Egg toxic compounds in the animal kingdom. A comprehensive review.

Covering: 1951 to 2022Packed with nutrients and unable to escape, eggs are the most vulnerable stage of an animal's life cycle. Consequently, many species have evolved chemical defenses and teamed up their eggs with a vast array of toxic molecules for defense against predators, parasites, or pathogens. However, studies on egg toxins are rather scarce and the available information is scattered. The aim of this review is to provide an overview of animal egg toxins and to analyze the trends and patterns with respect to the chemistry and biosynthesis of these toxins. We analyzed their ecology, distribution, sources, occurrence, structure, function, relative toxicity, and mechanistic aspects and include a brief section on the aposematic coloration of toxic eggs. We propose criteria for a multiparametric classification that accounts for the complexity of analyzing the full set of toxins of animal eggs. Around 100 properly identified egg toxins are found in 188 species, distributed in 5 phyla: cnidarians (2) platyhelminths (2), mollusks (9), arthropods (125), and chordates (50). Their scattered pattern among animals suggests that species have evolved this strategy independently on numerous occasions. Alkaloids are the most abundant and widespread, among the 13 types of egg toxins recognized. Egg toxins are derived directly from the environment or are endogenously synthesized, and most of them are transferred by females inside the eggs. Their toxicity ranges from ρmol kg-1 to mmol kg-1, and for some species, experiments support their role in predation deterrence. There is still a huge gap in information to complete the whole picture of this field and the number of toxic eggs seems largely underestimated.

Biochemical and morphological features of the uropygial gland of the Chilean Flamingo and their functional implications.

Flamingos inhabit specialized habitats and breed in large colonies, building their nests on islands that limit the access of terrestrial predators. Many aspects of their uropygial gland are still unknown. The uropygial gland, a sebaceous organ exclusive to birds, shares some histological features among species such as the presence of a capsule, adenomers with stratified epithelium and secondary and primary chambers. We found that the uropygial gland of the Chilean Flamingo (Phoenicopterus chilensis) displays most of these characteristics but lacks a primary storage chamber. This absence may be an adaptation to their aquatic environment. The uropygial secretion of this species has a variety of glycoconjugates while its lipid moiety is largely dominated by waxes and minor amounts of triacylglycerols and fatty acids. Mass spectrometry analysis of the preen wax showed branched fatty acids of varied chain length and unbranched fatty alcohols, resulting in a complex mixture of wax esters and no differences between sexes were observed. The glycoconjugates present in the preen secretion could play a role as antimicrobial molecules, as suggested for other bird species, while the absence of diester waxes in flamingos might be related with their nesting habits and limited exposure to predation. Our results were evaluated according to physiological and ecological aspects of the flamingo's biology.

A highly stable, non-digestible lectin from Pomacea diffusa unveils clade-related protection systems in apple snail eggs.

The acquisition of egg protection is vital for species survival. Poisonous eggs from Pomacea apple snails have defensive macromolecules for protection. Here we isolated and characterized a novel lectin called PdPV1 that is massively accumulated in the eggs of Pomacea diffusa and seems part of its protective cocktail. The native protein, an oligomer of ca 256 kDa, has high structural stability, withstanding 15 min boiling and denaturing by SDS. It resists in vitro proteinase digestion and displays structural stability between pH 2.0 and pH 12.0, and up to 85°C. These properties, as well as its subunit sequences, glycosylation pattern, presence of carotenoids, size and global shape resemble those of its orthologs from other Pomacea. Furthermore, like members of the canaliculata clade, PdPV1 is recovered unchanged in feces of mice ingesting it, supporting an anti-nutritive defensive function. PdPV1 also displays a strong hemagglutinating activity, specifically recognizing selected ganglioside motifs with high affinity. This activity is only shared with PsSC, a perivitelline from the same clade (bridgesii clade). As a whole, these results indicate that species in the genus Pomacea have diversified their egg defenses: those from the bridgesii clade are protected mostly by non-digestible lectins that lower the nutritional value of eggs, in contrast with protection by neurotoxins of other Pomacea clades, indicating that apple snail egg defensive strategies are clade specific. The harsh gastrointestinal environment of predators would have favored their appearance, extending by convergent evolution the presence of plant-like highly stable lectins, a strategy not reported in other animals.

Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg MACPF-Toxin.

Gastropod Molluscs rely exclusively on the innate immune system to protect from pathogens, defending their embryos through maternally transferred effectors. In this regard, Pomacea snail eggs, in addition to immune defenses, have evolved the perivitellin-2 or PV2 combining two immune proteins into a neurotoxin: a lectin and a pore-forming protein from the Membrane Attack Complex/Perforin (MACPF) family. This binary structure resembles AB-toxins, a group of toxins otherwise restricted to bacteria and plants. Many of these are enterotoxins, leading us to explore this activity in PV2. Enterotoxins found in bacteria and plants act mainly as pore-forming toxins and toxic lectins, respectively. In animals, although both pore-forming proteins and lectins are ubiquitous, no enterotoxins have been reported. Considering that Pomacea snail eggs ingestion induce morpho-physiological changes in the intestinal mucosa of rodents and is cytotoxic to intestinal cells in culture, we seek for the factor causing these effects and identified PmPV2 from Pomacea maculata eggs. We characterized the enterotoxic activity of PmPV2 through in vitro and in vivo assays. We determined that it withstands the gastrointestinal environment and resisted a wide pH range and enzymatic proteolysis. After binding to Caco-2 cells it promoted changes in surface morphology and an increase in membrane roughness. It was also cytotoxic to both epithelial and immune cells from the digestive system of mammals. It induced enterocyte death by a lytic mechanism and disrupted enterocyte monolayers in a dose-dependent manner. Further, after oral administration to mice PmPV2 attached to enterocytes and induced large dose-dependent morphological changes on their small intestine mucosa, reducing the absorptive surface. Additionally, PmPV2 was detected in the Peyer's patches where it activated lymphoid follicles and triggered apoptosis. We also provide evidence that the toxin can traverse the intestinal barrier and induce oral adaptive immunity with evidence of circulating antibody response. As a whole, these results indicate that PmPV2 is a true enterotoxin, a role that has never been reported to lectins or perforin in animals. This extends by convergent evolution the presence of plant- and bacteria-like enterotoxins to animals, thus expanding the diversity of functions of MACPF proteins in nature.

Hemocyanin of the caenogastropod Pomacea canaliculata exhibits evolutionary differences among gastropod clades.

Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tri-decameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal ß-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-ß1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin.

Egg perivitelline fluid proteome of a freshwater snail: Insight into the transition from aquatic to terrestrial egg deposition.

RATIONALE: Proteins from the egg perivitelline fluid (PVF) are assumed to play critical roles in embryonic development, but for many groups of animals their identities remain unknown. Identifying egg PVF proteins is a critical step towards understanding their functions including their roles in evolutionary transition in habitats. METHODS: We applied proteomic and transcriptomic analysis to investigate the PVF proteome of the eggs of Pomacea diffusa, an aerial ovipositing freshwater snail in the family Ampullariidae. The PVF proteins were separated with the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method, and proteomic analysis was conducted using an LTQ Velos ion trap mass spectrometer coupled with liquid chromatography. Comparison of PVF proteomes and evolution analyses was performed between P. diffusa and other ampullariids. RESULTS: In total, 32 egg PVF proteins were identified from P. diffusa. They were categorized as PV1-like subunits, immune-responsive proteins, protein degradation, signaling and binding, transcription and translation, metabolism, oxidation-reduction and proteins with unknown function. Interestingly, the proteome includes a calcium-binding protein important in forming the hard eggshell that enabled the terrestrial transition. However, it does not include PV2, a neurotoxic protein that was assumed to be present in all Pomacea species. CONCLUSIONS: The PVF proteome data from P. diffusa can help us better understand the roles that reproductive proteins played during the transition from underwater to terrestrial egg deposition. Moreover, they could be useful in comparative studies of the terrestrialization in several groups of animals that occurred independently during their evolution.

Signatures of Divergence, Invasiveness, and Terrestrialization Revealed by Four Apple Snail Genomes.

