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.

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.

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.

Distribution and current infection status of Biomphalaria straminea in Hong Kong.

BACKGROUND: Schistosomiasis, also generally known as snail fever, is a parasitic disease caused by trematode flatworms of the genus Schistosoma. In Hong Kong and mainland China, the freshwater snail Biomphalaria straminea has been introduced and has the potential to transmit intestinal schistosomiasis caused by S. mansoni, a parasite of man which has a wide distribution in Africa and parts of the New World, especially Brazil. The first identification of B. straminea in Hong Kong dates back to the 1970s, and its geographical distribution, phylogenetic relationships, and infection status have not been updated for more than 30 years. Thus, this study aims to reveal the distribution and current infection status of B. straminea in contemporary Hong Kong. METHODS: Snails were collected from different parts of Hong Kong from July 2016 to January 2017. Both anatomical and molecular methods were applied to identify B. straminea. Cytochrome c oxidase subunit 1 (cox1), internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and 16S ribosomal DNA (rDNA) were sequenced from individual snails and analyzed. To detect the presence of S. mansoni, both biopsy and PCR analyses were carried out. RESULTS: Using both anatomical and molecular analyses, this study demonstrated the existence of black- and red-coloured shell B. straminea in different districts in the New Territories in Hong Kong, including places close to the mainland China border. None of the B. straminea (n = 87) investigated were found to be infected with S. mansoni when tested by biopsy and PCR. The Hong Kong B. straminea are genetically indistinguishable, based on the chosen molecular markers (cox1, ITS1-5.8S-ITS2, and 16S rDNA), and are similar to those obtained in mainland China and South America. CONCLUSION: Biomphalaria straminea is now well established in freshwater habitats in Hong Kong. No evidence of infection with S. mansoni has been found. Surveillance should be continued to monitor and better understand this schistosomiasis intermediate host in mainland China and Hong Kong.

Update on the distribution and phylogenetics of Biomphalaria (Gastropoda: Planorbidae) populations in Guangdong Province, China.

In 1973 planorbid snails then identified as Biomphalaria straminea were discovered in Hong Kong, China. It was assumed that these snails had been introduced to Hong Kong via the import of tropical fish by air from South America. In 2012 Biomphalaria were found for the first time in Guangdong Province, China. In view of the renewed interest in these invasive snails, a morphological and DNA-sequence based phylogenetic study was undertaken for seven populations of Biomphalaria snails collected in Guangdong. Morphologically and phylogenetically, five of the populations clustered more closely with Biomphalaria kuhniana than with B. straminea. Levels of genetic diversity among the populations were about half those of autochthonous populations in Brazil, the phylogenetic relationships did not correlate with a radiation from any one international port in China, and different lineages appeared associated with different ports. Consequently in explaining the current distribution of the snails, multiple colonization events, each establishing a new local snail population near to maritime international container ports, were considered more likely than the spread of snails from Hong Kong to China. The displacement of B. straminea by B. kuhniana in Guangdong is considered as an explanation for the habitat changes observed among the snails between Hong Kong in the 1980s and the present. The conclusions of the study are that any risk of Schistosoma mansoni transmission in China is more likely to come from parasite importation in the intramolluscan stage, than from transmission by migrant workers from South America or Africa. In addition, although likely to be rare, sporadic outbreaks of imported schistosomiasis (caused by invading infected snails) could be a threat to public health in the vicinity of International container ports (not only in Guangdong Province). Further work is called for to investigate further the presence of B. kuhniana and its potential interactions with B. straminea (the former is thought to be incompatible with S. mansoni), and the responses of Chinese Biomphalaria to potential competitors such as Thiaridae. The current expansion of container ports in Brazil and Venezuela, and the increase in trade with China, is likely to accentuate any current risk of imported schistosomiasis, and surveillance around ports in China, together with further research, are necessary.

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.