Unveiling the ecotoxicological effects of azoxystrobin-based fungicides at realistic concentrations on the land snail, Theba pisana.

The ecotoxicological consequences of azoxystrobin on land snails have not yet been addressed. Therefore, the present study aims to provide novel data on the threat of a commercial grade azoxystrobin (AMISTAR) at two environmentally relevant concentrations (0.3 µg/ml) and tenfold (3 µg/ml) on the model species, Theba pisana by physiological, biochemical, and histopathological markers for 28 days. Our results showed a reduction in animal food consumption and growth due to exposure to both azoxystrobin concentrations. It also induced oxidative stress and led to a significant decrease in lipid peroxidation (LPO) levels after 7 days of exposure, while the opposite effect occurred after 28 days. Except for the 7-day exposure, all treated snails had significantly reduced glutathione (GSH) content and increased catalase (CAT) activity at all-time intervals. Glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, and protein content (PC) were elevated in treated snails at all-time intervals. Moreover, alterations in acetylcholinesterase (AChE) activity between a decrease and an increase were noticed. Additionally, azoxystrobin exerted changes in T. pisana hepatopancreas architecture. Our study suggests that azoxystrobin may have negative ecological consequences for T. pisana and highlights its potential risks to the natural environment.

Research on the Mechanism of Metaldehyde on Pomacea canaliculata.

Golden apple snail (Pomacea canaliculata), a major alien invasive organism in China, affects food production and poses a threat to human health. Metaldehyde is a highly effective, commonly used snail killer with low toxicity. Virulence determination, tissue section, iTRAQ and RNA interference were used to systematically study the toxicity of metaldehyde on P. canaliculata. The molluscicidal activity tests showed that metaldehyde exhibits strong toxicity against P. canaliculata. Physiological and biochemical data indicate that metaldehyde can cause damage to the gills, liver, pancreas, and kidneys of snails, also reduce the oxygen consumption rate and ammonia excretion rate of golden apple snails, and cause neurological diseases. The proteome of the gill region of the golden apple snail after exposure to metaldehyde was analyzed by using iTRAQ technology. A total of 360 differential proteins were identified, and four target proteins were screened, namely, alpha-protein kinase 1 (ALPK1), cubilin (CUBN), sodium- and chloride-dependent GABA transporter 2 (GAT2), and acetylcholinesterase (AChE). RNAi was used to target the four proteins. After the ALPK1 and CUBN protein genes were interfered with by metaldehyde treatment, it was found that the mortality rate of the golden apple snail significantly increased. However, interference of GAT2 and AChE protein genes by metaldehyde led to no significant change in the mortality rates of the snails. The histopathological observation of the gill showed that the rate of cilia shedding in the gill decreased after the interference of ALPK1 and CUBN protein genes.

How to Overcome a Snail? Identification of Putative Neurotoxins of Snail-Feeding Firefly Larvae (Coleoptera: Lampyridae, Lampyris noctiluca).

The larvae of some lampyrid beetles are highly specialized predators of snails. They have been observed to climb on the shells of their prey and use this exposed position to bite and inject secretions potentially originating from the midgut. Besides serving the purpose of extra-oral digestion (EOD), injected compounds also seem to have a paralyzing effect. Up to now, the toxins causing this paralyzing activity have not been identified. In the current study, we provide a first compositional analysis of the midgut secretion from lampyrid larvae, with a focus on identifying putative neurotoxins causing the observed paralyzing effect. For this purpose, we utilized a combined proteo-transcriptomic approach to characterize the compounds present in the midgut secretion of larval stages of Lampyris noctiluca. In terms of the absolute numbers of identified compounds, the midgut secretion is dominated by hydrolyzing enzymes comprising peptidases, carboxylesterases, and glycosidases. However, when considering expression levels, a few rather short cysteine-rich peptides exceed all other compounds. Some of these compounds show moderate similarity to putative neurotoxins identified in the venom of other arthropods and could be responsible for paralyzing effects. In addition to these potential toxins, we provide a list of peptides typical of the midgut secretion of L. noctiluca, supplemented by the corresponding precursor sequences.

