Albumen Gland Research Articles

Urate cells and tissues in the south american apple snail Pomacea canaliculata

Intracellular urate concretions are known to occur in some insects, crustaceans and gastropods. Crystalloid-containing urate cells and tissues are widespread in many organs of the apple-snail Pomacea canaliculata. The occurrence of three different urate tissues (perivascular, compact and nodular) correlated well with uric acid concentrations found in the organs containing them. The highest uric acid concentrations were found in the coiled gut (showing both perivascular and compact tissues), the style sac (compact tissue), the testis, the midgut gland and the lung (perivascular tissue) and the aortic ampulla (nodular tissue). Lower concentrations were found in organs with perivascular tissue associated only with their main supplying vessels (anterior kidney and ctenidium), while organs devoid of urate tissues had minimal concentrations (mantle skirt, posterior kidney, albumen gland and heart). Transmission electron microscopy of urate cells revealed a process of sequential differentiation and death, accompanied by the formation and resorption of urate crystalloids. This process occurred asynchronously in cells within the same tissue, suggesting an active turnover of uric acid in the organs containing them.

The population density effects on the reproductive biology of the snail Bradybaena similaris (Férussac, 1821) (Mollusca, Gastropoda)

The influence of population density on some aspects of the reproductive biology of the snail Bradybaena similaris was studied. Molluscs were maintained under 0.2 (isolated), 0.3, 0.6, 1.0, 1.3 and 1.7 snail/m(2) densities. The animals maintained under 0.3 and 0.6 snail/m(2) showed the lowest numbers of eggs laid/snail, being the highest value observed to the 1.7 snail/m(2). The hatching of the snails maintained under 0.3 snail/m(2) density, begun at the 21st day after laying, and the maximum time required to the hatching was 36 days was observed to the eggs came from snails maintained under the densities 0.6, 1.0, 1.3 snail/m(2), respectively. The highest percentage hatchability (55.56%) was observed to isolated snails. The galactogen content in the albumen gland did not seem to accompany the alterations occurred in the reproduction of B. similaris in response to the different population densities.

Effects of isolation and body size on the mating behaviour of the hermaphroditic land snail Succinea putris

We examined the combined effects of isolation and body size on (1) the likelihood of mating, (2) the activity role, (3) the precopulatory behaviour, (4) the copulation duration, (5) the reciprocity of sperm transfer and (6) the number of (reciprocally) transmitted sperm in the hermaphroditic land snail Succinea putris. Also, we assessed the effects of isolation on the sizes of the prostate and albumen gland. In contrast to a previous study, we observed no effect of body size on mating behaviour. However, there were strong effects of isolation: isolated individuals showed increased eagerness to mate, were more likely to adopt the active role and mounted shells of partners more than nonisolated individuals. Moreover, isolated individuals transferred more sperm than nonisolated individuals. Copulation duration and reciprocity of sperm transfer were unaffected by isolation. Isolation resulted in an increase in the size of the prostate gland and a decrease in the size of the albumen gland. We suggest that the motivation to mate after a period of isolation reflects a motivation to donate sperm rather than to receive sperm and that this motivation is regulated by the size of the prostate gland.

Histochemical correlations between egg capsule laminae and the female gonoduct reveal the process of capsule formation in the Muricidae (Neogastropoda: Mollusca)

Summary Limited information is currently available on the process of encapsulation and degree of variation in the micromorphology of egg capsules deposited by muricid gastropods. In this study, a histochemical approach was employed to determine the origin of egg capsule laminae within the female gonoduct of Dicathais orbita. Comparisons of capsule micromorphology from this Australian Rapaninae, with species from the Muricinae and Ocenebrinae, suggest that whole capsule and lamina 1 (L1) thickness, L2 fiber orientation and intracapsular fluid biochemistry display interspecific variability. Furthermore, the method of capsule laminae deposition was found to be substantially different to previous descriptions of muricid capsule formation. Simultaneous examination of capsule and gonoduct biochemistry in D. orbita revealed that the intracapsular fluid nd inner capsule wall (L3) contain secretions from the posterior lobe of the capsule gland; the thick middle lamina (L2) is derived from the lateral capsule gland lobes; and the outer layers (L1 and L0) include dorsal capsule lobe, albumen gland and pedal gland components. The insight gained into the processes involved in capsule formation imply that interspecific differences in capsule micromorphology are dependent on the timing of egg entry during capsule formation, and the protein content of posterior lobe secretory products coupled with the presence or absence of albumen gland secretions. This investigation supplements existing evidence on the origin of capsule components and variability in capsule micromorphology in the Muricidae by providing a simple method for deciphering the complex process of encapsulation in neogastropods.

