Toxicological effects of Saponin on the free larval stages of Schistosoma mansoni, infection rate, some biochemical and molecular parameters of Biomphalaria alexandrina snails.

Saponins have been used as biopesticides. The objective of the present study is to investigate the toxic effects of Saponin against Biomphalaria alexandrina snails. Results showed that Saponin exhibited a molluscicidal activity against adult B. alexandrina snails at LC50 (70.05 mg/l) and had a larvicidal effect on the free larval stages of Schistosoma mansoni. To evaluate the lethal effects, snails were exposed to either LC10 (51.8 mg/l) or LC25 (60.4 mg/l) concentrations of Saponin. The survival, the infection rates, protein, albumin, and total fat levels were decreased, while glucose levels were increased in exposed snails compared to control snails. Also, these concentrations significantly raised Malondialdehyde (MDA) and Glutathione S Transferase (GST) levels, whereas reduced Superoxide dismutase (SOD) activity and the total antioxidant capacity (TAC) in exposed snails. Furthermore, these concentrations resulted in endocrine disruptions where it caused a significant increase in testosterone (T) level; while a significant decrease in Estradiol (E2) levels were noticed. As for Estrogen (E) level, it was increased after exposure to LC10 Saponin concentration while after exposure to LC25 concentration, it was decreased. Also, LC10 and LC25 concentrations of Saponin caused a genotoxic effect and down-regulation of metabolic cycles in the snails. In conclusion, Saponins caused deleterious effects on the intermediate host of schistosomiasis mansoni. Therefore, B. alexandrina snails could be used as models to screen the toxic effects of Saponins in the aquatic environment and if it was used as a molluscicide, it should be used cautiously and under controlled circumstances.

Identification of Schistosoma mansoni miracidia attractant candidates in infected Biomphalaria glabrata using behaviour-guided comparative proteomics.

Schistosomiasis, caused by infection with Schistosoma digenetic trematodes, is one of the deadliest neglected tropical diseases in the world. The Schistosoma lifecycle involves the miracidial infection of an intermediate freshwater snail host, such as Biomphalaria glabrata. Dispersing snail host-derived Schistosoma miracidia attractants has been considered a method of minimising intermediate host infections and, by extension, human schistosomiasis. The attractiveness of B. glabrata to miracidia is known to be reduced following infection; however, the relationship between duration of infection and attractiveness is unclear. Excretory-secretory proteins (ESPs) most abundant in attractive snail conditioned water (SCW) are key candidates to function as miracidia attractants. This study analysed SCW from B. glabrata that were naïve (uninfected) and at different time-points post-miracidia exposure (PME; 16h, 1-week, 2-weeks and 3-weeks PME) to identify candidate ESPs mediating Schistosoma mansoni miracidia behaviour change, including aggregation and chemoklinokinesis behaviour (random motion, including slowdown and increased turning rate and magnitude). Miracidia behaviour change was only observed post-addition of naïve and 3W-PME SCW, with other treatments inducing significantly weaker behaviour changes. Therefore, ESPs were considered attractant candidates if they were shared between naïve and 3W-PME SCW (or exclusive to the former), contained a predicted N-terminal signal peptide and displayed low identity (<50%) to known proteins outside of the Biomphalaria genus. Using these criteria, a total of 6 ESP attractant candidates were identified, including acetylcholine binding protein-like proteins and uncharacterised proteins. Tissue-specific RNA-seq analysis of the genes encoding these 6 ESPs indicated relatively high gene expression within various B. glabrata tissues, including the foot, mantle and kidney. Acetylcholine binding protein-like proteins were highly promising due to their high abundance in naïve and 3W-PME SCW, high specificity to B. glabrata and high expression in the ovotestis, from which attractants have been previously identified. In summary, this study used proteomics, guided by behavioural assays, to identify miracidia attractant candidates that should be further investigated as potential biocontrols to disrupt miracidia infection and minimise schistosomiasis.

Molecular characterization of thioester-containing proteins in Biomphalaria glabrata and their differential gene expression upon Schistosoma mansoni exposure.