The family Ampullariidae includes both aquatic and amphibious apple snails. They are an emerging model for evolutionary studies due to the high diversity, ancient history, and wide geographical distribution. Insight into drivers of ampullariid evolution is hampered, however, by the lack of genomic resources. Here, we report the genomes of four ampullariids spanning the Old World (Lanistes nyassanus) and New World (Pomacea canaliculata, P. maculata, and Marisa cornuarietis) clades. The ampullariid genomes have conserved ancient bilaterial karyotype features and a novel Hox gene cluster rearrangement, making them valuable in comparative genomic studies. They have expanded gene families related to environmental sensing and cellulose digestion, which may have facilitated some ampullarids to become notorious invasive pests. In the amphibious Pomacea, novel acquisition of an egg neurotoxin and a protein for making the calcareous eggshell may have been key adaptations enabling their transition from underwater to terrestrial egg deposition.

Understanding the transition from water to land: Insights from multi-omic analyses of the perivitelline fluid of apple snail eggs.

Unlike most of the freshwater gastropod families, the family Ampullariidae includes members that exhibit both underwater and aerial oviposition, making it an ideal model for understanding mechanisms underlying the evolutionary transition from water to land. We applied SDS-PAGE and LC-MS/MS to analyse the proteome of the egg perivitelline fluid (PVF) of Marisa cornuarietis - an aquatic ovipositing ampullariid. Comparison with the reported PVF proteomes of two aerial ovipositing ampullariids (Pomacea canaliculata and P. maculata) showed that the three species all contain several major perivitellins that nourish the embryos. However, M. cornuarietis invests more heavily on immune-related proteins, which might be due to exposure to aquatic pathogens. Interestingly, only the PVF of out-of-water egg laying species have PV2 - a neurotoxin lethal to mice, and a calcium-binding protein which might be involved in the formation of calcareous eggshell. Integrated phylogenetic, evolutionary and gene expressional analyses detected the involvement of gene duplication, positive selection and neofunctionalisation in the formation of several major PVF proteins. Overall, our study provides multiple lines of evidence of adaptive evolution in the PVF proteins, and contributes to a better understanding of how aquatic gastropod ancestors invaded terrestrial habitats. SIGNIFICANCE: Aerial egg deposition has evolved in several groups of animals, but except for Vertebrata little is known about the mechanisms underlying this critical evolution process. We compared aquatic and aerial egg laying apple snails to understand the molecular mechanisms enabling such a transition in egg laying habitat. We found that the composition of perivitelline fluid proteomes of underwater and aerial egg depositors was remarkably different, and then gene duplication and positive selection were responsible for the formation of such novel proteins than enabled the evolutionary transition.

Egg perivitelline fluid of the invasive snail Pomacea canaliculata affects mice gastrointestinal function and morphology.

BACKGROUND: Species beloging to the genus Pomacea (Ampullariidae), often referred as apple snails, are freshwater, amphibious snails native to South, Central and North America. Some species such as P. canaliculata have become a driver of ecosystem changes in wetlands and an important rice and taro pest after its introduction to Asia and other parts of the world. Females deposit colored egg clutches above the waterline, a reproductive strategy that exposes the eggs to harsh conditions and terrestrial predation. However, eggs have no reported predators in their native range, probably because of the acquisition of unparalleled biochemical defenses provided by a set of proteins (perivitellins) that nourish embryos and protect them from predators and abiotic factors. Notably, ingestion of egg perivitelline fluid (PVF) decreases rat growth rate and alters their gastrointestinal morphology. The aim of the study is to determine the effect of apple snail egg PVF on mice gut digestive activity, morphology and nutrient absorption. METHODS: Carbohydrate digestion by intestinal disaccharidases (sucrase-isomaltase and maltase-glucoamylase) was evaluated ex vivo in mice gavaged with 1 or 4 doses of PVF. Changes in gut morphological and absorptive surface were measured. In addition, alteration on nutrient absorption rates, transport pathways and intestinal permeability was evaluated by luminal perfusions of small intestine with radiolabeled L-proline (absorbed by paracellular and transcellular pathways) and L-arabinose (absorbed exclusively by paracellular pathway). RESULTS: Perivitelline fluid affected mice displayed significant morphological changes in the small intestine epithelium inducing the appearance of shorter and wider villi as well as fused villi. This resulted in a diminished absorptive surface, notably in the proximal portion. Likewise, the activity of disaccharidases diminished in the proximal portion of the intestine. Total absorption of L-proline increased in treated mice in a dose-dependent manner. There were no differences neither in the ratio of paracellular-to-transcellular absorption of L-proline nor in gut permeability as revealed by the clearance of L-arabinose. DISCUSSION: Oral administration of apple snail PVF to mice adversely alters gut morphophysiology by reducing the intestinal absorptive surface, affecting enzymes of sugar metabolism and increasing the absorption rate of nutrients without affecting the relative contribution of the absorption pathways or gut permeability. These results further support the role of PVF in passive anti-predator defenses in Pomacea snail eggs that target the digestive system.