Impact of parasitic mite infection on a terrestrial snail

Summary Although parasitic infections have been shown to be critical for growth, reproduction and survival of many vertebrates, little is known about the impact of parasites on invertebrate hosts and particular on molluscs. Therefore, it is of interest how parasites may affect their invertebrate hosts and how hosts can manage the detrimental effect of infections. In the present study, the naturally widespread parasitic mite Riccardoella limacum, which has been suggested to play an important role in the ecology of the land snail Arianta arbustorum, was artificially transferred to A. arbustorum. We experimentally examined the effect of the parasite on the food consumption, shell growth, and survival of its host. Surprisingly, we found minor impacts in some traits, i.e. we found that infected and uninfected snails similarly completed their shell growth, attained sexual maturity, and allocated equal energy into their albumen glands. However, infected snails consumed less and showed a significantly higher mortality after winter than uninfected snails.

Characterization of Aplysia Alb-1, a candidate water-borne protein pheromone released during egg laying

Pheromones are known to be important to the innate behavior of marine animals. Attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, temptin and seductin, which are released from the albumen gland during egg laying. Other pheromones are predicted to act in concert with these pheromones, but their identities are unknown. To identify additional pheromone candidates, we employed differential library screening of an albumen gland cDNA library, RT-PCR, recombinant protein expression, rhinophore contraction bioassays and immunocytochemistry. Alb-1 is expressed in the Aplysia californica albumen gland and encodes a novel protein that does not share significant sequence identity with any proteins in the database. RT-PCR analysis detected Alb-1 transcripts in the albumen gland, exocrine atrial gland and ovotestis. The Alb-1 precursor has a signal peptide sequence followed by a predicted 101-residue protein sequence containing eight cysteine residues. Recombinant protein expression, RP-HPLC, microsequence analysis and MALDI mass spectrometry analyses demonstrated that mature recombinant Alb-1 was processed at a paired basic residue site to generate an N-terminal and C-terminal protein fragment; this was consistent with immunoblot observations on purified albumen gland extracts. In rhinophore contraction (twitch) bioassays, the recombinant N-terminal protein induced rhinophore contractions whereas the C-terminal protein did not. An antibody generated to the N-terminal protein was used for immunocytochemical and immunoblot analyses and demonstrated that this protein is present in albumen gland secretory cells, egg cordons and egg eluates. Overall, the data suggest that Alb-1 may be processed in the albumen gland and that the Alb-1 1–56 protein released during egg laying may serve a pheromonal function in concert with attractin, enticin, temptin and seductin.

Angiostrongylus cantonensis: Experimental study on the susceptibility of apple snails, Pomacea canaliculata compared to Pila polita

Six groups (15 snails/group) of Pomacea canaliculata and Pila polita were infected orally with 0 (control), 200, 400, 800, 1600 and 3200 first-stage Angiostrongylus cantonensis larvae (L1). The respective mean ± SD third stage larvae (L3) worm recovery 1-month post-infection (p.i.) for P. canaliculata was 0, 1.4 ± 5.42 (0.7%), 0.13 ± 0.35 (0.03%), 0.07 ± 0.26 (0.009%), 0.07 ± 0.26 (0.004%), 0, and for P. polita 0, 64.33 ± 21.38 (32.25%), 115.36 ± 36.82 (28.93%), 265.33 ± 90.01 (33.27%), 471.33 ± 92.98 (29.60%) and 849.00 ± 243.23 (26.61%). The susceptibility of A. cantonensis in P. polita was dose-dependent ( p < 0.001). In the three groups (nine snails/group) of P. polita given 500 L1, we studied the distribution of L3 in the internal organs (i.e., foot, head + esophagus, kidney, albumin gland, mantle, intestine, digestive gland) and found the highest density after 1, 2 and 3 months p.i. in the mantle at 29.37%, 31.09% and 37.45%. The infection rate in P. canaliculata was too low to study distribution rates.

Tandem Exploitation of Helix pomatia Glycosyltransferases: Facile Syntheses of H‐Antigen‐Bearing Oligosaccharides.

