Resistant Snails Research Articles

Speciation in the desert: A new endemic Iberus land snail restricted to the southeastern Iberian Peninsula

Arid habitats constitute harsh environments for hydrophilic animals such as land snails. Still, several land snail species appear to inhabit arid environments and such species present an array of adaptations needed to minimise water loss in dry habitats. However, different species may have found different solutions for the same problem. Here, we describe Iberus xerophilus sp. nov., a new species of arid-dwelling land snail from southeastern Spain, on the basis of phylogenetic analyses. Moreover, we show a detailed morphological description as well as a characterization of its habitat. A comparison with the shell morphology of other aridity resistant Iberus land snails casts the question of how this snail can succeed in such harsh arid environments. Southeastern Spain constitutes a hotspot for Iberus arid-dwelling species, displaying a plethora of shell morphologies. The description of this new species might contribute to our understanding of the evolution and diversification of land snails specifically adapted to drought and arid conditions.

Combining a transcriptomic approach and a targeted metabolomics approach for deciphering the molecular bases of compatibility phenotype in the snail Biomphalaria glabrata toward Schistosoma mansoni

Biomphalaria glabrata is a freshwater snail and the obligatory intermediate host of Schistosoma mansoni parasite, the etiologic agent of intestinal Schistosomiasis, in South America and Caribbean. Interestingly in such host-parasite interactions, compatibility varies between populations, strains or individuals. This observed compatibility polymorphism is based on a complex molecular-matching-phenotype, the molecular bases of which have been investigated in numerous studies, notably by comparing between different strains or geographical isolates or clonal selected snail lines. Herein we propose to decipher the constitutive molecular support of this interaction in selected non-clonal resistant and susceptible snail strain originating from the same natural population from Brazil and thus having the same genetic background. Thanks to a global RNAseq transcriptomic approach on whole snail, we identified a total of 328 differentially expressed genes between resistant and susceptible phenotypes among which 129 were up-regulated and 199 down-regulated. Metabolomic studies were used to corroborate the RNAseq results. The activation of immune genes and specific metabolic pathways in resistant snails might provide them with the capacity to better respond to parasite infection.

Inhibition of carbonic anhydrase using aspirin is a novel method to block schistosomiasis infection of the parasitic trematode, Schistosoma mansoni, in the intermediate snail host, Biomphalaria glabrata

Schistosomiasis is a major public health concern worldwide. Although praziquantel is currently available as the only treatment option for schistosomiasis, the absence of reliable diagnostic and prognostic tools highlights the need for the identification and characterization of new drug targets. Recently, we identified the B. glabrata homolog (accession number XP_013075832.1) of human CAXIV, showing 37% amino acid sequence identity, from a BLAST search in NCBI (National Center for Biotechnology Information). Carbonic Anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3. These enzymes are associated with many physiological processes, and their role in tumorigenesis has been widely implicated. CAs create an acidic extracellular environment that facilitates the survival, metastasis, and growth of cancer cells. In this study, we investigated the role of CA inhibition in B. glabrata snails exposed to S. mansoni miracidia. We analyzed the expression of the B. glabrata CA encoding transcript in juvenile susceptible and resistant snails, with and without exposure to S. mansoni. Our results showed that the expression of the CA mRNA encoding transcript was upregulated during early and prolonged infection in susceptible snails (BBO2), but not in the resistant BS-90 stock. Notably, sodium salicylate, a form of aspirin, inhibited the expression of CA, post-exposure, to the parasite. Increasing research between parasites and cancer has shown that schistosomes and cancer cells share similarities in their capacity to proliferate, survive, and evade host immune mechanisms. Here, we show that this model system is a potential new avenue for understanding the role of CA in the metastasis and proliferation of cancer cells. Further studies are needed to explore the potential of CA as a biomarker for infection in other schistosomiasis-causing parasites, including S. japonicum and S. haematobium.

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.

PIWI silencing mechanism involving the retrotransposon nimbus orchestrates resistance to infection with Schistosoma mansoni in the snail vector, Biomphalaria glabrata.

Schistosomiasis remains widespread in many regions despite efforts at its elimination. By examining changes in the transcriptome at the host-pathogen interface in the snail Biomphalaria glabrata and the blood fluke Schistosoma mansoni, we previously demonstrated that an early stress response in juvenile snails, manifested by induction of heat shock protein 70 (Hsp 70) and Hsp 90 and of the reverse transcriptase (RT) domain of the B. glabrata non-LTR- retrotransposon, nimbus, were critical for B. glabrata susceptibility to S. mansoni. Subsequently, juvenile B. glabrata BS-90 snails, resistant to S. mansoni at 25°C become susceptible by the F2 generation when maintained at 32°C, indicating an epigenetic response. To better understand this plasticity in susceptibility of the BS-90 snail, mRNA sequences were examined from S. mansoni exposed juvenile BS-90 snails cultured either at 25°C (non-permissive temperature) or 32°C (permissive). Comparative analysis of transcriptomes from snails cultured at the non-permissive and permissive temperatures revealed that whereas stress related transcripts dominated the transcriptome of susceptible BS-90 juvenile snails at 32°C, transcripts encoding proteins with a role in epigenetics, such as PIWI (BgPiwi), chromobox protein homolog 1 (BgCBx1), histone acetyltransferase (BgHAT), histone deacetylase (BgHDAC) and metallotransferase (BgMT) were highly expressed in those cultured at 25°C. To identify robust candidate transcripts that will underscore the anti-schistosome phenotype in B. glabrata, further validation of the differential expression of the above transcripts was performed by using the resistant BS-90 (25°C) and the BBO2 susceptible snail stock whose genome has now been sequenced and represents an invaluable resource for molecular studies in B. glabrata. A role for BgPiwi in B. glabrata susceptibility to S. mansoni, was further examined by using siRNA corresponding to the BgPiwi encoding transcript to suppress expression of BgPiwi, rendering the resistant BS-90 juvenile snail susceptible to infection at 25°C. Given transposon silencing activity of PIWI as a facet of its role as guardian of the integrity of the genome, we examined the expression of the nimbus RT encoding transcript at 120 min after infection of resistant BS90 piwi-siRNA treated snails. We observed that nimbus RT was upregulated, indicating that modulation of the transcription of the nimbus RT was associated with susceptibility to S. mansoni in BgPiwi-siRNA treated BS-90 snails. Furthermore, treatment of susceptible BBO2 snails with the RT inhibitor lamivudine, before exposure to S. mansoni, blocked S. mansoni infection concurrent with downregulation of the nimbus RT transcript and upregulation of the BgPiwi encoding transcript in the lamivudine-treated, schistosome-exposed susceptible snails. These findings support a role for the interplay of BgPiwi and nimbus in the epigenetic modulation of plasticity of resistance/susceptibility in the snail-schistosome relationship.