A lectin of a non-invasive apple snail as an egg defense against predation alters the rat gut morphophysiology.

The eggs of the freshwater Pomacea apple snails develop above the water level, exposed to varied physical and biological stressors. Their high hatching success seems to be linked to their proteins or perivitellins, which surround the developing embryo providing nutrients, sunscreens and varied defenses. The defensive mechanism has been unveiled in P. canaliculata and P. maculata eggs, where their major perivitellins are pigmented, non-digestible and provide a warning coloration while another perivitellin acts as a toxin. In P. scalaris, a species sympatric to the former, the defense strategy seems different, since no toxin was found and the major perivitellin, PsSC, while also colored and non-digestible, is a carbohydrate-binding protein. In this study we examine the structure and function of PsSC by sequencing its subunits, characterizing its carbohydrate binding profile and evaluating its effect on gut cells. Whereas cDNA sequencing and database search showed no lectin domain, glycan array carbohydrate binding profile revealed a strong specificity for glycosphingolipids and ABO group antigens. Moreover, PsSC agglutinated bacteria in a dose-dependent manner. Inspired on the defensive properties of seed lectins we evaluated the effects of PsSC on intestinal cells both in vitro (Caco-2 and IEC-6 cells) and in the gastrointestinal tract of rats. PsSC binds to Caco-2 cell membranes without reducing its viability, while a PsSC-containing diet temporarily induces large epithelium alterations and an increased absorptive surface. Based on these results, we propose that PsSC is involved in embryo defenses by altering the gut morphophysiology of potential predators, a convergent role to plant defensive lectins.

AmpuBase: a transcriptome database for eight species of apple snails (Gastropoda: Ampullariidae).

BACKGROUND: Gastropoda, with approximately 80,000 living species, is the largest class of Mollusca. Among gastropods, apple snails (family Ampullariidae) are globally distributed in tropical and subtropical freshwater ecosystems and many species are ecologically and economically important. Ampullariids exhibit various morphological and physiological adaptations to their respective habitats, which make them ideal candidates for studying adaptation, population divergence, speciation, and larger-scale patterns of diversity, including the biogeography of native and invasive populations. The limited availability of genomic data, however, hinders in-depth ecological and evolutionary studies of these non-model organisms. RESULTS: Using Illumina Hiseq platforms, we sequenced 1220 million reads for seven species of apple snails. Together with the previously published RNA-Seq data of two apple snails, we conducted de novo transcriptome assembly of eight species that belong to five genera of Ampullariidae, two of which represent Old World lineages and the other three New World lineages. There were 20,730 to 35,828 unigenes with predicted open reading frames for the eight species, with N50 (shortest sequence length at 50% of the unigenes) ranging from 1320 to 1803 bp. 69.7% to 80.2% of these unigenes were functionally annotated by searching against NCBI's non-redundant, Gene Ontology database and the Kyoto Encyclopaedia of Genes and Genomes. With these data we developed AmpuBase, a relational database that features online BLAST functionality for DNA/protein sequences, keyword searching for unigenes/functional terms, and download functions for sequences and whole transcriptomes. CONCLUSIONS: In summary, we have generated comprehensive transcriptome data for multiple ampullariid genera and species, and created a publicly accessible database with a user-friendly interface to facilitate future basic and applied studies on ampullariids, and comparative molecular studies with other invertebrates.

Convergent evolution of plant and animal embryo defences by hyperstable non-digestible storage proteins.