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Tandem Exploitation of Helix pomatia Glycosyltransferases: Facile Syntheses of H‐Antigen‐Bearing Oligosaccharides

Snails from the family Helicidae produce in their albumen glands a highly branched galactan, which consists almost exclusively of D- and L-galactose. The D-Gal residues are glycosydically beta(1-->6)- or beta(1-->3)-linked, whereas the L-Gal moieties are attached alpha(1-->2). Up until the present time, two beta(1-->6)-D-galactosyl transferases and one alpha(1-->2)-L-galactosyl transferase have been identified in a membrane preparation of these glands. These were used to synthesise various oligosaccharides by successive addition of the NDP-activated (NDP=nucleoside-5'-diphosphate) D-Gal or L-Fuc moieties, up to a heptasaccharide by starting from the disaccharide D-Gal-beta(1-->3)-D-Gal-beta(1-->OMe. Even larger oligosaccharides up to a tridecasaccharide were obtained by starting with the hexasaccharide D-Gal-[beta(1-->3)-D-Gal]4-beta(1-->4)-D-Glc as an acceptor substrate. This tandem exploitation process has high potential for the easy introduction of D-Gal and L-Fuc residues into a great variety of oligosaccharides, which can be used in ligand/acceptor studies.

Fine structure of reproductive glands in two primitive marine pulmonates (Basommatophora: Siphonariidae)

AbstractWithin the clade Euthyneura the marine basommatophorans are particularly neglected. More morphological and molecular studies are needed because their phylogenetic relationships with other pulmonates remain unresolved. The present study examines the most conspicuous reproductive gland, the glandular complex in two marine limpets, Siphonaria capensis and S. serrata (Pulmonata: Basommatophora) at both gross and fine structural levels. These two sympatric species with different developmental modes were selected to compare the structure and function of this enormous glandular structure. In both S. capensis and S. serrata, the glandular complex shows an undifferentiated state composed of an acidophilic albumen gland and a basophilic mucous gland. The glands contain secretory cells and supporting cells (= ciliated cells) that are highly ciliated. When the histochemical properties of the glandular complex were compared with those of siphonariid egg masses (of each species) it could be established that the albumen gland was responsible for the production of perivitelline fluid whereas the mucous gland secreted substances that help in the assembly of mucous layers surrounding the egg capsules. We suggest that the presence of a single glandular complex comprised of two glands is the most primitive organization of reproductive glands in pulmonates. Furthermore, the histology, fine structure and histochemistry of these glands are very similar to those of the reproductive glands of opisthobranchs.

Gross Morphology and Ultrastructural Study of Albumen Gland of the Land Snail Archachatina marginata ovum (Pfeiffer) (Pulmonata: Achatinidae)

The gross morphology of the albumen gland of the edible land snail Archachatina marginata ovum (Pfeiffer) was studied by measurement of the shell length of the snails and wet weight of albumen gland of specimens collected from wild. Additionally, the structure of the albumen gland was studied by light and electron microscopy. The gland is large during the breeding season and there is no correlation between its weigh and the shell length of the snail. The albumen gland is made up of tubules which consist of closely packed single layer of elongated columnar secretory cells arranged radially around a central lumen. The secretory cells have large nuclei, secretory granules and are densely packed with rough endoplasmic reticulum. The secretory cells of the albumen gland produce secretions which are massively stored in their cytoplasm. Two types of secretions were identified, small droplets and large globules. The large globule was so extensive that the diameter of the cell was greatly reduced. TEM shows that the secretory globules contain fine morular granules characteristic of glycogen in several cell types and probably beta-glycogen.

Molecular systematics of the Carinarion complex (Mollusca: Gastropoda: Pulmonata): a taxonomic riddle caused by a mixed breeding system