Schistosomiasis is a disease caused by trematode parasites of the genus Schistosoma that affects approximately 200 million people worldwide. Schistosomiasis has been a persistent problem in endemic areas as there is no vaccine available, currently used anti-helmintic medications do not prevent reinfection, and most concerning, drug resistance has been documented in laboratory and field isolates. Thus, alternative approaches to curtail this human disease are warranted. Understanding the immunobiology of the obligate intermediate host of these parasites, which include the freshwater snail Biomphalaria glabrata, may facilitate the development of novel methods to stop or reduce transmission to humans. Molecules from the thioester-containing protein (TEP) superfamily have been shown to be involved in immunological functions in many animals including corals and humans. In this study we identified, characterized, and compared TEP transcripts and their expression upon S. mansoni exposure in resistant and susceptible strains of B. glabrata snails. Results showed the expression of 11 unique TEPs in B. glabrata snails. These transcripts present high sequence identity at the nucleotide and putative amino acid levels between susceptible and resistant strains. Further analysis revealed differences in several TEPs' constitutive expression levels between resistant and susceptible snail strains, with C3-1, C3-3, and CD109 having higher constitutive expression levels in the resistant (BS90) strain, whereas C3-2 and TEP-1 showed higher constitutive expression levels in the susceptible (NMRI) strain. Furthermore, TEP-specific response to S. mansoni miracidia exposure reiterated their differential expression, with resistant snails upregulating the expression of both TEP-4 and TEP-3 at 2 h and 48 h post-exposure, respectively. Further understanding the diverse TEP genes and their functions in invertebrate animal vectors will not only expand our knowledge in regard to this ancient family of immune proteins, but also offer the opportunity to identify novel molecular targets that could aid in the efforts to develop control methods to reduce schistosomiasis transmission.

New Insights Into Biomphalysin Gene Family Diversification in the Vector Snail Biomphalaria glabrata.

Aerolysins initially characterized as virulence factors in bacteria are increasingly found in massive genome and transcriptome sequencing data from metazoans. Horizontal gene transfer has been demonstrated as the main way of aerolysin-related toxins acquisition in metazoans. However, only few studies have focused on their potential biological functions in such organisms. Herein, we present an extensive characterization of a multigene family encoding aerolysins - named biomphalysin - in Biomphalaria glabrata snail, the intermediate host of the trematode Schistosoma mansoni. Our results highlight that duplication and domestication of an acquired bacterial toxin gene in the snail genome result in the acquisition of a novel and diversified toxin family. Twenty-three biomphalysin genes were identified. All are expressed and exhibited a tissue-specific expression pattern. An in silico structural analysis was performed to highlight the central role played by two distinct domains i) a large lobe involved in the lytic function of these snail toxins which constrained their evolution and ii) a small lobe which is structurally variable between biomphalysin toxins and that matched to various functional domains involved in moiety recognition of targets cells. A functional approach suggests that the repertoire of biomphalysins that bind to pathogens, depends on the type of pathogen encountered. These results underline a neo-and sub-functionalization of the biomphalysin toxins, which have the potential to increase the range of effectors in the snail's immune arsenal.

Identification and localization of a gonadotropin-releasing hormone-related neuropeptide in Biomphalaria, an intermediate host for schistosomiasis.

Freshwater snails of the genus Biomphalaria serve as obligatory hosts for the digenetic trematode Schistosoma mansoni, the causative agent for the most widespread form of intestinal schistosomiasis. Within Biomphalaria, S. mansoni larvae multiply and transform into the cercariae form that can infect humans. Trematode development and proliferation is thought to be facilitated by modifications of host behavior and physiological processes, including a reduction of reproduction known as "parasitic castration." As neuropeptides participate in the control of reproduction across phylogeny, a neural transcriptomics approach was undertaken to identify peptides that could regulate Biomphalaria reproductive physiology. The present study identified a transcript in Biomphalaria alexandrina that encodes a peptide belonging to the gonadotropin-releasing hormone (GnRH) superfamily. The precursor and the predicted mature peptide, pQIHFTPDWGNN-NH2 (designated Biom-GnRH), share features with peptides identified in other molluscan species, including panpulmonates, opisthobranchs, and cephalopods. An antibody generated against Biom-GnRH labeled neurons in the cerebral, pedal, and visceral ganglia of Biomphalaria glabrata. GnRH-like immunoreactive fiber systems projected to all central ganglia. In the periphery, immunoreactive material was detected in the ovotestis, oviduct, albumen gland, and nidamental gland. As these structures serve crucial roles in the production, transport, nourishment, and encapsulation of eggs, disruption of the GnRH system of Biomphalaria could contribute to reduced reproductive activity in infected snails.

