the elongated distal component (Bock, 1923, p. 26). In the following years, the authors followed Bock’s interpretation of the male copulatory organ, adding unnecessary complexity to the microanatomy of the genus ( Faubel, 1983;

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Hyman, 1953a; 1953b; Kato, 1936; 1937; 1939; Marcus & Marcus, 1968).

The level of detail in descriptions of the Cryptocelis species varies, but most describe a long and tortuous, even a spiralized, secretory ejaculatory duct. Although a ciliated ejaculatory duct was described only for C. sinopae , we consider this may also be the case for other Cryptocelis species and that the proximal section of the ejaculatory duct contributes to the production of spermatophores. The prostatic secretions protect the sperm mass, and while this mass moves along the ejaculatory duct with the help of the metachronic movement of the cilia, the secretions of the ejaculatory duct are added, shaping the characteristic external striation ( Lang, 1884, p. 248-249). The spermatophores are stored in the distal part of the ejaculatory duct, which is probably the only portion of the duct that protrudes during mating. The partial projection of the distal ejaculatory duct from the male atrium could explain the difference in the Cryptocelis species between having a blunt penis papilla ( C. occidentalis Hyman, 1953 ), and single, or multiple penis sheaths ( C. glandulata , C. compacta Lang, 1884 ). Regarding the prostatic vesicle, except for C. glandulata , all other Cryptocelis species display a regularly folded glandular lining filled with eosinophilic secretions. Cryptocelis insularis Hyman, 1953 , also possesses the deeply folded inner lining with a proximal eosinophilous and a distal basophilic region. Cryptocelis sinopae is characterized by a rounded prostatic vesicle with a ridged glandular lining, only anteriorly showing some similarities to the prostatic vesicle of C. compacta , which has an inner glandular lining that is developed more antero-ventrally.

Bock (1923) describes the inner lining of the anteri- or portion of the prostatic vesicle of C. ijimae as an epithelium with a fine and inconspicuous basal membrane underlined by subepithelial gland cells, but in C. sinopae the glandular epithelium appears pseudostratified and ciliated. The reproductive system of the Cryptocelis species exhibits a striking number of glandular secretions. The glandular cells may be extravesicular, intrabulbar ( C. sinopae ), extrabulbar, ( C. alba ), or both ( C. ijimae ). The detailed description of the cirrus and the ejaculatory duct’s inner lining is not available for all species, but in the examined species, the glandular cells are located beneath the ciliated epithelium. The species of the Cryptocelis genus lack a true seminal vesicle, and in most of them, the vasa deferentia develop spermiducal bulbs. From that point, the vasa deferentia turn into slender ducts, which may join in a common vas deferens ( C. glandulata , C. compacta , C. insularis ), or continue to be paired ( C. lilianae , C. amakusensis , C. ijimae , C. littoralis , and C. orientalis ) before piercing the muscular bulb of the copulatory organ and opening to the prostatic vesicle. Like Cryptocelis occidentalis , in Cryptocelis sinopae , the vasa deferentia join into a common vas deferens that pierces the muscular bulb and projects into the prostatic vesicle, but in C. sinopae there are no spermiducal bulbs, and the common vas deferens is characterized by a well-developed circular muscle layer.

The female system seems more or less homogeneous

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in all species of the genus Cryptocelis . The oviducts open from the mid-lateral to the common female canal at the level of the female gonopore. This anterior part of the female canal is lined with ciliated non-glandular epithelium. Distally, the inner epithelium is ciliated and glandular. The canal runs dorsal and turns frontal, then turns backward, and finally ventral. The canal opens to the female atrium and gonopore. Along the vaginal canal, there are some distended areas, which vary among the different species. The female system is surrounded by a dense mass of glandular cells immersed in the parenchyma. Except for Cryptocelis glandulata , where the cement glands reach the distal margin of the animal, in all other Cryptocelis species, the glandular mass does not distally surpass the level of the female gonopore. The extreme development of the muscular fibers observed in Cryptocelis sinopae can also be observed in the female system,

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where the glandular mass seems limited by bundles of longitudinal muscle fibers.

There is little information available regarding the body wall of the Cryptocelis species. Cryptocelis amakusensis possesses a thick epidermis of 60 µm with a thin and delicate basement membrane, followed inwards by a circular, then a longitudinal, then an innermost diagonal muscular coat. The diagonal coat is divided into two layers. The ventral body wall is higher than the dorsal one since there is also a circular muscular layer between the diagonal layers. In Cryptocelis sinopae , the epidermis is lower; the basement membrane is well developed, and the dorsal and ventral body walls are different. While the dorsal epidermis is followed inwards by a circular, a longitudinal, and another circular muscle layer, ventrally there is an additional innermost longitudinal layer.