The original description of the slugs Arion (Carinarion) fasciatus, Arion (Carinarion) silvaticus and Arion (Carinarion) circumscriptus was based on subtle differences in body pigmentation and genital anatomy. However, body pigmentation in these slugs may be influenced by their diet, whereas the genital differences between the species could not be confirmed by subsequent multivariate morphometric analyses. Hence, the status of the three nominal morphospecies remains controversial, with electrophoretic studies based on albumen gland proteins and allozymes also providing conflicting results. These studies suggested that Carinarion species are difficult to reconcile with the biological species concept because there is evidence of interspecific hybridization in places where these predominantly self-fertilizing slugs apparently outcross. Therefore, in the present study, the three Carinarion species are evaluated under a phylogenetic species concept, using nucleotide sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS-1) and the mitochondrial 16S rDNA. ITS-1 showed no species specific variation. However, 16S rDNA yielded five haplotype groups. Three of these grouped haplotypes by species, whereas the two others joined haplotypes of different species and included all haplotypes that were shared by species (22% of all haplotypes). Hence, the three nominal Carinarion species appear to be inconsistent with a phylogenetic species concept. The present data also confirmed that North American Carinarion populations are genetically impoverished and may be not sufficiently representative with respect to the taxonomy of Carinarion. In conclusion, we currently regard Carinarion as a single species-level taxon, whose taxonomically deceiving, correlated phenotypic and genetic intraspecific variation is caused by sustained self-fertilization.

Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold

Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg 50 residue to generate a smaller 49-residue mature peptide. The Arg 50 cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I–II, III–IV, and V–VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly α-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.

Identification and expression of gene transcripts generated during an anti-parasitic response in Biomphalaria glabrata

In order to gain further insights into the molecular basis of gastropod anti-parasite immune responses, we investigated transcripts of Biomphalaria glabrata regulated during hemocytic encapsulation. Using a snail strain that is resistant to the parasite Echinostoma caproni, we performed suppression subtractive hybridization (SSH) to construct cDNA libraries of transcripts more abundantly expressed in unexposed or parasite-exposed snails. After sequence analysis and quantitative PCR analysis of expression, we identified 10 candidates of particular interest. They belonged to various functional groups such as detoxification enzymes (GST, SOD), antimicrobial proteins (LBI/BPI), protease inhibitors (cystatins), calcium-binding proteins, or C-type lectins. In situ hybridization (ISH) analysis revealed that one overexpressed cystatin-like candidate is specifically expressed in hemocytes participating in parasite encapsulation or aggregating at the site of infection. Two other candidates (C-type lectin and a LBP/BPI) were expressed in the albumen gland, further supporting the role of this organ in immunity and/or host–parasite interaction.

Biochemical and Structural Analysis of Helix pomatia Agglutinin: A HEXAMERIC LECTIN WITH A NOVEL FOLD

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) M range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharides.

Anatomy of an adaptive radiation: a unique reproductive strategy in the endemic freshwater gastropod Tylomelania (Cerithioidea: Pachychilidae) on Sulawesi, Indonesia and its biogeographical implications

The patterns of adaptive radiations in ancient lakes provide valuable clues to mechanisms of speciation and adaptation. In contrast to vertebrate radiations, for instance in fishes or finches, invertebrate species flocks have been largely neglected. While the increase in molecular data narrows this gap, the anatomical basis for interpreting these data against the background of evolutionary hypotheses is still widely lacking. Here we evaluate anatomical findings in the live-bearing pachychilid freshwater gastropod Tylomelania, which has radiated extensively in ancient lakes in the Indonesian island, Sulawesi; we have aimed at reconciling these data with recently obtained molecular phylogenetic evidence. Discovered more than a century ago, the speciose and phenotypically diverse species flock with 34 currently described taxa was only occasionally cited as an example of adaptive radiation in ancient lakes, while anatomical data were entirely lacking. Our study of anatomical characters reveals very low qualitative variation at the species level. Thus, contrary to earlier views we suggest the existence of a single monophyletic lineage endemic to this island. The most conspicuous feature of Tylomelania is its uterine brooding strategy, i.e. retaining eggs and embryos in the pallial oviduct. This is unique among South-East Asian pachychilids. Within the uterine brood pouch the offspring is surrounded by considerable amounts of nutritive material produced by a very large albumin gland, and the embryos are produced continuously. The shelled juveniles of some species are the largest known so far in viviparous gastropods, measuring almost 2 cm in length when hatching. This combination of reproductive features in Tylomelania, characterized by a high amount of maternal investment, is considered to be ovoviviparous, rendering its brooding strategy unique also among other gastropods. In addition, our data reject a previously assumed close relationship to other South-East Asian pachychilids and instead suggest the North Australian Pseudopotamis as sister group to Tylomelania. These findings have significant consequences for the phylogenetic interpretation of morphological characters of Tylomelania in an evolutionary and biogeographical context, leading to the hypothesis that the common ancestor of both genera originated somewhere on the northern Australian continental margin. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 85, 513–542.