Toxicity evaluation of the active ingredient acetamiprid and a commercial formulation (Assail® 70) on the non-target gastropod Biomphalaria straminea (Mollusca: Planorbidae).

Neonicotinoids emerged as an environmentally safe alternative to previous generations of insecticides becoming one of the most widely applied in modern agriculture. Nevertheless, they have been reported to affect several non-target organisms. Most toxicity studies focus on the effects on pollinators or terrestrial invertebrates and evaluate either the active ingredient or the commercial formulation. In the present study, we aimed to assess the long-term effects of the active ingredient acetamiprid and a broadly used commercial formulation (Assail® 70) on the non-target freshwater gastropod Biomphalaria straminea using a battery of biomarkers. A 14 day-exposure of adult organisms to both active ingredient and commercial formulation increased carboxylesterase activity and glutathione content, inhibited superoxide dismutase activity and decreased reactive oxygen species levels. The commercial formulation additionally increased glutathione S-transferase activity and inhibited catalase activity. The results indicate a greater toxicity of the commercial formulation than that of the active ingredient alone. Cholinesterase activity, development and offspring survival of B. straminea were not impaired. We conclude that the toxicity of acetamiprid on this gastropod species is mainly related to effects on detoxification and oxidative metabolism responses. This study provides novel information about the adverse effects of the active ingredient and a commercial formulation of a widely used neonicotinoid on a non-target aquatic species.

Evaluation of changes in the carbohydrate metabolism of Biomphalaria glabrata Say, 1818 exposed to experimental coinfection by Angiostrongylus cantonensis (Nematoda) and Echinostoma paraensei (Trematoda).

The interaction between intermediate snail hosts and helminths can cause metabolic changes in the former. The snails use their reserves for maintenance of their vital processes, by activating the internal defense system and repairing tissue damage, while also supplying necessary energy for the parasites' development. Our aims were to evaluate the lactate dehydrogenase activity and the glucose concentration in the hemolymph of Biomphalaria glabrata experimentally coinfected by Angiostrongylus cantonensis and Echinostoma paraensei. Besides these aspects, the glycogen content in the digestive gland complex and cephalopedal mass along with histochemical changes in parasitized snails were analyzed. The snails were divided in group A (infected by 1200 L1 of A. cantonensis), group E (infected by 20 E. paraensei miracidia), group A + E (co-infected with A. cantonensis first and after a week by E. paraensei), group E + A (co-infected with E. paraensei first and then by A. cantonensis) and control group (not infected). During four weeks after exposure, samples were collected for biochemical and histochemical analyses. In the infected snails, glucose levels and glycogen content in the digestive gland complex and cephalopedal mass were significantly lower, in contrast with an increase of lactate dehydrogenase activity. These results indicate that the intense energy demand resulting from the presence of parasites causes the host snail to accelerate the anaerobic degradation of carbohydrates to obtain energy, in an attempt to maintain homeostasis. Both parasites were observed in histochemical analysis to cause tissue damages in the snails. So, although the snails were able to sustain the coinfection, several metabolic and tissue changes occurred, mainly in those infected with E. paraensei and then with A. cantonensis.

Localization of keyhole limpet hemocyanin-like immunoreactivity in the nervous system of Biomphalaria alexandrina.