Novel insights into the glucose metabolic alterations of freshwater snails: a pathway to molluscicide innovation and snail control strategies.

As ecosystem disruptors and intermediate hosts for various parasites, freshwater snails have significant socioeconomic impacts on human health, livestock production, and aquaculture. Although traditional molluscicides have been widely used to mitigate these effects, their environmental impact has encouraged research into alternative, biologically based strategies to create safer, more effective molluscicides and diminish the susceptibility of snails to parasites. This review focuses on alterations in glucose metabolism in snails under the multifaceted stressors of parasitic infections, drug exposure, and environmental changes and proposes a novel approach for snail management. Key enzymes within the glycolytic pathway, such as hexokinase and pyruvate kinase; tricarboxylic acid (TCA) cycle; and electron transport chains, such as succinate dehydrogenase and cytochrome c oxidase, are innovative targets for molluscicide development. These targets can affect both snails and parasites and provide an important direction for parasitic disease prevention research. For the first time, this review summarises the reverse TCA cycle and alternative oxidase pathway, which are unique metabolic bypasses in invertebrates that have emerged as suitable targets for the formulation of low-toxicity molluscicides. Additionally, it highlights the importance of other metabolic pathways, including lactate, alanine, glycogenolysis, and pentose phosphate pathways, in snail energy supply, antioxidant stress responses, and drug evasion mechanisms. By analysing the alterations in key metabolic enzymes and their products in stressed snails, this review deepens our understanding of glucose metabolic alterations in snails and provides valuable insights for identifying new pharmacological targets.

Elevated CO2 exacerbates the risk of methylmercury exposure in consuming aquatic products: Evidence from a complex paddy wetland ecosystem.

Elevated CO2 levels and methylmercury (MeHg) pollution are important environmental issues faced across the globe. However, the impact of elevated CO2 on MeHg production and its biological utilization remains to be fully understood, particularly in realistic complex systems with biotic interactions. Here, a complete paddy wetland microcosm, namely, the rice-fish-snail co-culture system, was constructed to investigate the impacts of elevated CO2 (600 ppm) on MeHg formation, bioaccumulation, and possible health risks, in multiple environmental and biological media. The results revealed that elevated CO2 significantly increased MeHg concentrations in the overlying water, periphyton, snails and fish, by 135.5%, 66.9%, 45.5%, and 52.1%, respectively. A high MeHg concentration in periphyton, the main diet of snails and fish, was the key factor influencing the enhanced MeHg in aquatic products. Furthermore, elevated CO2 alleviated the carbon limitation in the overlying water and proliferated green algae, with subsequent changes in physico-chemical properties and nutrient concentrations in the overlying water. More algal-derived organic matter promoted an enriched abundance of Archaea-hgcA and Deltaproteobacteria-hgcA genes. This consequently increased the MeHg in the overlying water and food chain. However, MeHg concentrations in rice and soil did not increase under elevated CO2, nor did hgcA gene abundance in soil. The results reveal that elevated CO2 exacerbated the risk of MeHg intake from aquatic products in paddy wetland, indicating an intensified MeHg threat under future elevated CO2 levels.

Effects of arsenic on gut microbiota and its bioaccumulation and biotransformation in freshwater invertebrate.

This study aimed to investigate the impact of arsenic stress on the gut microbiota of a freshwater invertebrate, specifically the apple snail (Pomacea canaliculata), and elucidate its potential role in arsenic bioaccumulation and biotransformation. Waterborne arsenic exposure experiments were conducted to characterize the snail's gut microbiomes. The results indicate that low concentration of arsenic increased the abundance of gut bacteria, while high concentration decreased it. The dominant bacterial phyla in the snail were Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota. In vitro analyses confirmed the critical involvement of the gut microbiota in arsenic bioaccumulation and biotransformation. To further validate the functionality of the gut microbiota in vivo, antibiotic treatment was administered to eliminate the gut microbiota in the snails, followed by exposure to waterborne arsenic. The results demonstrated that antibiotic treatment reduced the total arsenic content and the proportion of arsenobetaine in the snail's body. Moreover, the utilization of physiologically based pharmacokinetic modeling provided a deeper understanding of the processes of bioaccumulation, metabolism, and distribution. In conclusion, our research highlights the adaptive response of gut microbiota to arsenic stress and provides valuable insights into their potential role in the bioaccumulation and biotransformation of arsenic in host organisms. ENVIRONMENTAL IMPLICATION: Arsenic, a widely distributed and carcinogenic metalloid, with significant implications for its toxicity to both humans and aquatic organisms. The present study aimed to investigate the effects of As on gut microbiota and its bioaccumulation and biotransformation in freshwater invertebrates. These results help us to understand the mechanism of gut microbiota in aquatic invertebrates responding to As stress and the role of gut microbiota in As bioaccumulation and biotransformation.

