Morphology and activities of cell populations of haemocytes in Oncomelania hupensis following Schistosoma japonicum infection.

Oncomelania hupensis is the only obligatory intermediate host of Schistosoma japonicum, the pathogen of zoonosis schistosomiasis. Haemocytes play a critical role in the cellular immune defence of O. hupensis against S. japonicum challenge. Here, the morphology and classification of haemocytes of O. hupensis were investigated by Giemsa staining and light microscopy, combining with the scanning and transmission electron microscopy and flow cytometry. Granulocytes and hyalinocytes were confirmed as two main types of haemocytes, account for ~ 10% and ~ 90% of all haemocytes, with size varying in 4.3-10.9 µm and 0.4-30.8 µm, respectively. Subpopulations can be identified further by granule feature, shape, size, and surface and inner structure of cells. The heterogeneity in morphology implied varied developmental process and function of haemocyte subpopulations. After the S. japonicum challenge, haemocytes of O. hupensis respond to S. japonicum invasion immediately. The dynamic change of haemocyte subpopulations indicates that the small hyalinocyte could differentiate into a larger one or granulocyte after S. japonicum challenge, and the granulocytes and larger hyalinocytes play leading roles in early defence reaction, but in different ways. Phagocytosis and apoptosis of haemocytes in O. hupensis were proved to be related to immune defence against S. japonicum, with the combined effect of granulocytes and larger hyalinocytes. However, the main pathway of each subpopulation to take effect in different periods need further investigation.

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails.

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 Å resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed ß-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

Effect of Copper Sulphate (CuSO4) on Freshwater Snail, Physa acuta Draparnaud, 1805: A Histopathological Evaluation.

In this study, freshwater snail (Physa acuta) was investigated to determine histopathological effects of CuSO4 on digestive gland, foot, mantle and ovotestis under laboratory conditions. The snails were exposed to different sublethal concentrations of CuSO4 (0.05 mg/L, 0.1 mg/L and 0.2 mg/L) periods of 10, 20 and 30 days. The relationship between CuSO4 concentration and mortality rate in snails was calculated as Y = 8.8 + 125.14X, R2 = 0.9444. The histopathological examinations revealed that CuSO4 caused significant histopathological changes in all the tissues of the snail. The severity of these lesions in tissues increased with increasing CuSO4 concentration and duration of exposure. The results showed that freshwater snail, Physa acuta can be considered to be a suitable bioindicator to demonstrate the toxic effect of copper in aquatic environments.

A molecular phylogeny of the Indo-West Pacific species of Haloa sensu lato gastropods (Cephalaspidea: Haminoeidae): Tethyan vicariance, generic diversity, and ecological specialization.

The cephalaspidean gastropod genus Haminoea has been considered a worldwide radiation with species living in intertidal and shallow areas with algae, seagrass sandy-mud, mangroves, and coral reefs. Recently this monophyletic status was questioned and it was suggested that Haminoea proper only included Atlantic plus eastern Pacific species, whereas the Indo-West Pacific species were a separate radiation belonging to the genus Haloa. In this work we used an extended dataset of Haminoea sensu lato including to our best knowledge representatives of all Indo-West Pacific species, plus a large representation of Atlantic and eastern Pacific species. Bayesian and maximum likelihood molecular phylogenetic analyses using three mitochondrial (COI, 12S rRNA, 16S rRNA) and two nuclear genes (28S rRNA and Histone-3) were employed to study relationships and diversification including also the closely related genus Smaragdinella. Our results support a monophyletic Atlantic + eastern Pacific Haminoea clade and a monophyletic Indo-West Pacific radiation with five genera all defined by unique morphological characters and ecological requirements, namely Haloa proper with tropical and sub-tropical species spread across the Indo-West Pacific, Lamprohaminoea including only colourful species of tropical and sub-tropical affinity, Bakawan gen. nov. with species associated with mangrove habitats across the tropical eastern Indian Ocean and western Pacific, Papawera gen. nov. restricted to temperate waters of Australasia, and Smaragdinella the only cephalaspidean genus inhabiting intertidal hard substrates across sub-tropical and tropical regions of the Indo-West Pacific. This result suggests the role of the closure of the Tethys seaway structuring the phylogeny of worldwide "Haminoea" snails and of ecology driving the phylogenetic structure of the Indo-West Pacific radiation.