Recent years have led to increased effort to describe and understand the peripheral nervous system and its influence on central mechanisms and behavior in gastropod molluscs. This study revealed that an antibody raised against keyhole limpet hemocyanin (KLH) cross-reacts with an antigen(s) found extensively in both the central and the peripheral nervous systems of Biomphalaria alexandrina. The results revealed KLH-like immunoreactive (LIR) neurons in the cerebral, pedal, buccal, left pleural, right parietal, and visceral ganglion within the CNS with fibers projecting throughout all the peripheral nerves. Numerous KLH-LIR peripheral sensory neurons located in the foot, lips, tentacles, mantle, esophagus, and penis exhibited a bipolar morphology with long tortuous dendrites. KLH-LIR cells were also present in the eye and statocyst, thus suggesting the labeling of multiple sensory modalities/cell types. KLH-LIR cells did not co-localize with tyrosine hydroxylase (TH)-LIR cells, which have previously been described in this and other gastropods. The results thus provide descriptions of thousands of peripheral sensory neurons, not previously described in detail. Future research should seek to pair sensory modalities with peripheral cell type and attempt to further elucidate the nature of KLH-like reactivity. These findings also emphasize the need for caution when analyzing results obtained through use of antibodies raised against haptens conjugated to carrier proteins, suggesting the need for stringent controls to help limit potential confounds caused by cross-reactivity. In addition, this study is the first to describe neuronal cross-reactivity with KLH in Biomphalaria, which could provide a substrate for host-parasite interactions with a parasitic trematode, Schistosoma.

Biomphalaria glabrata Granulin Increases Resistance to Schistosoma mansoni Infection in Several Biomphalaria Species and Induces the Production of Reactive Oxygen Species by Haemocytes.

Gastropod molluscs, which have co-evolved with parasitic digenean trematodes for millions of years, utilize circulating heamocytes as the primary method of containing and killing these invading parasites. In order to do so, they must generate suitable amounts of haemocytes that are properly armed to kill parasitic worms. One method by which they generate the haemocytes required to initiate the appropriate cell mediated immune response is via the production and post-translational processing of granulins. Granulins are an evolutionarily conserved family of growth factors present in the majority of eukaryotic life forms. In their pro-granulin form, they can elicit cellular replication and differentiation. The pro-granulins can be further processed by elastase to generate smaller granulin fragments that have been shown to functionally differ from the pro-granulin precursor. In this study, we demonstrate that in vivo addition of Biomphalaria glabrata pro-granulin (BgGRN) can reduce Schistosoma mansoni infection success in numerous Biomphalaria sp. when challenged with different S. mansoni strains. We also demonstrate that cleavage of BgGRN into individual granulin subunits by elastase results in the stimulation of haemocytes to produce reactive oxygen species.

Environmental concentrations of azinphos-methyl cause different toxic effects without affecting the main target (cholinesterases) in the freshwater gastropod Biomphalaria straminea.

Organophosphate insecticides (OPs) are commonly used in Argentina and around the world for pest control in food crops. They exert their toxicity through the inhibition of the enzyme acetylcholinesterase. In the present study, we aimed to evaluate biochemical and reproductive effects in Biomphalaria straminea, a freshwater gastropod naturally distributed in Argentina, of subchronic exposures to environmental azinphos-methyl concentrations (20 and 200 µg L-1). For biochemical parameters, adult organisms were exposed for 14 days and the activity of cholinesterases (ChEs), carboxylesterases (CEs), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), the production of reactive oxygen species (ROS), the total antioxidant capacity (TAC), glycogen and proteins were determined. For reproductive parameters, the egg masses of B. straminea were exposed to azinphos-methyl for one month, and the hatching time and success as well as the offspring survival were registered. We found different toxic effects elicited by the insecticide on the studied biomarkers. CEs activity was significantly inhibited while CAT and GST activities, ROS production and TAC were significantly increased, with respect to the solvent control group. ChE and SOD activities and protein and glycogen contents were not altered by azinphos-methyl. The hatching time and success were not statistically different from control. Nevertheless, the offspring survival was severely affected by the insecticide. Our results show that the primary target of the insecticide (ChE) was not inhibited but CEs, GST, CAT, ROS, TAC and offspring survival were sensitive biomarkers and valuable endpoints for subchronic toxicity assessments in this species.

Molluscicidal impacts of Anagallis arvensis aqueous extract on biological, hormonal, histological and molecular aspects of Biomphalaria alexandrina snails.

