The shell formation mechanism of Turbo argyrostomus based on ultrastructure and transcriptome analysis.

The gold inner shell of Turbo argyrostomus is an important morphological classification characteristic in Gastropoda. However, the gene sets responsible for shell formation in gastropods remain poorly explored. In this study, we investigated the microstructure using scanning electron microscopy (SEM), hematoxylin-eosin (HE) and Alcian blue staining-periodic acid-Schiff (AB-PAS) staining. The SEM results illustrated that the T. argyrostomus shell exhibited a special "sandwich" microstructure. The results of histological observation demonstrated two major cell types: adipocytes and mucin cells. A total of 318 differentially expressed genes were identified between edge mantle and central mantle, among which whey acidic protein, N66, and nacre-like proteins, and Lam G and EGF domains may be related to shell microstructure. 22.39% - 25.20% of the mucin genes had biomineralization related domains, which supported for the relationship between mucins and shell formation. Moreover, this study revealed energy distribution differences between the edge mantle and central mantle. These results provide insights for further understanding of the biomineralization mechanism in Gastropoda.

The multilevel organismal diversity approach deciphers difficult to distinguish nudibranch species complex.

Species identification is a key procedure for broad-scoped ecological, phylogeographic and evolutionary studies. However, to perform a taxonomic study in the molecular era is a complicated task that has many pitfalls. In the present study we use particular examples of common but difficult to distinguish European species within the genus of Polycera (Nudibranchia, Mollusca) to discuss the general issues of the "cryptic species" problem that has broad biological and interdisciplinary importance and can significantly impede ecological, evolutionary, and other biodiversity-related research. The largest dataset of molecular and morphological information for European nudibranchs ever applied encompasses a wide geographical area and shapes a robust framework in this study. Four species are recognized in the species complex, including a new one. It is shown that a lack of appropriate taxonomic analysis led recently to considerable errors in species identity assessment of this complex. Chromatic polymorphism for each species is mapped in a periodic-like framework and combined with statistical analysis of the diagnostic features that considerably facilitates identification of particular species in the complex for biologists and practitioners. The present study evidently shows that "cryptic" and "non-cryptic" components are present within the same species. Therefore, this species complex is well suited for the exploring and testing of general biological problems. One of the main conclusions of this study is that division of biological diversity into "cryptic" and "non-cryptic" components is counterproductive. We propose that the central biological phenomenon of a species can instead be universally designated as multilevel organismal diversity thereby provide a practical set of methods for its investigation.

Feeding experiments on Vittina turrita (Mollusca, Gastropoda, Neritidae) reveal tooth contact areas and bent radular shape during foraging.

The radula is the food gathering and processing structure and one important autapomorphy of the Mollusca. It is composed of a chitinous membrane with small, embedded teeth representing the interface between the organism and its ingesta. In the past, various approaches aimed at connecting the tooth morphologies, which can be highly distinct even within single radulae, to their functionality. However, conclusions from the literature were mainly drawn from analyzing mounted radulae, even though the configuration of the radula during foraging is not necessarily the same as in mounted specimens. Thus, the truly interacting radular parts and teeth, including 3D architecture of this complex structure during foraging were not previously determined. Here we present an experimental approach on individuals of Vittina turrita (Neritidae, Gastropoda), which were fed with algae paste attached to different sandpaper types. By comparing these radulae to radulae from control group, sandpaper-induced tooth wear patterns were identified and both area and volume loss could be quantified. In addition to the exact contact area of each tooth, conclusions about the 3D position of teeth and radular bending during feeding motion could be drawn. Furthermore, hypotheses about specific tooth functions could be put forward. These feeding experiments under controlled conditions were introduced for stylommatophoran gastropods with isodont radulae and are now applied to heterodont and complex radulae, which may provide a good basis for future studies on radula functional morphology.

Functional morphology of the sperm-containing chambers of the sea slug Okenia polycerelloides in the context of sexual selection.

