General morphology (Fig. 1)next section
Fig. 1. Parotoplana crassispina sp. n. General organisation as seen in a slightly squeezed specimen. Scale bar: 100 µm.
All species of the Parotoplana jondelii species-group have a flattened body, comparatively short and broad for the genus. Fixed specimens average 1 mm in length. The anterior end is provided with a ciliated sensory furrow, several tactile bristles and a few adhesive papillae. The encapsulated, oval-shaped brain (about 64 µm in diameter in P. carthagoensis sp. n.) abuts the statocyst, which contains two nuclei. The fan-shaped caudal end is provided with numerous adhesive papillae. A ciliated creeping sole runs from the sensory furrow to the level of the genital pore. The length of the cilia ranges from about 5 μm (P. carthagoensis) to 7 μm (P. pulchrispina sp. n.).
The epithelial cells, with intraepithelial nuclei, are cubical ventrally and more flattened dorsally. Differences between ventral and dorsal surfaces are most marked in P. pulchrispina sp. n., where their height is 5 μm and 2-2.5 μm, respectively.
Rhabdoids are arranged in numerous (15-20), more or less regular, rows. They are narrow and rod shaped, 5-12 μm long.
Body musculature consists of a very thin outer circular layer, and of a much more developed inner longitudinal layer. This latter is particularly strong ventrally. Only in P. jondelii and P pulchrispina dorsal longitudinal fibres are well developed. Dorso-ventral muscle fibres are present anteriorly, just in front of the statocyst.
The pharynx, located in the middle of the body, is short and tubular. Its orientation can quickly shift from being placed horizontally to vertically, when it appears collar-shaped. Fixed specimens may present either condition. The pharynx is ciliated both internally and externally. External cilia are shorter than the inner cilia, ranging 1-1.5 μm and 2-2.5 μm in length, respectively. The distal tip, where pharyngeal glands discharge, is unciliated. No oesophageal area could be detected.
Fig. 2. Parotoplana crassispina sp. n. Reconstruction of the genital organs from sagittal sections. Scale bar: 20 µm.
Species of the complex have few (five to ten) pairs of testes in the anterior half of the body, arranged into two regular rows. The copulatory organ consists of an ovoid seminal vesicle and a prostatic vesicle which is distally connected to the sclerotised apparatus. The seminal vesicle is lined by a thin nucleated epithelium, and is surrounded by a fine, spiral musculature. The shape of the prostatic vesicle is ovoid to elongated, depending upon the contraction during fixation. It ranges from 30-40 μm in length in P. crassispina sp. n. to 20 µm in P. carthagoensis. Its epithelium is ciliated, high and glandular, and is surrounded by very weak circular muscles.
The sclerotised apparatus consists of a girdle of spines, which, in most cases, are unusually broad and flat compared to other species of the genus Parotoplana. The morphology and the number of these spines proved to be diagnostic features for species recognition within the complex, and will be described in detail in the appendix. In general, the spines are symmetrically arranged in pairs. The dorsal pairs of spines (D) are straight and have a simple, needle-like morphology. Usually, they consist of a central thinner, needle-like pair (indicated in species descriptions as D1 ). These are flanked by members of a much broader pair (D2 ), tapering distally into a point.
Ventrally, a central pair of spines (V) is present. In most instances, these spines are quite distinct from the rest of the set, and are provided with a subterminal apophysis which, unlike in other species of Parotoplana, can be very large and flat.
Between the dorsal and ventral pairs, a series of spines, usually with a different morphology, are present. They are termed in the descriptions that follow as spines of the girdle (G), and are numbered progressively, starting from the pair (G1 ) closest to the dorsal pairs. In some of the species, G1 spines are somewhat intermediate in morphology between dorsal spines and the rest of the spines of the girdle. In these instances, distinguishing between the dorsal and girdle spines is clearly arbitrary. Similarly, the girdle spines close to the ventral pair may be differently shaped than the others – particularly so in P. pulchrispina, where V spines are similar in morphology to the inner spines of the girdle.