Plants have evolved sophisticated embryo defences by kinetically-stable non-digestible storage proteins that lower the nutritional value of seeds, a strategy that have not been reported in animals. To further understand antinutritive defences in animals, we analysed PmPV1, massively accumulated in the eggs of the gastropod Pomacea maculata, focusing on how its structure and structural stability features affected its capacity to withstand passage through predator guts. The native protein withstands >50 min boiling and resists the denaturing detergent sodium dodecyl sulphate (SDS), indicating an unusually high structural stability (i.e., kinetic stability). PmPV1 is highly resistant to in vitro proteinase digestion and displays structural stability between pH 2.0-12.0 and 25-85 °C. Furthermore, PmPV1 withstands in vitro and mice digestion and is recovered unchanged in faeces, supporting an antinutritive defensive function. Subunit sequence similarities suggest a common origin and tolerance to mutations. This is the first known animal genus that, like plant seeds, lowers the nutritional value of eggs by kinetically-stable non-digestible storage proteins that survive the gut of predators unaffected. The selective pressure of the harsh gastrointestinal environment would have favoured their appearance, extending by convergent evolution the presence of plant-like hyperstable antinutritive proteins to unattended reproductive stages in animals.

Dataset for the proteomic and transcriptomic analyses of perivitelline fluid proteins in Pomacea snail eggs.

This article describes how the proteomic and transcriptomic data were produced during a study of the reproductive proteins of Pomacea maculata, an aquatic apple snail laying colorful aerial eggs, and provides public access to the data. The data are related to a research article titled 'An integrated proteomic and transcriptomic analysis of perivitelline fluid proteins in a freshwater gastropod laying aerial eggs' (Mu et al., 2017) [1]. RNA was extracted from the albumen gland and other tissues and sequenced on an Illumina Hiseq. 2000. The assembled transcriptome was translated into protein sequences and then used for protein identification. Proteins from the perivitelline fluid of P. maculata were separated in SDS-PAGE and analyzed by LTQ-Orbitrap Elite coupled to an Easy-nLC. The translated transcriptome data are provided in this article. Proteomic data (.raw file format) are available via ProteomeXchange with the identifier PXD006718.

Apple Snail Perivitellin Precursor Properties Help Explain Predators' Feeding Behavior.

In contrast with vitellogenin maturation, it is unknown whether gastropod perivitellin precursors are subject to large structural changes. The gastropod reproductive tract includes an accessory organ, the albumen gland (AG), that produces and secretes perivitelline fluid. In the apple snail Pomacea canaliculata, the large, reddish-pink AG provides eggs with perivitellins that are defensive against predators. Although the AG makes a considerable contribution to apple snail biomass, field observations indicate that it is rejected by avian and mammalian predators, although the underlying reason remains unknown. By analyzing the structure-function properties of P. canaliculata perivitellin precursors, we provide insight into perivitellin maturation and its relationship with apple snail predator feeding behavior. Structural analysis using small-angle X-ray scattering, absorption and fluorescence spectroscopy, circular dichroism, electrophoresis, chromatography, and partial proteolysis showed that the size, shape, and structure of perivitellin precursors resemble those of egg mature forms. Functional analysis indicates that the precursors of the defensive perivitellins ovorubin (PcOvo) and perivitellin-2 (PcPV2) are highly stable and antinutritive, withstanding proteinase digestion and displaying structural stability of their quaternary structure under a wide pH range (4.0-10.0). Furthermore, AG extracts limit a predator's ability to digest nutrients and are toxic to mice (median lethal concentration 96 h after administration: 5.9 mg/kg). Treated mice displayed neurologic signs similar to those produced by egg PcPV2. Results indicate that apple snails store active precursors of egg proteins inside the AG, providing evidence that gastropod perivitellin precursors do not experience the large structural processing of invertebrate vitellogenin maturation. These defensive proteins provide the apple snail AG with neurotoxic, antinutritive, and antidigestive activity, a likely explanation for the predators' feeding behavior.

An integrated proteomic and transcriptomic analysis of perivitelline fluid proteins in a freshwater gastropod laying aerial eggs.