Sea slugs are interesting models to study post-copulatory sexual selection in simultaneous hermaphrodites due to the enormous variation of their reproductive systems. However, the knowledge of the functional morphology of their reproductive system is limited to few species, and it is rarely discussed in the context of sexual selection theory. In this study, we investigated the functional morphology of the sperm-containing chambers (i.e., ampulla, seminal receptacle, and bursa copulatrix) of the reproductive system of Okenia polycerelloides (Ortea & Bouchet, 1983), based on light, confocal, and electron microscopy. Although the morphology of the ampulla is similar to other species, indicating that it is a site for autosperm storage, we found some sperm facing the ampullar epithelium, a feature commonly regarded as characteristic of the seminal receptacle of sea slugs. The seminal receptacle of O. polycerelloides showed secretory activity and contained sperm with distribution and orientation suggestive of stratification of allosperm from distinct mating events, a feature that would affect sperm competition. The bursa copulatrix had epithelial cells with secretory and absorptive characteristics, and contained degraded sperm and yolk granules within its lumen. Comparative analyses of the contents of each organ demonstrated that sperm digestion occurs in the bursa copulatrix and affects sperm heads first, changing their morphology from slender and curved to shorter and ellipsoid before complete lysis. Although digestion and absorption of surplus sperm are currently the main hypothesized functions for the bursa copulatrix, its role in cryptic female choice should not be ruled out. The close structural connection between the seminal receptacle and bursa copulatrix, as well as their muscular walls, would enable control over the fate of the sperm received in each mating event, that is, storage or digestion.

Radula formation in two species of Conoidea (Gastropoda).

The radula is the basic feeding structure in gastropod molluscs and exhibits great morphological diversity that reflects the exceptional anatomical and ecological diversity occurring in these animals. This uniquely molluscan structure is formed in the blind end of the radular sac by specialized cells (membranoblasts and odontoblasts). Secretion type, and the number and shape of the odontoblasts that form each tooth characterize the mode of radula formation. These characteristics vary in different groups of gastropods. Elucidation of this diversity is key to identifying the main patterns of radula formation in Gastropoda. Of particular interest would be a phylogenetically closely related group that is characterized by high variability of the radula. One such group is the large monophyletic superfamily Conoidea, the radula of which is highly variable and may consist of the radular membrane with five teeth per row, or the radular membrane with only two or three teeth per row, or even just two harpoon-like teeth per row without a radular membrane. We studied the radulae of two species of Conoidea (Clavus maestratii Kilburn, Fedosov & Kantor, 2014 [Drilliidae] and, Lophiotoma acuta (Perry, 1811) [Turridae]) using light and electron microscopy. Based on these data and previous studies, we identify the general patterns of the radula formation for all Conoidea: the dorsolateral position of two groups of odontoblasts, uniform size, and shape of odontoblasts, folding of the radula in the radular sac regardless of the radula configuration. The morphology of the subradular epithelium is most likely adaptive to the radula type.

Phylogenetic relationships of ghost slugs (Selenochlamys) and overlooked instances of limacization in Western Palaearctic Limacoidei (Gastropoda: Stylommatophora).

We analysed the phylogenetic relationships of the enigmatic ghost slug Selenochlamys with other Western Palaearctic Limacoidei based on mitochondrial and nuclear sequences. Selenochlamys is a carnivorous slug group from the Caucasus region. Until now, it has been classified in Trigonochlamydidae, which includes several carnivorous slug groups from the Caucasus region. However, the molecular phylogenetic analyses revealed that Selenochlamys originated from omnivorous snails belonging to Oxychilidae. The similarities of Selenochlamys and Trigonochlamydidae are convergences resulting from limacization, the evolutionary transition from shelled snails to slugs, and from the transition from herbivory or detritivory via omnivory to obligate carnivory. These two evolutionary trends occurred repeatedly within the Limacoidei. Phylogenetic analyses based on morphological characters are prone to underestimate the instances with which such recurrent evolutionary trends occurred, as they depend on the parsimony criterion. The molecular phylogenetic analyses revealed that Limacoidea and Parmacelloidea, which were at least partly based on characters associated with limacization, are polyphyletic. The molecular phylogenetic tree implied that 5-7 independent limacizations occurred in the Western Palaearctic Limacoidei. Half of these limacizations remained undetected in a previous parsimony analysis based on morphological characters. Moreover, the analyses revealed that Godwiniinae belong to Gastrodontidae, not Oxychilidae.

Characterization of the Arion vulgaris pedal gland system.

