3-dimensional visualisation of the anatomy of Pontohedyle milaschewitchii (Kowalevsky, 1901) (Gastropoda, Opisthobranchia, Acochlidia) with remarks on its reproductive biology
Knowledge on the anatomy of acochlidian opisthobranch gastropods is still scarce, and successful phylogenetic analysis is hindered by the poor set of reliable data on most acochlidian species. In former studies, one member of each of the major traditional subgroups, i.e. Hedylopsacea and Microhedylacea, has been examined in detail. The use of few model organisms, however, cannot satisfactorily reflect the enormous structural and biological variation known to occur among acochlidian species. Previous studies also indicated that the current classifications of Acochlidia are contradictory and do not reflect phylogenetic relationships.
The present study supplements the knowledge on the anatomy of acochlidians by the detailed anatomical, histological and ultrastructural examination of the marine interstitial microhedylacean Pontohedyle milaschewitchii (Kowalevsky, 1901). This is an abundant member of shallow subtidal sands from the Black Sea throughout the Mediterranean Sea and is easily identifiable due to externally visible features such as the lack of rhinophores and the presence of eyes. It was thus mentioned in several ecological papers and commonly thought to be a likewise well-known acochlidian species. However, biological and anatomical knowledge was fragmentary, little detailed and hardly reliable, and contradictory information (e.g. on the radula formula) in different data sources required reinvestigation. Moreover, the taxonomic status of the south-western Atlantic Pontohedyle brasilensis (Rankin, 1979) is uncertain; some potential differences to P. milaschewitchii needed critical re-examination and comparison with the previously unknown adult and ontogenetic variation found in Mediterranean populations.
About 200 individuals of P. milaschewitchii from 0.4 to 3 mm body length were extracted from coarse subtidal sands from Istria (Croatia, Mediterranean) and used for feeding, life-span and reproduction experiments. Several entire specimens were embedded in Spurr’s resin and semithin serial sections were prepared. All major organ systems of P. milaschewitchii were reconstructed 3-dimensionally using AMIRA software. Additional comparative anatomical examination of a Brazilian individual and of some early juvenile stages from the Mediterranean was conducted. The radula and the pattern of ciliation of the body wall of entire specimens was analysed by SEM. The ultrastructure of the epidermis and the spermatozoa was studied using TEM. The ultrastructure of an acochlidian spermatophore was analysed for the first time. Cutaneous fertilisation via spermatophores could be directly observed on intruding DAPI-stained spermatozoa.
Pontohedyle milaschewitchii is externally characterised by the lack of rhinophores and by having bow-shaped to elongated triangular oral tentacles. In contrast to most other acochlidian species the foot of P. milaschewitchii is very short and has a rounded posterior end. SEM-examination of the body wall revealed a constant ciliary pattern, comprised of two ciliary bands on the oral tentacles, one transverse ciliary band just behind of the tentacles and scatted bundles of cilia on the entire head-food complex, excluding the posterodorsal part. Thus, SEM-examination of entire specimens might be a useful technique and ciliary patterns a helpful additional character for separation and definition of acochlidian species in the future.
The pre-pharyngeal central nervous system (cns) of P. milaschewitchii is highly concentrated and shows a euthyneurous and probably epiathroid condition. In contrast to earlier reports, the cerebral and pleural ganglia are not fused, as considered characteristic for all acochlidian taxa. The aggregations of accessory ganglia in the anterior region of the cns could be grouped into three complexes that are innervated by three distinct cerebral nerves. Considering the additional static nerve, P. milaschewitchii possesses four pairs of cerebral nerves, rejecting earlier assumptions of a constant reduction to three pairs of cerebral nerves within the Acochlidia. The presence of a Hancock’s organ, in form of a conspicuous fold in the epidermis just posterior to the oral tentacles could be confirmed for P. milaschewitchii.
The fairly large oral gland opens via an unpaired duct to the exterior just anteriorly to the mouth opening. A radula formula of 41-54 x 1.1.1 could be determined for adult specimens from the Mediterranean. The original light-microscopic description of 2 lateral teeth from the Brazilian specimen is explained by having misinterpreted a median notch and denticle as a separation of the single, broad lateral tooth. A large, spherical stomach could not be detected in the material studied herein and its description for the Brazilian specimen is explained by a misinterpretation of biological or artificial factors. The position of the anal opening varies during ontogeny; being located on the right side of the body in the posterior region of the head-foot complex in early juvenile stages and in the anterior part of the visceral hump in adult specimens. The heart of P. milaschewitchii is reduced; no clear division into auricle and ventricle could be detected.
Sexes are separate. The presence of an unusual cephalic male genital opening could be confirmed. The described “intraepidermal duct” in male specimens can be functionally considered as ciliated part of the vas deferens and, from a histological view, is sub-epidermal. The ultrastructure of the spermatozoa closely resembles the one described for M. remanei in having an exceptionally large, helically keeled nucleus and one glycogen helix coursing around the mid-piece. However, considerable differences occur to the sperm described for Hedylopsis ballantinei Sommerfeldt & Schrödl, 2005 (Hedylopsacea) regarding the development of the mid-piece and, probably, the length and morphology of the sperm nucleus. The female genital system of P. milaschewitchii shows large similarities with the one described for M. remanei, concerning shape, relative size and histology of the nidamental glands. In comparison to other acochlidians, the mature eggs of P. milaschewitchii are comparably small and suggest a free veliger larva, rather than an intra-capsular development as known for Asperspina riseri (Morse, 1976). Thus, at this point of knowledge it can be concluded that at least two different reproductive strategies might have evolved in the acochlidians.
The high intraspecific variation of some characters (e.g. shape of oral tentacles) in adults, plus the observed ontogenetic variation (e.g. position of anal opening) weaken the seperation of P. milaschewitchii and P. brasilensis into two different species. Moreover, re-examination could not confirm the described differences (e.g. radula formula, fusion of ganglia, pattern of cilia). Thus, no more distinguishing features are left, which justify the separation of the two populations at this point of knowledge. Therefore, the results of the present study on the Mediterranean P. milaschewitchii and an additional specimen from Brazil strongly support earlier assumptions that P. brasilensis is a junior synonym of P. milaschewitchii. The monotypic genus Gastrohedyle Rankin, 1979 that was based on P. brasilensis, thus is a synonym of Pontohedyle Golikov & Starobogatov, 1972. Two different species of Pontohedyle remain: the Atlantic P. milaschewitchii and the Pacific P. verrucosa (Challis, 1970), easily distinguishable as the latter lacks eyes and spicules.