Proteins of the egg perivitelline fluid (PVF) that surrounds the embryo are critical for embryonic development in many animals, but little is known about their identities. Using an integrated proteomic and transcriptomic approach, we identified 64 proteins from the PVF of Pomacea maculata, a freshwater snail adopting aerial oviposition. Proteins were classified into eight functional groups: major multifunctional perivitellin subunits, immune response, energy metabolism, protein degradation, oxidation-reduction, signaling and binding, transcription and translation, and others. Comparison of gene expression levels between tissues showed that 22 PVF genes were exclusively expressed in albumen gland, the female organ that secretes PVF. Base substitution analysis of PVF and housekeeping genes between P. maculata and its closely related species Pomacea canaliculata showed that the reproductive proteins had a higher mean evolutionary rate. Predicted 3D structures of selected PVF proteins showed that some nonsynonymous substitutions are located at or near the binding regions that may affect protein function. The proteome and sequence divergence analysis revealed a substantial amount of maternal investment in embryonic nutrition and defense, and higher adaptive selective pressure on PVF protein-coding genes when compared with housekeeping genes, providing insight into the adaptations associated with the unusual reproductive strategy in these mollusks. SIGNIFICANCE: There has been great interest in studying reproduction-related proteins as such studies may not only answer fundamental questions about speciation and evolution, but also solve practical problems of animal infertility and pest outbreak. Our study has demonstrated the effectiveness of an integrated proteomic and transcriptomic approach in understanding the heavy maternal investment of proteins in the eggs of a non-model snail, and how the reproductive proteins may have evolved during the transition from laying underwater eggs to aerial eggs.

Insights into embryo defenses of the invasive apple snail Pomacea canaliculata: egg mass ingestion affects rat intestine morphology and growth.

BACKGROUND: The spread of the invasive snail Pomacea canaliculata is expanding the rat lungworm disease beyond its native range. Their toxic eggs have virtually no predators and unusual defenses including a neurotoxic lectin and a proteinase inhibitor, presumably advertised by a warning coloration. We explored the effect of egg perivitellin fluid (PVF) ingestion on the rat small intestine morphology and physiology. METHODOLOGY/PRINCIPAL FINDINGS: Through a combination of biochemical, histochemical, histopathological, scanning electron microscopy, cell culture and feeding experiments, we analyzed intestinal morphology, growth rate, hemaglutinating activity, cytotoxicity and cell proliferation after oral administration of PVF to rats. PVF adversely affects small intestine metabolism and morphology and consequently the standard growth rate, presumably by lectin-like proteins, as suggested by PVF hemaglutinating activity and its cytotoxic effect on Caco-2 cell culture. Short-term effects of ingested PVF were studied in growing rats. PVF-supplemented diet induced the appearance of shorter and wider villi as well as fused villi. This was associated with changes in glycoconjugate expression, increased cell proliferation at crypt base, and hypertrophic mucosal growth. This resulted in a decreased absorptive surface after 3 days of treatment and a diminished rat growth rate that reverted to normal after the fourth day of treatment. Longer exposure to PVF induced a time-dependent lengthening of the small intestine while switching to a control diet restored intestine length and morphology after 4 days. CONCLUSIONS/SIGNIFICANCE: Ingestion of PVF rapidly limits the ability of potential predators to absorb nutrients by inducing large, reversible changes in intestinal morphology and growth rate. The occurrence of toxins that affect intestinal morphology and absorption is a strategy against predation not recognized among animals before. Remarkably, this defense is rather similar to the toxic effect of plant antipredator strategies. This defense mechanism may explain the near absence of predators of apple snail eggs.

Effects of sewage discharges on lipid and fatty acid composition of the Patagonian bivalve Diplodon chilensis.

Lipid and fatty acid (FA) composition and selected oxidative stress parameters of freshwater clams (Dipolodon chilensis), from a sewage-polluted (SMA) and a clean site, were compared. Trophic markers FA were analyzed in clams and sediment. Saturated FA (SAFA), and bacteria and sewage markers were abundant in SMA sediments, while diatom markers were 50% lower. Proportions of SAFA, branched FA, 20:5n-3 (EPA) and 22:6n-3 (DHA) were higher in SMA clams. Chronic exposure of D. chilensis to increasing eutrophication affected its lipid and FA composition. The increase in EPA and DHA proportions could be an adaptive response, which increases stress resistance but could also lead to higher susceptibility to lipid peroxidation TBARS, lipofuscins (20-fold) and GSH concentrations were higher in SMA clams. FA markers indicated terrestrial plant detritus and bacteria are important items in D. chilensis diet. Anthropogenic input in their food could be traced using specific FA as trophic markers.

Novel animal defenses against predation: a snail egg neurotoxin combining lectin and pore-forming chains that resembles plant defense and bacteria attack toxins.