The most common European gastropod species, Arion vulgaris, is one of the most troublesome pests for private garden owners and commercial agriculturists. The sticky and hard to remove secretion produced by these animals allows them to overcome most artificial and natural barriers. However, this highly adherent biopolymer has recently shown great potential for novel wound-healing applications in medicine. Nevertheless, our knowledge of the underlying gland system is still limited and few studies on the ventral gland system are available. We studied the lateral and ventral pedal glands in Arion vulgaris to determine their secretory content histochemically and through lectin assays. Using these histological and histochemical methods we differentiate five gland types with different mucus composition in the lateral pedal region of the foot of Arion vulgaris. These contain sulphated and carboxylated mucosubstances (positive Alcian blue staining) but lack hexose-containing mucosubstances (negative PAS staining). In the ventral pedal region, four gland types can be differentiated producing sulphated and carboxylated mucosubstances. Within the ventral mucus, a high affinity for the lectins PNA and WGA is observed. While the lateral glands are histochemically negative for PAS, a positive staining with the lectin JAC is observed. Arion vulgaris shows clear morphological differences from other arionid species. This raises the question whether the variation in the chemistry of the secretory material and mucus composition is the result of different functions and/or is related to the animals' different environmental conditions. A comparison of some glands of Arion vulgaris with those of the helicid species Helix pomatia and Cepaea hortensis indicates morphological similarities.

General morphology and ultrastructure of the radula of Testudinalia testudinalis (O. F. Müller, 1776) (Patellogastropoda, Gastropoda).

The radular morphology of the patellid species Testudinalia testudinalis (O. F. Müller, 1776) from the White Sea was studied using light, electron, and confocal microscopy. The radula is of the docoglossan type with four teeth per row and consisting of six zones. We characterize teeth formation in T. testidinalis as follows: one tooth is formed by numerous and extremely narrow odontoblasts through apocrine secretion; this initially formed tooth consists of numerous vesicles; the synthetic apparatus of the odontoblasts is localized in the apical and central parts of the cells throughout the cytoplasm and is penetrated by microtubules which are involved in the transport of the synthesized products to the apical part of the odontoblast; the newly formed teeth consist of unpolymerized chitin. Mitotic activity is located in the lateral parts of the formation zone. The first four rows contain an irregular arrangement of teeth, but the radular teeth are regularly arranged after the fifth row. The irregularly arranged teeth early on could be a consequence of the asynchronous formation of teeth and the distance between the odontoblasts and the membranoblasts. The morphological data obtained significantly expands our knowledge of the morphological diversity of the radula formation in Gastropoda.

Morphology of the digestive gland of the marine panpulmonate limpet Siphonaria lessonii: A cytological, histochemical, and ultrastructural description.

The molluskan digestive gland has been widely studied and its structural and ultrastructural descriptions have allowed the understanding of its several functions. Despite siphonarids are broadly distributed around the world, morphological studies on their digestive system are poorly represented. The panpulmonate limpet Siphonaria lessonii is the most abundant gastropod and the dominant herbivore in the rocky intertidal coast of Buenos Aires. The aim of this study was to describe the morphology, histology, ultrastructure, and histochemistry of the digestive gland of this gastropod as well as the cycle of activity of digestion. For that, different histochemical techniques along with light microscopy, transmission electron microscopy, and scanning electron microscopy were employed. This study revealed a complex epithelium, composed of a simple layer with five cell types. Digestive cells and vacuolated cells are responsible for intracellular digestion and energy accumulation; basophilic cells, secrete substances that would be involved in extracellular digestion; pigmented cells might have an excretory function and thin cells would correspond to undifferentiated cells. In addition, the tubules present a changing morphology according to the digestive activity that they undergo. As S. lessonii is a grazer that feeds continuously, the cycle of activity of the digestive gland seems to be daily.

Multilevel fine-scale diversity challenges the 'cryptic species' concept.

'Cryptic' species are an emerging biological problem that is broadly discussed in the present study. Recently, a cryptic species definition was suggested for those species which manifest low morphological, but considerable genetic, disparity. As a case study we present unique material from a charismatic group of nudibranch molluscs of the genus Trinchesia from European waters to reveal three new species and demonstrate that they show a dual nature: on one hand, they can be considered a 'cryptic' species complex due to their overall similarity, but on the other hand, stable morphological differences as well as molecular differences are demonstrated for every species in that complex. Thus, this species complex can equally be named 'cryptic', 'pseudocryptic' or 'non-cryptic'. We also present evidence for an extremely rapid speciation rate in this species complex and link the species problem with epigenetics. Available metazoan-wide data, which are broadly discussed in the present study, show the unsuitability of a 'cryptic' species concept because the degree of crypticity represents a continuum when a finer multilevel morphological and molecular scale is applied to uncover more narrowly defined species making the 'cryptic' addition to 'species' redundant. Morphological and molecular methods should be applied in concordance to form a fine-scale multilevel taxonomic framework, and not necessarily implying only an a posteriori transformation of exclusively molecular-based 'cryptic' species into morphologically-defined 'pseudocryptic' ones. Implications of the present study have importance for many fields, including conservation biology and fine-scale biodiversity assessments.