Ultrastructural study of neuronal cells and localization of ghrelin-like peptide and its receptor in the ganglia of the golden apple snail (Pomacea canaliculata).

Pomacea canaliculata is an invasive snail species causing major problems in agriculture. The snail biology was then investigated. The main objective of the present study was to investigate the nervous system of the snail. The nervous system comprises pairs of cerebral, buccal, pedal, pleural, parietal ganglia and an unpaired visceral ganglion. Most neurons were concentrated at the periphery of the ganglia. The neurons were classified into four types: NR1, NR2, NR3, and NR4. The percentages of the NR3 and NR4 in the pleural and pedal ganglia were significantly higher than those of other ganglia. Ultrastructural study revealed that nuclei of all neuronal types exhibited mostly euchromatins. Many organelles including ribosomes and endoplasmic reticulum were found in their cytoplasm. However, various mitochondria were found in the NR2 and NR3. The immunohistochemistry revealed immunoreactivity of ghrelin-like peptide in the neurons of the cerebral, pleural and pedal ganglia. However, immunoreactivity of GHS-R1a-like peptide existed only in the neurons of the pleural and pedal ganglia. The present study is the first to demonstrate the existence of ghrelin-like peptide and its receptor in P. canaliculata nervous system.

Metals and metallothionein evolution in snails: a contribution to the concept of metal-specific functionality from an animal model group.

This is a critical review of what we know so far about the evolution of metallothioneins (MTs) in Gastropoda (snails, whelks, limpets and slugs), an important class of molluscs with over 90,000 known species. Particular attention will be paid to the evolution of snail MTs in relation to the role of some metallic trace elements (cadmium, zinc and copper) and their interaction with MTs, also compared to MTs from other animal phyla. The article also highlights the important distinction, yet close relationship, between the structural and metal-selective binding properties of gastropod MTs and their physiological functionality in the living organism. It appears that in the course of the evolution of Gastropoda, the trace metal cadmium (Cd) must have played an essential role in the development of Cd-selective MT variants. It is shown how the structures and Cd-selective binding properties in the basal gastropod clades have evolved by testing and optimizing different combinations of ancestral and novel MT domains, and how some of these domains have become established in modern and recent gastropod clades. In this context, the question of how adaptation to new habitats and lifestyles has affected the original MT traits in different gastropod lineages will also be addressed. The 3D structures and their metal binding preferences will be highlighted exemplarily in MTs of modern littorinid and helicid snails. Finally, the importance of the different metal requirements and pathways in snail tissues and cells for the shaping and functionality of the respective MT isoforms will be shown.

Biomonitoring of PAHs and PCBs in industrial, suburban, and rural areas using snails as sentinel organisms.

There is a worldwide concern about the presence of persistent organic pollutants (POPs) in the environment because of their toxicity, bioaccumulation, and resistance to degradation. Various conventional monitoring techniques have been used to assess their presence in diverse environmental compartments. Most currently available methods, however, have limitations with regards to long-term monitoring. In the present work, juvenile Cornu aspersum (O. F. Müller, 1774) snails were tested in field microcosms as biomonitors for two major classes of organic pollutants, namely, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). The study assessed their deployment in one suburban, one rural, and two industrial sites over an 18-week period and monitored for temporal variations of 16 PAHs and 22 PCBs. Sampling was conducted once every 3 weeks. Targeted pollutants were extracted from the caged snails using the QuEChERS extraction procedure and subsequently analyzed using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). The results showed that the bioaccumulation of specific pollutants was site dependent; significantly higher levels of PCBs were observed at the industrial sites as compared to the suburban and rural ones. PAHs were bioaccumulated by the snails via ingestion of air and soil whereas PCBs were mainly bioaccumulated via soil contact and ingestion. The findings of this study indicate that C. aspersum is a reliable model organism for the biomonitoring of organic pollutants in air and soil compartments and can be used as part of an integrated environmental assessment.