Although most eggs are intensely predated, the aerial egg clutches from the aquatic snail Pomacea canaliculata have only one reported predator due to unparalleled biochemical defenses. These include two storage-proteins: ovorubin that provides a conspicuous (presumably warning) coloration and has antinutritive and antidigestive properties, and PcPV2 a neurotoxin with lethal effect on rodents. We sequenced PcPV2 and studied whether it was able to withstand the gastrointestinal environment and reach circulation of a potential predator. Capacity to resist digestion was assayed using small-angle X-ray scattering (SAXS), fluorescence spectroscopy and simulated gastrointestinal proteolysis. PcPV2 oligomer is antinutritive, withstanding proteinase digestion and displaying structural stability between pH 4.0-10.0. cDNA sequencing and protein domain search showed that its two subunits share homology with membrane attack complex/perforin (MACPF)-like toxins and tachylectin-like lectins, a previously unknown structure that resembles plant Type-2 ribosome-inactivating proteins and bacterial botulinum toxins. The protomer has therefore a novel AB toxin combination of a MACPF-like chain linked by disulfide bonds to a lectin-like chain, indicating a delivery system for the former. This was further supported by observing PcPV2 binding to glycocalix of enterocytes in vivo and in culture, and by its hemaggutinating, but not hemolytic activity, which suggested an interaction with surface oligosaccharides. PcPV2 is able to get into predator's body as evidenced in rats and mice by the presence of circulating antibodies in response to sublethal oral doses. To our knowledge, a lectin-pore-forming toxin has not been reported before, providing the first evidence of a neurotoxic lectin in animals, and a novel function for ancient and widely distributed proteins. The acquisition of this unique neurotoxic/antinutritive/storage protein may confer the eggs a survival advantage, opening new perspectives in the study of the evolution of animal defensive strategies.

Agglutinating activity and structural characterization of scalarin, the major egg protein of the snail Pomacea scalaris (d'Orbigny, 1832).

Apple snail perivitellins are emerging as ecologically important reproductive proteins. To elucidate if the protective functions of the egg proteins of Pomacea canaliculata (Caenogastropoda, Ampullariidae), involved in embryo defenses, are present in other Pomacea species we studied scalarin (PsSC), the major perivitellin of Pomacea scalaris. Using small angle X-ray scattering, fluorescence and absorption spectroscopy and biochemical methods, we analyzed PsSC structural stability, agglutinating activity, sugar specificity and protease resistance. PsSC aggluttinated rabbit, and, to a lesser extent, human B and A erythrocytes independently of divalent metals Ca(2+) and Mg(2+) were strongly inhibited by galactosamine and glucosamine. The protein was structurally stable between pH 2.0 to 10.0, though agglutination occurred only between pH 4.0 to 8.0 (maximum activity at pH 7.0). The agglutinating activity was conserved up to 60 °C and completely lost above 80 °C, in agreement with the structural thermal stability of the protein (up to 60 °C). PsSC was able to withstand in vitro gastrointestinal digestion, and showed no trypsin inhibition activity. The presence of lectin activity has been reported in eggs of other Pomacea snails, but here we link for the first time, this activity to an apple snail multifunctional perivitellin. This novel role for a snail egg storage protein is different from closely related P.canaliculata defensive proteins.

Vitellogenin and lipovitellin from the prawn Macrobrachium borellii as hydrocarbon pollution biomarker.

During reproduction vitellogenin (VTG) is transported to vitellogenic oocytes as a precursor of egg yolk lipovitellin (LV). As VTG synthesis is affected by environmental stressors, it is widely used as biomarker in endocrine disruption studies. However, it has seldom been employed to evaluate invertebrate hydrocarbon pollution. An ELISA with anti-LV antibody was developed to evaluate the impact of water-soluble fraction of crude oil (WSF) on Macrobrachium borellii vitellogenesis. Prawn VTG concentration was within the range reported for other crustaceans; LV values were positively correlated with gonadosomatic index (GSI). Females at different vitellogenic stages were exposed to a sub-lethal concentration of WSF for 7 days. Exposed animals with GSI>7 increased their VTG and LV titer as compared to control organisms (190% and 140%, respectively). VTG levels in M. borellii were upregulated and highly sensitive to WSF exposure. This assay could be employed as a biomarker for freshwater hydrocarbon pollution.