Spermatogenesis and lobular cyst type of testes organization in marine gastropod Littorina saxatilis (Olivi 1792).

Although individual stages of spermatogenesis in Littorina saxatilis are well studied at the electron microscopic level, the gonad structure and the spatial localization of gametes at different stages of maturation remain unclear. Using differential-interference contrast (DIC) for observations of fresh tissue we show that the mature testis consists of numerous ovoid lobules forming larger lobes. The lobules of intact mature testes of L. saxatilis are filled with randomly arranged multicellular cysts containing gametes at different stages of maturation. Gametes within a cyst are highly synchronized in respect of the differentiation degree. At the same time, no spatial gradient in the arrangement of cysts according to the maturation degree of gametes in them was observed in any of the studied lobules. The male gonads contain cysts with early spermatids, mid, late spermatids, and spermatozoa. Using silver-staining, DAPI, and chromomycin A3 (CMA3) staining, we identify 20 main types of nucleus organization in differentiating sperm. Premature and mature male gonads contain cysts with a mosaic arrangement as well as rare solitary cyst cells, goniablast cysts, or separate spermatogonia in between them. Our data indicate that the testis structure in L. saxatilis cannot be attributed to the tubular type, as previously thought. It corresponds to the lobular cyst type but individual lobules contain cysts with gametes at the same stage of development. It is similar to the testis structure of several fishes, amphibians, and Drosophila melanogaster. This type of the gonad organization has never been described in gastropods before.

Siphonariid development: Quintessential euthyneuran larva with a mantle fold innovation (Gastropoda; Panpulmonata).

Recent phylogenetic revisions of euthyneuran gastropods ("opisthobranchs" and "pulmonates") suggest that clades with a planktotrophic larva, the ancestral life history for euthyneurans, are more widely distributed along the trunk of the euthyneuran tree than previously realized. There is some indication that the planktotrophic larva of euthyneurans has distinctive features, but information to date has come mainly from traditional "opisthobranch" groups. Much less is known about planktotrophic "pulmonate" larvae. If planktotrophic larvae of "pulmonates" share unique traits with those of "opisthobranchs," then a distinctive euthyneuran larval-type has been the developmental starting template for a spectacular amount of evolved morphological and ecological disparity among adult euthyneurans. We studied development of a siphonariid by preparing sections of larval and postmetamorphic stages for histological and ultrastructural analysis, together with 3D reconstructions and data from immunolabeling of the larval apical sensory organ. We also sought a developmental explanation for the unusual arrangement of shell-attached, dorso-ventral muscles relative to the mantle cavity of adult siphonariids. Adult siphonariids ("false limpets") have a patelliform shell but their C-shaped shell muscle partially embraces a central mantle cavity, which is different from the arrangement of these components in patellogastropods ("true limpets"). It is not obvious how shell muscles extending into the foot become placed anterior to the mantle cavity during siphonariid development from a veliger larva. We found that planktotrophic larvae of Siphonaria denticulata are extremely similar to previously described, planktotrophic "opisthobranch" larvae. To emphasize this point, we update a list of distinctive characteristics of planktotrophic euthyneuran larvae, which can anchor future studies on the impressive evolvability of this larval-type. We also describe how premetamorphic and postmetamorphic morphogenesis of larval mantle fold tissue creates the unusual arrangement of shell-muscles and mantle cavity in siphonariids. This result adds to the known postmetamorphic evolutionary innovations involving mantle fold tissue among euthyneurans.

Drilling in the dorid species Vayssierea cf. elegans (Gastropoda: Nudibranchia): Functional and comparative morphological aspects.