Controlling of Biomphalaria alexandrina snails by plant molluscicides is the cornerstone in treating schistosomiasis in Egypt. The objective of this study is, to evaluate the molluscicidal activity of the aqueous leaves extract of Anagallis arvensis against B. alexandrina snails. The present results showed that this aqueous extract was lethal for B. alexandrina snails at (LC50 37.9 mg/l; LC90 48.3 mg/l), and caused reduction in survival; reproductive rates and hormonal activity (testosterone (T) and 17ß-estradiol (E)) of these snails. Histopathological changes occurred in the hermaphrodite glands of snails exposed to the sub lethal concentrations of this aqueous extract are detected, where, there were degeneration in both eggs and sperms and there were losses of connective tissues between acini. The present investigation revealed that this plant had a genotoxic effect especially with its concentration (LC10 and LC25), where, the length of olive tail moment was significantly increased than control group. These observations prove the potent molluscicidal activity of aqueous leaves extract of A. arvensis against the intermediate hosts of Schistosoma mansoni and provide natural biodegradable resources for snails' molluscicidal agents.

GABA-like immunoreactivity in Biomphalaria: Colocalization with tyrosine hydroxylase-like immunoreactivity in the feeding motor systems of panpulmonate snails.

The simpler nervous systems of certain invertebrates provide opportunities to examine colocalized classical neurotransmitters in the context of identified neurons and well defined neural circuits. This study examined the distribution of γ-aminobutyric acid-like immunoreactivity (GABAli) in the nervous system of the panpulmonates Biomphalaria glabrata and Biomphalaria alexandrina, major intermediate hosts for intestinal schistosomiasis. GABAli neurons were localized in the cerebral, pedal, and buccal ganglia of each species. With the exception of a projection to the base of the tentacle, GABAli fibers were confined to the CNS. As GABAli was previously reported to be colocalized with markers for dopamine (DA) in five neurons in the feeding network of the euopisthobranch gastropod Aplysia californica (Díaz-Ríos, Oyola, & Miller, 2002), double-labeling protocols were used to compare the distribution of GABAli with tyrosine hydroxylase immunoreactivity (THli). As in Aplysia, GABAli-THli colocalization was limited to five neurons, all of which were located in the buccal ganglion. Five GABAli-THli cells were also observed in the buccal ganglia of two other intensively studied panpulmonate species, Lymnaea stagnalis and Helisoma trivolvis. These findings indicate that colocalization of the classical neurotransmitters GABA and DA in feeding central pattern generator (CPG) interneurons preceded the divergence of euopisthobranch and panpulmonate taxa. These observations also support the hypothesis that heterogastropod feeding CPG networks exhibit a common universal design.

The protein pheromone temptin is an attractant of the gastropod Biomphalaria glabrata.

The freshwater snail Biomphalaria glabrata has drawn much research interest by virtue of it being one of the intermediate hosts of the parasitic flatworm Schistosoma mansoni, a causative agent of human schistosomiasis. Schistosomiasis is a chronic disease that affects over 260 million people globally, particularly in tropical and sub-tropical regions. One strategy that has been proposed as a way to prevent human infection by the parasite, involves the use of pheromone traps to lure the snail host away from areas of human activity. This requires an understanding of chemosensory communication in B. glabrata, especially of the chemoattractive factors. Although evidence indicates that specific chemical communication takes place, little is known about chemoattractants produced by the snail itself. Here, we report on the functional characterization of an endogenously produced temptin-like protein (BgTemptin) from B. glabrata and demonstrate that recombinant BgTemptin is attractive to this snail. Exposure of B. glabrata to BgTemptin results in 81% (lane maze) and 70% (T-maze) time spent near to the BgTemptin source. This effect, which is dependent on the concentration of the protein, provides another tool that can be further developed and used in efforts to control and eliminate schistosomiasis.

Biomphalaria glabrata Metallothionein: Lacking Metal Specificity of the Protein and Missing Gene Upregulation Suggest Metal Sequestration by Exchange Instead of through Selective Binding.