Phylogeography of Bellamya (Mollusca: Gastropoda: Viviparidae) snails on different continents: contrasting patterns of diversification in China and East Africa.

BACKGROUND: Species diversity is determined by both local environmental conditions that control differentiation and extinction and the outcome of large-scale processes that affect migration. The latter primarily comprises climatic change and dynamic landscape alteration. In the past few million years, both Southeast Asia and Eastern Africa experienced drastic climatic and geological oscillations: in Southeast Asia, especially in China, the Tibetan Plateau significantly rose up, and the flow of the Yangtze River was reversed. In East Africa, lakes and rivers experienced frequent range expansions and regressions due to the African mega-droughts. To test how such climatic and geological histories of both regions relate to their respective regional species and genetic diversity, a large scale comparative phylogeographic study is essential. Bellamya, a species rich freshwater snail genus that is widely distributed across China and East Africa, represents a suitable model system to address this question. We sequenced mitochondrial and nuclear DNA for members of the genus from China and used published sequences from Africa and some other locations in Asia to investigate their phylogeny and distribution of genetic diversity. RESULTS: Our phylogenetic analysis revealed two monophyletic groups, one in China and one in East Africa. Within the Chinese group, Bellamya species show little genetic differentiation. In contrast, we observe fairly deep divergence among the East African lakes with almost every lake possessing its unique clade. Our results show that strong divergence does not necessarily depend on intrinsic characteristics of a species, but rather is related to the landscape dynamics of a region. CONCLUSION: Our phylogenetic results suggest that the Bellamya in China and East Africa are independent phylogenetic clades with different evolutionary trajectories. The different climate and geological histories likely contributed to the diverging evolutionary patterns. Repeated range expansions and regressions of lakes likely contributed to the great divergence of Bellamya in East Africa, while reversal of the river courses and intermingling of different lineages had an opposite effect on Bellamya diversification in China.

[Transmission electron microscopic observation on gonad of Oncomelania hupensis offspring bred in Weishan Lake areas].

OBJECTIVE: To observe the morphological changes in the testes and ovaries of adult 12th-generation Oncomelania hupensis bred for 12 winters in Weishan Lake areas. METHODS: The offspring of the adult O. hupensis snails bred in the Weishan Lake that were originated from the Yangzhou section of the Yangtze River was defined as the experiment group, while uninfected, adult O. hupensis snails captured from the marshland of the Yangzhou section of the Yangtze River served as the control group. Snails were dissected and intact testicular and ovarian specimens were sampled, routinely fixed, dehydrated, embedded, polymerized in an oven and sliced on an ultramicrotome. The sections were visualized under a transmission electron microscope, and the ultrastructure of the snail gonad was compared between the experiment and control groups. RESULTS: Transmission electron microscopy showed "9 + 2" microtubules on the transverse sections of the tails of sperm cells in the testes of male snails in the control group, with triangular acrosomes and spiral, dense nuclei seen in the tip, while in the experiment group, the "9 + 2" microtubules disappeared on the transverse sections of the tails of sperm cells in the testes of male snails, with low chromatin density found in the tip. Transmission electron microscopy revealed clear nucleolus and nuclear membranes in the ova of female snail ovaries, and displayed yolk body, liposomes and endoplasmic reticulum in the cytoplasm, bilayer twists of nuclear membrane and a uniform nucleolus in the control group, while in the experiment group, smooth nuclear membrane and unclear nucleolus were observed in the ova of female snail ovaries, with few contents seen within cells. CONCLUSIONS: Following breeding for 12 winters in the Weishan Lake, the 12th-generation O. hupensis snails fail to fully adapt to the natural environment in northern China, and the remarkable changes in the ultrastructure of the snail gonad may be a cause of gradual decline and even extinction of O. hupensis in the Weishan Lake areas.

Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos.

The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24-28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

The structure of the cutaneous pedal glands in the banded snail Cepaea hortensis (Müller, 1774).

Although gastropods have been crawling through the ocean and on the land for 60 million years, we still know very little about the sticky mucus produced in their foot. Most research has been focused on marine species in particular and, to a lesser extent, on the well-known terrestrial species Arion vulgaris and Cornu aspersum. Within this study, we aim to characterize the foot anatomy of a smaller representative of the family Helicidae, the banded snail Cepaea hortensis. We are particularly interested in the microanatomy of the foot glands, their position, and the histochemical nature of their secretory content. Characterization of the dorsal foot region of Cepaea hortensis reveals four glands, differing in their size and in the granules produced. Histochemically, three of them react positively for sugars (PAS staining and lectin affinity tests for mannose, glucose and N-acetyl-d-glucosamine) and acidic proteins (positive Alcian blue and Toluidine blue staining), indicating the presence of acidic glycosaminoglycans. The fourth gland type does not react to any of these dyes. The ventral pedal region includes two different gland types, which are positive for the presence of acidic glycoproteins, with a lectin affinity for mannose only. A comparison with Helix pomatia indicates differences regarding the number of glands and their contents. In Helix, only three gland types are described in the dorsal region of the foot, which show a similar granular appearance but nevertheless differ in their chemical composition. Congruently, there are two gland types in the ventral region in both species, whereas in Helix an additional sugar moiety is found. This raises the question whether these differences between the pedal glandular systems of both helicid species are the result of protection or size-related adaptations, as they occur in the same habitat.

Nerve-granular cell communication in the atrium of the snail Achatina achatina occurs via the cardioexcitatory transmitters serotonin and FMRFamide.

In the present study, the anatomical association and functional interaction between nerve fibres and granular cells in the atrium of the snail Achatina achatina are investigated using a combination of scanning electron microscopy (SEM), pharmacological and immunofluorescence techniques. The SEM studies support a close anatomical association of axons with granular cells and new features of surface morphology are revealed. Pharmacological experiments showed that both serotonin and FMRFamide were able to induce degranulation of granular cells and the release of cysteine-rich atrial secretory protein. Serotonin- and FMRFamide-immunoreactive nerve fibres were observed at variable distances from granular cells, ranging from close contact to distances as far as the diameter of a muscle bundle. These results suggest that serotonin and FMRFamide play a role as paracrine excitatory transmitters in nerve-to-granular cell communication.

Novel shell device for gas exchange in an operculate land snail.

The operculum of terrestrial snails tightly seals the shell aperture providing protection from predators and body-water loss. To allow respiration with a closed operculum, operculate land snails repeatedly evolved shell devices such as tubes or channels that open to the air. In all Asian members of the Alycaeidae, an externally closed tube lies along the suture behind the aperture that possesses a small internal opening into the last whorl at the tube's anterior end. However, this structure presents a paradox: how is gas exchanged through an externally closed tube? Here we show that many microtunnels open into the tube and run beneath radial ribs along the growth line of the last whorl in Alycaeus conformis These tunnels open to the outside of the shell surface near the umbilicus. Examination under high magnification revealed that the outermost shell layer forms these tunnels only in the whorl range beneath the sutural tube. Each tunnel (ca 16 µm diameter) is far narrower than any known metazoan parasite. These findings support our hypothesis that the externally closed sutural tube functions with microtunnels as a specialized apparatus for predator-free gas exchange with minimal water loss when the operculum seals the aperture.

On the Ultrastructure and Function of Rhogocytes from the Pond Snail Lymnaea stagnalis.