The drilling mode of feeding is known from two clades of Gastropoda: Caenogastropoda and Heterobranchia. However, the level of convergence and parallelism or homology among these two lineages is unclear. The morphology of the buccal complex is well studied for drilling caenogastropods, but poorly known for drilling nudibranchs. It is also unclear whether the drilling feeding mechanism is similar between inside gastropods. Accordingly, a comparison between the feeding mechanisms of drilling nudibranchs and caenogastropods can help to understand the evolutional trends inside gastropods. In this study, we redescribe the morphology of the buccal complex of drilling dorid nudibranch Vayssierea cf. elegans, and compare it to that of previous investigations on this species and closely related dorid species. We describe the feeding mechanism of this species based on the obtained morphological and literature data and compare it to the feeding mechanisms described for drilling caenogastropods. The feeding apparatus of Vayssierea cf. elegans corresponds to the general morphology of the dorid buccal complex; that is, it has a similar arrangement of the buccal musculature and pattern of radular morphology. However, there are also adaptations to the drilling feeding mode similar to those found in Caenogastropoda: that is, specialized dissolving glands and lateral teeth with elongated pointed cusps; and even Sacoglossa: the specialized muscle for sucking. The feeding process of Vayssierea cf. elegans includes the same two stages as those described for drilling caenogastropods: (a) the boring stage, which is provided by mechanical and chemical activity, and (b) the swallowing stage.

Microstructures in relation to temperature-induced aragonite-to-calcite transformation in the marine gastropod Phorcus turbinatus.

Mollusk shells represent important archives for paleoclimatic studies aiming to reconstruct environmental conditions at high temporal resolution. However, the shells, made of calcium carbonate in the form of aragonite and /or calcite, can be altered through time which may undermine the suitability for any reconstruction based on geochemical proxies (i.e., stable isotopes, radiocarbon). At present, the diagenetic processes involved in this chemical and physical deterioration are still poorly understood. The present study aims to shed light on the onset and development of diagenetic alteration in the aragonitic shell of Phorcus turbinatus. To artificially mimic diagenesis, shells of P. turbinatus were exposed to elevated temperatures. The transformation of the mineral phase was monitored by means of Confocal Raman Microscopy whereas the structural changes were investigated using Scanning Electron Microscopy and Atomic Force Microscopy. The results indicate that the two distinct shell layers (prismatic layer and nacre) respond differently to the elevated temperatures, suggesting that the different microstructural organization and organic content may drive the onset and spread of the aragonite-to-calcite transformation. Furthermore, changes in the microstructural arrangement became visible prior to the mineralogical transition. Our results demonstrate that the specific physico-chemical characteristics of structurally different areas within the biogenic carbonates have to be taken into account when studying the phase transformation occurring during diagenesis.

The Microstructure, Proteomics and Crystallization of the Limpet Teeth.

Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase-Rabs, and clathrin, are identified by proteomics. A goethite-binding experiment coupled with proteomics and RNA-seq highlights six chitin-binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.

Digestive gland inclusion bodies in queen conch (Lobatus gigas) are non-parasitic.

Unusual inclusion bodies occur within the epithelial cells of the digestive gland of queen conch, Lobatus gigas, and have previously been described as apicomplexan parasites. The aim of this study was to investigate the parasitic features of these inclusion bodies in queen conch. L. gigas from St. Kitts (Caribbean Sea) consistently (100% of n = 61) showed large numbers of ovoid to tri-bulbous dark brown inclusion bodies (15 × 30 µm) within vacuolar cells. Histochemical stains demonstrated iron, melanin, and glycoprotein and/or mucopolysaccharide within the inclusion bodies. Microscopic features indicative of a host response to injury were lacking in every case, as were consistent morphological forms to indicate distinct parasitic stages. Transmission electron microscopy failed to reveal cellular organelles of parasitic organisms and DNA extractions of purified inclusion bodies did not yield sufficient concentrations for successful PCR amplification. Scanning electron microscopy with energy dispersive X-ray analysis revealed a number of elements, particularly iron, within the inclusion bodies. We conclude that the inclusion bodies are not an infectious agent, and hypothesize that they represent a storage form for iron, and potentially other elements, within a protein matrix. Similar structures have been described in the digestive glands of other invertebrates, including prosobranchs.

Sparkling Reflective Stacks of Purine Crystals in the Nudibranch Flabellina iodinea.