The wild-type metallothionein (MT) of the freshwater snail Biomphalaria glabrata and a natural allelic mutant of it in which a lysine residue was replaced by an asparagine residue, were recombinantly expressed and analyzed for their metal-binding features with respect to Cd2+, Zn2+ and Cu⁺, applying spectroscopic and mass-spectrometric methods. In addition, the upregulation of the Biomphalaria glabrataMT gene was assessed by quantitative real-time detection PCR. The two recombinant proteins revealed to be very similar in most of their metal binding features. They lacked a clear metal-binding preference for any of the three metal ions assayed-which, to this degree, is clearly unprecedented in the world of Gastropoda MTs. There were, however, slight differences in copper-binding abilities between the two allelic variants. Overall, the missing metal specificity of the two recombinant MTs goes hand in hand with lacking upregulation of the respective MT gene. This suggests that in vivo, the Biomphalaria glabrata MT may be more important for metal replacement reactions through a constitutively abundant form, rather than for metal sequestration by high binding specificity. There are indications that the MT of Biomphalaria glabrata may share its unspecific features with MTs from other freshwater snails of the Hygrophila family.

Changes in energetic metabolism of Biomphalaria glabrata (Mollusca, Planorbidae) in response to exogenous calcium / Alterações no metabolismo energético de Biomphalaria glabrata em resposta ao cálcio exógeno

Abstract Calcium is considered an essential element for the metabolism of aquatic snail Biomphalaria glabrata (Say, 1818), intermediate host of Schistosoma mansoni Sambon, 1907 in Brazil, and represents a limiting factor to its distribution and adaptation to the environment. This study investigated the effect of different concentrations of exogenous CaCO3 on the energetic metabolism of B. glabrata for better understanding the physiological interference of chemical elements dissolved in the environment with the physiology of this species. Sixty-day-old snails were distributed into six groups, five exposed to different concentrations of CaCO3 (20, 40, 60, 80 and 100 mg/L) and a control group. The exposure to CaCO3 was assessed over time, with analysis of 15 snails of each group in the following intervals: 1, 14, 21 or 30 days for hemolymph extraction. Concentrations of calcium and glucose in the hemolymph were determined by commercial kits, and organic acids were extracted using an ion exchange column and analyzed by high-performance liquid chromatography. Concentration of calcium in the hemolymph showed no significant difference (p>0.05) from the control group and between the concentrations tested. Concentration of glucose decreased (p<0.05) in the treatments of exposure to 20 and 40 mg/L and increased when exposed to 80 and 100 mg/L CaCO3 compared to control and to other concentrations tested over 30 days. The organic acids pyruvate, oxaloacetate, citrate, succinate, fumarate, beta-hydroxybutyrate and lactate presented increased concentrations, while propionate and acetoacetate, decreased concentrations, when exposed to CaCO3 compared to control. Considering the influence of different periods of exposure to CaCO3, on the 14th day, there were stronger alterations in the metabolism of B. glabrata. In conclusion, exposure to CaCO3 reduced the concentration of glucose, which is metabolized into pyruvate, the final product of glycolysis, and also influenced the energetic metabolism pathways, indicating an aerobic or partially anaerobic functioning.

Resumo O cálcio é considerado um elemento essencial no metabolismo do molusco aquático Biomphalaria glabrata (Say, 1818), principal hospedeiro intermediário de Schistosoma mansoni Sambon, 1907 no Brasil e, tem sido descrito como um fator limitante na distribuição e adaptação desse molusco no ambiente. O presente trabalho avaliou o efeito de diferentes concentrações de carbonato de cálcio (CaCO3) exógeno ao metabolismo energético de B. glabrata, a fim de subsidiar uma melhor compreensão da interferência de elementos químicos dissolvidos no meio aquático na fisiologia destes moluscos. Foram utilizados moluscos com sessenta dias de vida, distribuídos em seis grupos, cinco expostos a diferentes concentrações de CaCO3 (20, 40, 60, 80 e 100mg/L) e um controle. A exposição ao CaCO3 foi avaliada em função do tempo, sendo retirados 15 moluscos de cada grupo nos seguintes intervalos: 1, 14, 21 ou 30 dias para extração da hemolinfa. As concentrações de cálcio e glicose na hemolinfa foram determinadas usando-se kits comercial e os ácidos orgânicos foram extraídos por meio da coluna de troca iônica e analisados através cromatografia líquida de alta eficiência. Os resultados demonstraram que a concentração de cálcio na hemolinfa não apresentou diferença significativa (p>0,05) em relação ao controle e nas concentrações testadas. A concentração de glicose diminuiu (p<0,05) nas exposições a 20 mg e 40 mg/L e aumentou nas exposições a 80 mg e 100 mg/L de CaCO3 em relação ao controle e demais concentrações testadas ao longo de 30 dias. Os ácidos orgânicos piruvato, oxaloaceato, citrato, succinato, fumarato, β-hidroxibutirato e lactato tiveram suas concentrações aumentadas, enqunato, propionato e acetoacetato tiveram suas concentrações diminuídas na exposição ao CaCO3 comparada ao controle. Quanto a influência dos diferentes períodos de exposição ao CaCO3, aos 14 dias, as alterações no metabolismo de B. glabrata foram mais expressivas. Conclui-se que as exposições ao CaCO3 influenciaram na redução de glicose, sendo esta metabolizada a piruvato, produto final da glicólise e alteraram as vias de metabolismo energético, indicando um funcionamento aeróbio ou parcialmente anaeróbio.