Benzodiazepine Delorazepam Induces Locomotory Hyperactivity and Alterations in Pedal Mucus Texture in the Freshwater Gastropod Planorbarius corneus.

Benzodiazepines, psychotropic drugs, are ubiquitous in the aquatic environment due to over-consumption and inefficient removal by sewage treatment plants. Bioaccumulation with consequent behavioral and physiological effects has been reported in many aquatic species. However, the responses are species-specific and still poorly understood. To improve the knowledge, we exposed the freshwater snail Planorbarius corneus to 1, 5, or 10 µg/L of delorazepam, the most widely consumed benzodiazepine in Italy. Conventional behavioral tests were used to assess the effects on locomotor and feeding behavior. Histological and biochemical analyses were also performed to detect possible changes in the structure and composition of the foot mucus and glands. The results show a paradoxical response with reduced feeding activity and locomotor hyperactivity. Pedal mucus was altered in texture but not in composition, becoming particularly rich in fibrous collagen-like material, and a significant change in the protein composition was highlighted in the foot. In conclusion, exposure to delorazepam induces disinhibited behavior in Planorbarius corneus, potentially increasing the risk of predation, and an increase in mucus protein production, which, together with reduced feeding activity, would severely compromise energy resources.

Comparative study on Lake Manzala in Egypt after and before development and purifications: Ecosystem and biomarkers; biochemical, histopathological and immunohistochemical.

The present work aims to evaluate the impact of Lake Manzala development, started in 2017, on lake water quality and biomarkers of Lake Oreochromis niloticus and Biomphalaria alexandrina samples from Dakahlia and Port Said during 2021 and compare it with the results of a series of studies concerning the same criteria in Lake Manzala during 2015. Results showed a remarkable increase in water EC, indicating a higher water exchange with the sea, a significant decrease in Pb, Cd, Cu, and Zn levels in water samples, and a remarkable decline in Cd and Pb bioaccumulation in all fish and snail samples. Macroinvertebrate samples showed higher taxa richness than in 2015, indicating biologically improved lake water quality. Results showed no trematode transmission, while there were natural infections in B. alexandrina snails during 2015. Biochemically, liver enzymes and hematological criteria in fish and snail samples during 2021 showed levels nearer to control at Port Said, indicating a less stressed liver and more healthy specimens than in 2015. Histopathological examination of fish organs (except spleen) and snail tissues pointed to their improved tissue architecture in Port Said than that of Dakahlia (2021). However, the 2021 samples were better than those of 2015. The immunohistochemical study showed higher expression of IL-6 in Dakahlia samples than the other samples, denoting higher tissue inflammation and humoral immune response. So, all the examined criteria indicated that Manzala Lake is positively impacted by the developmental and purification process, especially in Port Said.

Purification and characterisation of heparin-like sulfated polysaccharides with potent anti-SARS-CoV-2 activity from snail mucus of Achatina fulica.

Heparin-like sulfated polysaccharide, acharan sulfate, was purified from the mucus of an African giant snail with unique sulfated glycosaminoglycans (GAGs). This study reported on finding novel and safe heparin resources from Achatina fulica for further use as well as easy isolation and purification of the active fraction from the initial raw material. Its structure was characterised by a strong-anion exchange combined with high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that the potential acharan sulfate fraction is a glycosaminoglycan composed of several repeating disaccharide units, namely, of →4)-α-IdoA(2S)(1→4)-α-GlcNAc/GlcNAc(6S)/GlcNSO3(6S)(1→, and hence, presents heterogeneity regarding negative net charge density. Furthermore, the heparinase digests inhibit the binding of SARS-CoV-2 spike protein to the ACE2 receptor. In summary, the acharan sulfate presented in this work has shown its great potential for application in the preparation of sulfated polysaccharides as an alternative to heparin with important biological activity.

Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica).

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax were estimated to be 0.11 mM and 55.6 µmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications.

Biomineralization biomarkers to assess microplastics toxic effects in the freshwater snail Pomacea canaliculata.

Microplastics (MPs) pollution has increased the number of reports on the toxic effects on biota, especially aquatic organisms. Recently, studies highlighted changes in ion transport and concentration, especially Ca2+, in organisms exposed to MPs. For calcifying organisms, such as mollusks, Ca2+ homeostasis is critical for their shells construction. We investigated the effects of polyethylene (PE) MPs at 20 µg/L on biomineralization biomarkers (Ca2+ATPase, carbonic anhydrase, hemolymph [Ca2+], and shell regeneration) of the freshwater gastropod Pomacea canaliculata. Two experimental sets were performed: (1) animals in physiological condition and (2) animals with their shells excised. The results of the first set showed that within 24 h, the hemolymph [Ca2+] decreased, and the Ca2+ATPase activity increased in the mantle edge. For carbonic anhydrase (CA), the activity decreased in the gland and increased in the mantle. By 72 h, the hemolymph [Ca2+] had not changed, whereas both enzymes had increased in both tissues. In the second set, the hemolymph [Ca2+] increased after 72 h, whereas Ca2+ATPase activity decreased in both tissues. For AC, the opposite results were observed. At 120 h, calcium pumping was still reduced and CA values increased in the digestive gland. Additionally, MPs exposure increased the capacity of the gastropods to recover their shells. Based on this, our work provides novel data associating PE microplastic exposures (at 20 µg/L) and their potential to stimulate biomineralization enzymes of P. canaliculata, as well as increase shell regeneration in excised animal; a good prerogative for further investigations on both subjects that still lacks of more robust evidence.

Combined toxic effects of environmental predominant microplastics and ZnO nanoparticles in freshwater snail Pomaceae paludosa.

In the past few years, microplastics are one of the ubiquitous threatening pollutants in aquatic habitats. These persistent microplastics interact with other pollutants, especially nanoparticles were adherent on the surface, which causes potential hazards in the biota. In this study, the toxic effects of individual and combined (28 days) exposure with zinc oxide nanoparticles and polypropylene microplastics were assessed in freshwater snail Pomeacea paludosa. After the experiment, the toxic effect was evaluated by the estimation of vital biomarkers activities including antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), oxidative stress in carbonyl protein (CP), lipid peroxidation (LPO), and digestive enzymes (esterase and alkaline phosphatase). Chronic exposure to pollutants in snails causes increased reactive oxygen species level (ROS) and generates free radicals in their body which leads to impairment and alterations of biochemical markers. Where alteration in acetylcholine esterase (AChE) activity and decreased digestive enzymes (esterase and alkaline phosphatase) activities were observed in both individual and combined exposed groups. Further, histology results revealed the reduction of haemocyte cells, the disintegration of blood vessels, digestive cells, calcium cells, and DNA damage was also detected in the treated animals. Overall, when compared to individual exposures, combined exposure of pollutants (zinc oxide nanoparticles and polypropylene microplastics) causes more serious harms including decline and increased antioxidant enzyme parameters, damage the protein and lipids by oxidative stress, increased neurotransmitter activity, decrease digestive enzyme activities in the freshwater snail. The outcome of this study concluded that polypropylene microplastics along with nanoparticles cause severe ecological threats and physio-chemical effects on the freshwater ecosystem.

Ion channel chameleons: Switching ion selectivity by alternative splicing.

Voltage-gated sodium and calcium channels are distinct, evolutionarily related ion channels that achieve remarkable ion selectivity despite sharing an overall similar structure. Classical studies have shown that ion selectivity is determined by specific binding of ions to the channel pore, enabled by signature amino acid sequences within the selectivity filter (SF). By studying ancestral channels in the pond snail (Lymnaea stagnalis), Guan et al. showed in a recent JBC article that this well-established mechanism can be tuned by alternative splicing, allowing a single CaV3 gene to encode both a Ca2+-permeable and an Na+-permeable channel depending on the cellular context. These findings shed light on mechanisms that tune ion selectivity in physiology and on the evolutionary basis of ion selectivity.