The apophyses are attached to bundles of muscles. In P. crassispina, these bundles are particularly strong, and the movement of the apophyses is easily appreciable in semi-squashed living specimens.
Oocytes were observed in living specimens and in sections just posterior to the testis follicles. The vitellaria were almost equally distributed anteriorly and posteriorly to the pharynx. Most specimens of P. crassispina and P. carthagoensis, for example, had about 10-14 follicles in each area. P. pulchrispina had more posterior follicles (about 10) than the anterior ones (six). Only P. ichnusae sp. n. had markedly more anterior (about 20) than posterior (seven) vitellarian follicles; the living specimen studied appeared somewhat immature, however.
The female duct, formed by the fusion of the two oviducts, could be traced only close to its outlet in the lowermost, posterior area of the genital atrium. Its opening is surrounded by the outlets of female glands, whose bodies extend considerably into the caudal region. Both in P. crassispina and P. carthagoensis, the opening of the female duct is located in a pocket of the atrium, lined by a high epithelium.
The bursal system is complex and appears heterogeneous in morphology in the species-group. In P. crassispina, where the quality and quantity of the sectioned material allowed for a detailed reconstruction, the bursal system is particularly remarkable. Observations made on living specimens revealed an unusual, inverted-V shaped, striated structure close to the male copulatory organ (Figs 5C, D). Hardly squeezed karyological slides revealed the presence of a series of small triangular spines (‘bursal nozzles’), often discernible as roughly arranged into two circles (Fig. 5A). The lack of surrounding nuclei suggests that these spines are basal lamina derivates, and not of an intracellular origin as the copulatory spines. In sectioned specimens, the striated, V-shaped structure is resolved as the two paired openings into the genital atrium of a wide bursal canal, provided with spines in its distalmost portion (Figs 2; 5E, F). The openings are separated by a transverse ridge, the core of which consists of a thick muscle bundle. These structures, including the bursal canal, are lined by a peculiar, deeply furrowed, infra-nucleated epithelium. It somehow resembles the ‘pseudociliation’ described for other Platyhelminthes (see Artois and Schockaert, 2001), but is much more regular in appearance, and with evenly spaced striae. These furrows correspond to the striae visible in living specimens. The outer bursal duct leads to a smooth inner portion, lined by what appears to be basal lamina, without overlaying epithelium, and which progressively narrows into a small, irregularly ovoid bursa, with a few chambers. The innermost chamber contained degenerating sperm, and may act as a bursa resorbiens.
Bursal spines are also found in P. jondelii (see Delogu and Curini-Galletti, 2007). In this species, they are arranged in two distinct blocks, suggesting that they may line the outlets of two independent bursal openings. However, in the sectioned material, only the outer, deeply furrowed portion of the bursal canal could be confidently traced.
Observation of living specimens of P. carthagoensis revealed a single, tubular bursal structure. In the karyological slides, a girdle of spines, sometimes fused basally into a tube (as in the specimen in Fig. 5B) could be seen. Sectioned material was not adequate to reconstruct the fine details of the structure. However, the presence of a single opening, surrounded by spines, leading to an outer bursal canal, deeply and evenly furrowed, could be detected with confidence. Morphology of the inner bursal area appears similar to P. crassispina.
P. pulchrispina lacks bursal spines. Its bursal canal is formed by an outer, undivided portion, lined by corrugated epithelium, and an inner area lined by basal lamina, leading to a small bursa, similar to that of P. crassispina.
Observation made on living specimens of P. gracilispina sp. n. revealed the presence of a clearly distinguishable, striated, inverted V-shaped bursal system, similar to previous species. In the karyological slides, however, no bursal spines were detected.
No information is available on the bursal system for the other species, nor could bursal spines be detected in karyological slides. It should be noted, however, that at least in P. crassispina, where a larger sample could be studied, bursal canal and spines were only visible in fully mature specimens. The lack of these structures may thus be related to immaturity.