Although pigments contribute to much of the brilliant purple and orange coloration of the aeolid nudibranch Flabellina iodinea, the optical appearance of the animal was found to be augmented by dynamically sparkling, brightly reflective material in cells located throughout its epidermis. Electron microscopy revealed that specialized cells most abundant near the epithelial basal lamina contain numerous multilayer stacks of crystals, each within a fragile membrane capsule. High-resolution light microscopy of tissue sections showed that these crystalline stacks intermittently reflect light, with a temporally dynamic, sparkling appearance, suggesting that they are free to move-a phenomenon also observed in the live, intact whole animal and in the purified crystal stacks as well. Thin-layer chromatography and ultraviolet spectrometry show that the crystals isolated from all epithelial tissues are identical in composition, with guanine being the major component and its derivative, hypoxanthine, a minor component, regardless of the tissue's pigmentary color. Electron diffraction of the crystals purified separately from the orange and purple tissues exhibits nearly identical lattice parameters that closely match those measured for guanine crystals, which are widely distributed in other biophotonic systems ranging from marine invertebrates to terrestrial vertebrates. Heterogeneity of the thickness and spacing of the crystals within their stacks accounts for their broadband silvery reflectance. The optical appearance of the epidermis of this nudibranch thus results from the interaction of incident light with mobile stacks of purine crystals, augmenting the effects of its pigmentary colors.

Possible Mechanisms of Hatching from Egg Capsules in the Gastropods Crepipatella dilatata and Crepipatella peruviana, Species with Different Modes of Early Development.

Many invertebrates enclose their embryos within egg capsules, from which the offspring hatch. In marine gastropods that brood their egg capsules, hatching could involve radular activity by the mother or by unhatched stages, increased osmotic concentration of the intracapsular fluid, or production of hatching enzymes. The present research sought to determine whether mechanical action by the brooding female or by the encapsulated embryos was involved in the hatching for two sympatric and closely related species of calyptraeid: Crepipatella dilatata, which exhibits direct development without free-living larvae, and Crepipatella peruviana, which releases free-living veliger larvae. We also considered the role that enzymatic action or osmotic changes in the intracapsular fluid might play in hatching. Using scanning electron micrograph analyses, we found no evidence that the well-developed, pre-hatching juvenile radula of C. dilatata played any role in the hatching process and that the radula of C. peruviana did not even develop until long after hatching; so there was no evidence of radular activity involved in the hatching of either species. For C. peruviana, the intracapsular fluid osmolality was always higher than that of the surrounding seawater, suggesting that there is a strong natural water inflow during development. Moreover, when egg capsules of C. peruviana were exposed to lower ambient salinities, the substantial entry of water correlated well with high percentages of hatching, particularly for egg capsules containing advanced veligers, suggesting that an osmotic mechanism may be involved in the hatching process of this species. In contrast, hatching in C. dilatata appeared to be enzymatically mediated.

Mannitol oxidase and polyol dehydrogenases in the digestive gland of gastropods: Correlations with phylogeny and diet.

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet.

Histological, histochemical, and ultrastructural investigation of the male copulatory apparatus of Haminoea navicula (Gastropoda, Cephalaspidea).

Due to its biological and systematic importance, the morphology and function of the male copulatory apparatus of Haminoea navicula, a Cephalaspidea gastropod mollusk, was investigated by light and electron microscopy. These systems are poorly understood in haminoids, but are often used in the taxonomy of the genus. In H. navicula, the male copulatory apparatus comprises the penis within a penial sheath, a seminal duct and the prostate with two lobes. The penis is a muscular structure with a tip covered by spikes formed by muscular cells. The penial sheath consists of muscular tissue folds lined by an epithelium. Below this epithelium, polysaccharide-secreting cells and pigment cells were observed. A large number of vacuolar cells were found below the ciliated epithelium of the seminal duct. The proximal lobe of the prostate was formed by tubules that could be divided in basal, middle and apical zones, containing cells that secrete polysaccharides and proteins. The tubules of the prostate distal lobe contained a single type of secretory cells with vesicles that were stained by histochemical techniques for detection of polysaccharides and proteins. Ciliated cells were present along the tubules in both lobes of the prostate. This study revealed a complex prostate with five types of secretory cells, which secrete substances that should be involved in the maintenance and eventually also in the maturation of spermatozoa. A comparison with previous publications, shows that the male copulatory apparatus can differ substantially among cephalaspideans, even between H. navicula and non-European species attributed to this genus.