Proteomic analysis of Biomphalaria glabrata plasma proteins with binding affinity to those expressed by early developing larval Schistosoma mansoni.

Interactions between early developing Schistosoma mansoni larval stages and the hemolymph of its snail intermediate host represent the first molecular encounter with the snail's immune system. To gain a more comprehensive understanding of this early parasite-host interaction, biotinylated sporocyst tegumental membrane (Mem) proteins and larval transformation proteins (LTP) were affixed to streptavidin-agarose beads and used as affinity matrices to enrich for larval-reactive plasma proteins from susceptible (NMRI) and resistant (BS-90) strains of the snail Biomphalaria glabrata. Nano-LC/MS-MS proteomic analyses of isolated plasma proteins revealed a diverse array of 94 immune-and nonimmune-related plasma proteins. Included among the immune-related subset were pattern recognition receptors (lectins, LPS-binding protein, thioester-containing proteins-TEPs), stress proteins (HSP60 and 70), adhesion proteins (dermatopontins), metalloproteases (A Disintegrin And Metalloproteinase (ADAM), ADAM-related Zn proteinases), cytotoxins (biomphalysin) and a Ca2+-binding protein (neo-calmodulin). Variable immunoglobulin and lectin domain (VIgL) gene family members, including fibrinogen-related proteins (FREPs), galectin-related proteins (GREPs) and C-type lectin-related proteins (CREPs), were the most prevalent of larval-reactive immune lectins present in plasma. FREPs were highly represented, although only a subset of FREP subfamilies (FREP 2, 3 and 12) were identified, suggesting potential selectivity in the repertoire of plasma lectins recognizing larval glycoconjugates. Other larval-binding FREP-like and CREP-like proteins possessing a C-terminal fibrinogen-related domain (FReD) or C-type lectin binding domain, respectively, and an Ig-fold domain also were identified as predicted proteins from the B. glabrata genome, although incomplete sequence data precluded their placement into specific FREP/CREP subfamilies. Similarly, a group of FReD-containing proteins (angiopoeitin-4, ficolin-2) that lacked N-terminal Ig-fold(s) were identified as a distinct group of FREP-like proteins, separate from the VIgL lectin family. Finally, differential appearance of GREPs in BS-90 plasma eluates, and others proteins exclusively found in eluates of the NMRI strain, suggested snail strain differences in the expression of select larval-reactive immune proteins. This hypothesis was supported by the finding that differential gene expression of the GREP in BS-90 and ADAM in NMRI snail strains generally correlated with their patterns of protein expression. In summary, this study is the first to provide a global comparative proteomic analysis of constitutively expressed plasma proteins from susceptible and resistant B. glabrata strains capable of binding early-expressed larval S. mansoni proteins. Identified proteins, especially those exhibiting differential expression, may play a role in determining immune compatibility in this snail host-parasite system. A complete listing of raw peptide data are available via ProteomeXchange using identifier PXD004942.

HSP70 expression in Biomphalaria glabrata snails exposed to cadmium.