Rhogocytes, also termed "pore cells", occur as solitary or clustered cells in the connective tissue of gastropod molluscs. Rhogocytes possess an enveloping lamina of extracellular matrix and enigmatic extracellular lacunae bridged by cytoplasmic bars that form 20 nm diaphragmatic slits likely to act as a molecular sieve. Recent papers highlight the embryogenesis and ultrastructure of these cells, and their role in heavy metal detoxification. Rhogocytes are the site of hemocyanin or hemoglobin biosynthesis in gastropods. Based on electron microscopy, we recently proposed a possible pathway of hemoglobin exocytosis through the slit apparatus, and provided molecular evidence of a common phylogenetic origin of molluscan rhogocytes, insect nephrocytes and vertebrate podocytes. However, the previously proposed secretion mode of the respiratory proteins into the hemolymph is still rather hypothetical, and the possible role of rhogocytes in detoxification requires additional data. Although our previous study on rhogocytes of the red-blooded (hemoglobin-containing) freshwater snail Biomphalaria glabrata provided much new information, a disadvantage was that the hemoglobin molecules were not unequivocally defined in the electron microscope. This made it difficult to trace the exocytosis pathway of this protein. Therefore, we have now performed a similar study on the rhogocytes of the blue-blooded (hemocyanin-containing) freshwater snail Lymnaea stagnalis. The intracellular hemocyanin could be identified in the electron microscope, either as individual molecules or as pseudo-crystalline arrays. Based on 3D-electron microscopy, and supplemented by in situ hybridization, immunocytochemistry and stress response experiments, we provide here additional details on the structure and hemocyanin biosynthesis of rhogocytes, and on their response in animals under cadmium and starvation stress. Moreover, we present an advanced model on the release of synthesized hemocyanin molecules through the slit apparatus into the hemolymph, and the uptake of much smaller particles such as cadmium ions from the hemolymph through the slit apparatus into the cytoplasm.

Ultrastructural localization of NADPH diaphorase and nitric oxide synthase in the neuropils of the snail CNS.

Comparative studies on the nervous system revealed that nitric oxide (NO) retains its function through the evolution. In vertebrates NO can act in different ways: it is released solely or as a co-transmitter, released from presynaptic or postsynaptic site, spreads as a volumetric signal or targets synaptic proteins. In invertebrates, however, the possible sites of NO release have not yet been identified. Therefore, in the present study, the subcellular distribution of the NO synthase (NOS) was examined in the central nervous system (CNS) of two gastropod species, the terrestrial snail, Helix pomatia and the pond snail, Lymnaea stagnalis, which are model species in comparative neurobiology. For the visualization of NOS NADPH-diaphorase histochemistry and an immunohistochemical procedure using a universal anti-NOS antibody were applied. At light microscopic level both techniques labeled identical structures in sensory tracts ramifying in the neuropils of central ganglia and cell bodies of the Lymnaea and Helix CNS. At ultrastructural level NADPH-d reactive/NOS-immunoreactive materials were localized on the nuclear envelope and membrane segments of the rough and smooth endoplasmic reticulum, as well as the cell membrane and axolemma of positive perikarya. NADPH-d reactive and NOS-immunoreactive varicosities connected to neighboring neurons with both unspecialized and specialized synaptic contacts. In the varicosities, the majority of the NADPH-d reactive/NOS-immunoreactive membrane segments were detected in round and pleomorph agranular vesicles of small size (50-200 nm). However, only a small portion (16%) of the vesicles displayed the NADPH-d reactivity/NOS-immunoreactivity. No evidence for the postsynaptic location of NOS was found. Our results suggest that the localization of NADPH-diaphorase and NOS is identical in the snail nervous system. In contrast to vertebrates, however, NO seems to act exclusively in an anterograde way possibly released from membrane segments of the presynaptic transmitter vesicle surface. Based on the subcellular distribution of NOS, NO could be both a volume and a synaptic mediator, in addition NO may function as a co-transmitter.

Radix natalensis: the effect of Fasciola hepatica infection on the reproductive activity of the snail.