Aplysia capsulin is localized to egg capsules and egg cordon sheaths and shares sequence homology with Drosophila dec-1 gene products

Differential library screening of an albumen gland cDNA library, Western blot analysis, protein expression, immunolocalization studies, comparative genomics, and secretion assays identified a major Aplysia californica albumen gland protein (‘capsulin’) that is localized to egg capsules and to the sheaths of the egg cordon. Capsulin shared sequence homology with eggshell proteins encoded by the Drosophila dec-1 gene. The 1790-amino acid A. californica precursor contains 17 repeat sequences that are flanked by basic residue processing sites. The numerous proteolytic processing sites may facilitate the breakdown of capsulin prior to when veliger larvae break out of egg capsules as free-swimming larvae. An Aplysia brasiliana capsulin repeat sequence was 97% identical to its A. californica homolog. Capsulin fragments were not detected in the eluates of egg cordons, suggesting that capsulin is not a candidate water-borne pheromone precursor.

Aplysia seductin is a water-borne protein pheromone that acts in concert with attractin to stimulate mate attraction

Mate attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, and temptin which are released during egg-laying. Other water-borne pheromones are predicted to act in concert with attractin, enticin, and temptin, but their identities were unknown. We recently identified a highly expressed Aplysia californica albumen gland gene ( Alb-23) that encoded a novel protein by differential library screening of an albumen gland cDNA library. To determine whether Alb-23 (‘seductin’) was a water-borne pheromone, we employed Western blot analysis, purification and expression of albumen gland proteins, immunolocalization studies, pheromone secretion assays, comparative genomics, and behavioral bioassays. Immunoreactive seductin was detected in eluates of egg cordons, indicating that seductin was secreted onto the cordon during egg laying. Aplysia brasiliana seductin was 94% identical to its A. californica homolog. In T-maze attraction assays, the combination of attractin and seductin was significantly attractive to potential mates, whereas either protein alone was not. Data from this and previous studies support the hypothesis that seductin is a water-borne protein pheromone that acts in concert with attractin, enticin, and temptin to attract Aplysia to form and maintain mating aggregations.

Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni

Snail immune responses towards a trematode infection are known to rely on both plasmatic and cellular host factors. As an approach to further investigate the suspected involvement of plasmatic factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns of plasma collected from susceptible and resistant snails. This proteomic approach revealed that 13 plasmatic proteins exhibited significant differences in their apparent representativity. The genes corresponding to five of them were characterised by a combination of mass spectrometry and molecular cloning. They encode two isoforms of a glycolytic enzyme, two isoforms of a calcium binding protein and an inhibitor of cysteine protease. Furthermore, we investigated gene expression in parasite-exposed or -unexposed snails as well as in various tissues by quantitative PCR. This study showed that: (i) differential representation of plasma proteins between the snail strains was correlated with a differential level of transcripts; (ii) expression of these genes after parasite exposure was differentially regulated in the two strains; and (iii) these genes were expressed predominantly in the albumen gland.

Maturation timing in the land snails Archachatina marginata ovum (Pfeiffer) and Limicolaria flammea (Müller) (Pulmonata: Achatinidae)

Summary The timing of gonadal maturation was studied in two edible species of West African achatinid snails, Archachatina marginata ovum (Pfeiffer) and Limicolaria flammea (Müller), using light microscopic observation of gametogenesis in the ovotestis and oviposition. Additionally, the growth of the reproductive system was analysed by weekly measurement of body weight, shell length and reproductive system in both laboratory-bred and wild snails of both species. The ovotestis in A. marginata ovum and L. flammea produces oocytes and spermatozoa simultaneously. The five active and two inactive gametogenic stages observed in the two species are described and compared. Genitalia development start after the onset of gametogenesis in both species with the male system developing before the female system. The growth of the albumen gland follows the maturation of the gonad in L. flammea, but in A. marginata ovum the growth is cyclical and seasonal. The first oviposition occurs at approximately 19 months of age in A. marginata ovum and 5 months in L. flammea bred in the laboratory. L. flammea breeds almost throughout the year, whereas this process is restricted to March-October in A. marginata ovum. In L. flammea, the reproductive investment is nearly 200% of parental body weight and the species is estimated to produce 375 young with full size biomass of 1,725 g, while the 140 young produced by A. marginata ovum represent 97% of parental body weight in 3 years and have at full size a biomass of 54,740 g.