The dorsal and ventral epidermis of Cryptocelis sin-

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opae have similar thicknesses. The only recorded differences agree with the observations made by Bock (1923) for Cryptocelis ijimae , where the dorsal and ventral epidermis of the body differs from the marginal and the ventral epidermis of the genital area.

The presence of masses of spermatozoa in the parenchyma of Cryptocelis sinopae agrees with the observation made by Lang (1884) for Cryptocelis alba , and the production of spermatophores and the hypodermic insemination may be a usual mating behavior of the Cryptocelis species.

Figures

Fig. 2: Cryptocelis sinopae sp. nov. A, dorsal view of fixed specimen; B, detail of the spermatophores (arrow); C, sagittal section of the dorsal body wall; D, sagittal section of the ventral body wall; E, sagittal section of the ventral body wall at genital openings’ area; F, sagittal section of the marginal body wall. Abbreviations to the figures: bm, basement membrane; bw, body wall; cm, circular muscle layer; e, eyes; eo, eosinophilic glands; gbw, body wall around the genital area; gc, marginal glandular cell (glandular corpuscles [Bock, 1923]); lm, longitudinal muscle layer; mbw, marginal body wall; mci, marginal cilia; me, marginal eye; mml, marginal muscle layer; mrh, marginal rhabdites; tm, transversal muscle fibres. Scale bars: A: 2 mm, B: 1 mm; C: 50 µm; D: 50 µm; E: 200 µm; F: 20 µm.

Fig. 3: Cryptocelis sinopae sp. nov. A, sagittal section of the male copulatory organ (vas deferens and prostatic vesicle); B, sagittal section of the ejaculatory duct, the arrow points the projection of the ejaculatory duct into the proximal prostatic vesicle; C, sagittal section of the male copulatory organ (cirrus and male atrium); D, sagittal section of the male copulatory apparatus (cirrus); E and E´, sagittal sections of the musculo-glandular folds; F, transversal diagram of the male atrium. Abbreviations to the figures: co, cirrus opening; cr, cirrus; ed, ejaculatory duct; gce, glandular cells; gf, musculo-glandular fold; gf 1, right musculo-glandular fold; gf 2, left musculo-glandular fold; ma, male atrium; mg, male gonopore; ppv, proximal prostatic vesicle; vd, vas deferens. Scale bars: A: 100 µm; B: 30 µm; C: 200 µm; D: 200 µm; E: 200 µm; E΄: 30 µm.

Fig. 4: Cryptocelis sinopae sp. nov. A-C, sagittal section of the female reproductive system; D, sagittal section of the uterus and vas deferens; E, sagittal section of an oocyte; F, sperm cells immersed in body parenchyma. The black arrows point sperm cells. Abbreviations to the figures: cg, cement glands; ev, external vagina; fa, female atrium; i, intestine; iv, internal vagina; mg, male gonopore; mv, median vagina; ov, oviduct; vd, vas deferens; vf, vaginal fold. Scale bars: A: 100 µm; B: 100 µm; C: 100 µm; D: 100 µm; E: 30 µm; F: 15 µm.

Fig. 8: Pleioplana okusi. A, sagittal section of the prostatic vesicle; B, sagittal section of the ovary; C-D, sagittal section of the vagina and female atrium. The arrows point the sperm mass. Abbreviations to the figures: ch, tubular chamber of the prostatic vesicle; ed, ejaculatory duct; fg, female gonopore; o, ovary; pi, epidermal pigment; pp, penis papilla; pv, prostatic vesicle. Scale bars: A: 100 µm; B: 50 µm; C: 30 µm; D: 50 µm.

Fig. 5: Cryptocelis sinopae sp. nov. Sagittal reconstruction of reproductive system. Abbreviations to the figures: cg, cement gland; cmw, cirrus muscular wall; ed, ejaculatory duct; ep, epidermis; fa, female atrium; fg, female gonopore: gf, musculo-glandular fold; ma, male atrium; mb, muscular bulb; mg, male gonopore; pv, prostatic vesicle; se, secretory epithelium; vd, vas deferens; vf, vaginal fold. Scale bar: 1000 µm.