In this study, the effects of the heavy metal cadmium on the stress protein HSP70 are investigated in freshwater mollusks Biomphalaria glabrata. Adult snails were exposed for 96h to CdCl2 at concentrations ranging from 0.09 to 0.7mgL-1 (LC50/96h=0.34 (0.30-0.37). Time and concentration-dependent increases in the expression of HSP70 were observed at sub-lethal levels in the immunoblotting assay. Further, an increased survival to a lethal heat shock was observed in animals pre-exposed to a nonlethal concentration of cadmium, evidencing the induction of acquired tolerance. The present study demonstrated the inducibility of B. glabrata HSP70 by cadmium, a relevant environmental contaminant, at non-lethal levels, providing evidences that the assessment of HSP70 in B. glabrata can be regarded as a suitable biomarker for ecotoxicological studies.

Changes in energetic metabolism of Biomphalaria glabrata (Mollusca, Planorbidae) in response to exogenous calcium.

Calcium is considered an essential element for the metabolism of aquatic snail Biomphalaria glabrata (Say, 1818), intermediate host of Schistosoma mansoni Sambon, 1907 in Brazil, and represents a limiting factor to its distribution and adaptation to the environment. This study investigated the effect of different concentrations of exogenous CaCO3 on the energetic metabolism of B. glabrata for better understanding the physiological interference of chemical elements dissolved in the environment with the physiology of this species. Sixty-day-old snails were distributed into six groups, five exposed to different concentrations of CaCO3 (20, 40, 60, 80 and 100 mg/L) and a control group. The exposure to CaCO3 was assessed over time, with analysis of 15 snails of each group in the following intervals: 1, 14, 21 or 30 days for hemolymph extraction. Concentrations of calcium and glucose in the hemolymph were determined by commercial kits, and organic acids were extracted using an ion exchange column and analyzed by high-performance liquid chromatography. Concentration of calcium in the hemolymph showed no significant difference (p>0.05) from the control group and between the concentrations tested. Concentration of glucose decreased (p<0.05) in the treatments of exposure to 20 and 40 mg/L and increased when exposed to 80 and 100 mg/L CaCO3 compared to control and to other concentrations tested over 30 days. The organic acids pyruvate, oxaloacetate, citrate, succinate, fumarate, beta-hydroxybutyrate and lactate presented increased concentrations, while propionate and acetoacetate, decreased concentrations, when exposed to CaCO3 compared to control. Considering the influence of different periods of exposure to CaCO3, on the 14th day, there were stronger alterations in the metabolism of B. glabrata. In conclusion, exposure to CaCO3 reduced the concentration of glucose, which is metabolized into pyruvate, the final product of glycolysis, and also influenced the energetic metabolism pathways, indicating an aerobic or partially anaerobic functioning.

The Planorbid Snail Biomphalaria glabrata Expresses a Hemocyanin-Like Sequence in the Albumen Gland.

The parasitic flatworm Schistosoma mansoni, causative agent of human intestinal schistosomiasis in South America, relies importantly on the freshwater snail Biomphalaria glabrata as intermediate host to achieve development of cercariae that infect humans. The recommendation from the World Health Organization (WHO) to integrate snail control in efforts to counter schistosomiasis transmission provides impetus for in depth study of B. glabrata biology. Our analysis indicates that two distinct hemocyanin-like genes (hcl-1 and hcl-2) are present in B. glabrata, a snail that uses hemoglobin for oxygen transport. Characterization of BAC clones yielded the full length hcl-1 gene, which is comprised of three functional unit (FU) domains at the amino acid level. Database searches and in silico analyses identified the second hcl gene (hcl-2), composed of six FU domains. Both genes are unusual for lacking canonical residues and having fewer FU domains than typical molluscan hemocyanins that contain 7-8 FUs. Reverse transcription PCR demonstrated that Hcl-1 is expressed in a manner that correlates with reproductive maturity in the albumen gland (AG), an immune- and reproduction-relevant organ. Immune cross-reactivity with anti-keyhole limpet hemocyanin (α-KLH) antiserum and tandem-mass spectrometry validated the presence of Hcl-1 protein in the AG and egg mass fluid (EMF). The evolutionary conservation of hemocyanin-like sequences in B. glabrata in the presence of the oxygen carrier hemoglobin, combined with our results, suggest that the Hcl-1protein has a functional role in general and/or reproductive biology. Further investigations are needed to explore Hcl-1 as a potential target for snail control.