Coping with harsh heat environments: molecular adaptation of metabolic depression in the intertidal snail Echinolittorina radiata.

Harsh thermal environments in the rocky intertidal zone pose serious physiological and molecular challenges to the inhabitants. Metabolic depression is regarded as an energy-conserving feature of intertidal species. To understand the molecular mechanism of metabolic depression, we investigated physiological and transcriptomic responses in the intertidal snail Echinolittorina radiata. The metabolic rate and expression of most genes were insensitive to temperatures ranging from 33 to 45 °C and then increased with further heating to 52 °C. Different from other genes, the genes involved in heat shock response (HSR) and oxidative stress response (OSR) (e.g., genes encoding heat shock protein 70 (HSP70) and cytochrome P450 protein (CYP450)) kept upregulating during metabolic depression. These high levels of HSR and OSR genes should be important for surviving the harsh thermal environments on the rocky shore. In the population experiencing more frequent moderate heat events, the depression breadth was larger, and the change in magnitude of upregulation was insensitive for HSR genes (e.g., HSP70s) but heat-sensitive for OSR genes (e.g., CYP450s) at the temperature of 37 to 45 °C. These findings indicate that both the thermal sensitivity of HSR and OSR genes and the insensitivity of metabolic genes are crucial for surviving extreme intertidal environments, and different populations of the same species rely on various physiological mechanisms to differing extents to deal with heat stress. The cellular stress response is not a "one size fits all" response across populations largely depending on local thermal regimes.

Spatio-temporal changes in oxidative stress physiology parameters in apple snail Pila globosa as a function of soil Mg, Ca, organic carbon and aquatic physico-chemical factors.

Information on the oxidative stress physiology parameters (OSPP) in general and as a function of the fluctuation of Mg, Ca and organic carbon present in soil and aquatic physico-chemical factors such as pH, temperature and salinity in particular are scanty in the amphibious snail Pila globosa. A spatio-temporal analysis of redox metabolism (as OSPP) followed by discriminant function analysis of the obtained data were performed in P. globosa sampled from the east-coasts of Odisha state, India (mostly along the Bay of Bengal) for environmental health assessment purposes. Results revealed that the OSPP are susceptible to seasonal synergistic variation of soil and physico-chemical factors. Overall, lipid peroxidation, total antioxidant capacity, activities of catalase, glutathione reductase had positive correlation whereas ascorbic acid, the reduced glutathione and the activity of superoxide dismutase had non-significant correlation with the soil Mg, Ca, organic carbon, and pH, temperature and salinity of water. In the summer season, the snails had a marked 51.83% and 26.41% higher lipid peroxidation level and total antioxidative activity as compared to the other seasons. Spatial variation of OSPP indicates that snails residing away from the Bay of Bengal coast had at least 4.4% lower antioxidant level in winter and 30% higher lipid peroxide levels in summer as compared to the rest of the sampling sites. Results on OSPP in P. globosa may be useful for monitoring the ecotoxic effects of environment using molluscs in general and P. globosa in particular.

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17ß-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

From a Cone Snail Toxin to a Competitive MC4R Antagonist.

The melanocortin 4 receptor (MC4R) plays a role in energy homeostasis and represents a target for treating energy balance disorders. For decades, synthetic ligands have been derived from MC4R endogenous agonists and antagonists, such as setmelanotide used to treat rare forms of genetic obesity. Recently, animal venoms have demonstrated their capacity to provide melanocortin ligands with toxins from a scorpion and a spider. Here, we described a cone snail toxin, N-CTX-Ltg1a, with a nanomolar affinity for hMC4R but unrelated to any known toxins or melanocortin ligands. We then derived from the conotoxin the linear peptide HT1-0, a competitive antagonist of Gs, G15, and ß-arrestin2 pathways with a low nanomolar affinity for hMC4R. Similar to endogenous ligands, HT1-0 needs hydrophobic and basic residues to bind hMC4R. Altogether, it represents the first venom-derived peptide of high affinity on MC4R and paves the way for the development of new MC4R antagonists.