Experimental infections of Egyptian Radix natalensis (shell height at miracidial exposure: 4 mm) with a French isolate of Fasciola hepatica were carried out under laboratory conditions at 22 °C to specify the characteristics and follow the dynamics of their egg-laying. Controls constituted unexposed R. natalensis of the same size. No significant difference between controls and the uninfected snails of the exposed group was noted, whatever the parameter considered. In controls and exposed snails, the dates of the first egg masses were close to each other (56.4-65.3 days). In contrast, the life span of snails and the length of the egg-laying period were significantly shorter and egg production was significantly lower in infected R. natalensis than in controls and uninfected snails. In infected R. natalensis, but without cercarial shedding (NCS snails), egg production was irregular throughout the egg-laying period. In cercarial-shedding (CS) snails, the first egg masses were laid before the first cercarial emergence (at a mean of 56 days and 67 days, respectively). Thereafter, egg mass production of CS snails was irregular up to day 72 of the experiment, stopped during the following two weeks and started again after day 88 for a single snail. In conclusion, the F. hepatica infection of R. natalensis reduced the reproductive activity in both NCS and CS snails. The pattern noted for egg production in infected R. natalensis seems to be species-specific because of the high shell size of this lymnaeid and its role as an atypical intermediate host in the life cycle of the parasite.

Effect of cardiac glycosides from Nerium indicum on feeding rate, digestive enzymes activity and ultrastructural alterations of hepatopancreas in Pomacea canaliculata.

Cardiac glycosides from Nerium indicum showed potent molluscicide activity against Pomacea canaliculata (GAS), but the toxicological mechanism is still far less understood. Effects of sublethal treatments of cardiac glycosides on feeding rate, digestive enzymes and ultrastructural alterations of the hepatopancreas in GAS were evaluated in this study. Exposure of GAS to sublethal concentrations of cardiac glycosides resulted in a significant reduction of feeding rate of GAS. The amylase, cellulose and protease activity were increase significantly at the end of 24 h followed by significant inhibition after 48 h of exposure while lipase activity was not affected significantly at the end of 24 h followed by a significant inhibition after 48 h of exposure during experimental period. The main ultrastructural alterations of hepatopancreas observed in snails under cardiac glycosides treatment comprised disruption of nuclear membrane, increased vesiculation and dilatation of endoplasmic reticulum, and vacuolization and swelling of mitochondrial compared to the untreated GAS. These results, for the first time, provide systematic evidences showing that cardiac glycosides seriously impairs the hepatopancreas tissues of GAS, resulting in inhibition of digestive enzymes activity and feeding rate and cause GAS death in the end.

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata.

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches. Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.

Oxychilus (Drouetia) viridescens (Gastropoda: Pulmonata: Oxychilidae), a new species from Santa Maria, Açores, and a review of the subgenus.

Oxychilus (Drouetia) viridescens n.sp. is described from Santa Maria Island, Açores. It is conchologically similar to the sympatric Oxychilus (Drouetia) brincki Riedel, 1964, from which it is distinguished by the greenish coloration, the flatter spire and the slightly smaller number of whorls. Anatomically, the new species differs from all consubgenerics by the genital morphology: the penis is very thin, the distal half is wrapped in a very thick penial sheath; the epiphallus has two distinct portions, the proximal one attached to the edge of the penial sheath, the distal one attached to a constriction near mid-length of the penis; the atrial end of the vagina has a spongy, glandular appearance. A detailed morphological and anatomical comparison of the new species with the non-umbilicated Oxychilus species of Santa Maria is presented. The species of Drouetia are reviewed and an identification key is provided.

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model.

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.

A hairy business-periostracal hair formation in two species of helicoid snails (gastropoda, stylommatophora, helicoidea).

In molluscs, the calcareous shell is covered externally by a thin organic layer, the periostracum. The periostracum of some pulmonate species is of special taxonomic interest because it bears distinct microscale architectures. Where and how these structures are formed is as yet unknown. Using histological sections through their shells, gelatin cuts, and live observations I studied the pattern by which the periostracal hair-like projections in two helicoid land snail species are secreted and evenly arranged on the shell. The results indicate a complex mechanism: a hair is formed in the periostracal groove independently of the periostracum, after which it is attached to the edge of the shell, drawn out of the tissue, and finally swivelled to the upper side of the periostracum. Upon further growth of the periostracum, the hairs are finally fixed upright on the shell.