Hemocyte siRNA uptake is increased by 5' cholesterol-TEG addition in Biomphalaria glabrata, snail vector of schistosome.

Biomphalaria glabrata is one of the snail intermediate hosts of Schistosoma mansoni, the causative agent of intestinal schistosomiasis disease. Numerous molecular studies using comparative approaches between susceptible and resistant snails to S. mansoni infection have helped identify numerous snail key candidates supporting such susceptible/resistant status. The functional approach using RNA interference (RNAi) remains crucial to validate the function of such candidates. CRISPR-Cas systems are still under development in many laboratories, and RNA interference remains the best tool to study B. glabrata snail genetics. Herein, we describe the use of modified small interfering RNA (siRNA) molecules to enhance cell delivery, especially into hemocytes, the snail immune cells. Modification of siRNA with 5′ Cholesteryl TriEthylene Glycol (Chol-TEG) promotes cellular uptake by hemocytes, nearly eightfold over that of unmodified siRNA. FACS analysis reveals that more than 50% of hemocytes have internalized Chol-TEG siRNA conjugated to Cy3 fluorophores, 2 hours only after in vivo injection into snails. Chol-TEG siRNA targeting BgTEP1 (ThioEster-containing Protein), a parasite binding protein, reduced BgTEP1 transcript expression by 70–80% compared to control. The level of BgTEP1 protein secreted in the hemolymph was also decreased. However, despite the BgTEP1 knock-down at both RNA and protein levels, snail compatibility with its sympatric parasite is not affected suggesting functional redundancy among the BgTEP genes family in snail-schistosoma interaction.

Indonesia Schistosoma japonicum: Origin, Genus Oncomelania and Elimination of the Parasite With Cluster Genes Innoculated Into Female Oncomelania hupensis lindoensis Via CRISPR/Cas9 System

Background: Schistosoma japonicum in Central Sulawesi derived from Japan and China. Oncomelania hupensis lindoensis (Oncomelania lorelindoensis) is the intermediate host of S. japonicum in Central Sulawesi, Indonesia. Southeast Asia’s Oncomelania consists of O. lorelindoensis and O. quadrasi. Schistosoma japonicum has become endemic in certain areas of Central Sulawesi. To eliminate this parasite in this region, genetic manipulation on its intermediate host can be very beneficial. It can help eliminate it without killing its intermediate host. In this study, I report the progress of schistosomiasis that focused on dispersion of S. japonicum, genus Oncomelania, and use of genetic manipulations on O. lorelindoensis for eliminating S. japonicum schistosomiasis in Central Sulawesi as the objectives of this study. Methods: I accessed GenBank in NCBI for determining origin of S. japonicum and the Oncomelania. GenBank data for S. japonicum were from NCBI Nucleotide such as KU196306.1, KU196398.1, and KU196348.1 and GenBank data for Oncomelania were from NCBI Nucleotide such as KR002673.1, KR002674.1, and KR002675.1. Results: Results of Nucleotide BLAST showed that S. japonicum in Central Sulawesi originated from Japan and China. These nucleotide sequences showed that S. japonicum in Sulawesi could be from West China or China and Japan. Nucleotide BLAST results showed that Oncomelania of Southeast Asia was close to O. hupensis robertsoni (Oncomelania robertsoni) than O. hupensis and O. minima. Elimination of the parasite in O. lorelindoensis could occur in the field using anti-Schistosoma, inoculated into female ovary O. lorelindoensis with CRISPR/Cas9 system. Each transgenic snail could result in 100% of snails resistant to schistosomiasis in F3 generation. This approach could not damage O. lorelindoensis. Conclusions: Nucleotide BLAST results showed that ancestors of S. japonicum in Central Sulawesi originated from Japan and China. Oncomelania lorelindoensis is the intermediate host of S. japonicum in Sulawesi, Indonesia. Each transgenic female O. lorelindoensis resulted in resistant snails against S. japonicum infection in F3 generation.

Morphology and kinetics of susceptible and resistant Biomphalaria alexandrina hemocytes during the first week of exposure to Schistosoma mansoni miracidia

Background: Few studies concerning Biomphalaria alexandrina (B. alexandrina) snail hemocytes’subpopulations, and their relation to the compatibility with Schistosoma mansoni (S. mansoni)’ miracidiawere performed. Manipulation of parasite development inside these snails could be applied as a controlmeasure against schistosomiasis.Objectives: Knowing that the snail hemocytes temporarily bind to the parasites, allowing the developmentof cercariae that are infective to the definitive host. This study aimed at studying the morphology andkinetics of diverse hemocytes of susceptible and resistant B. alexandrina and their participation in thesnail early immune response after challenge by S. mansoni.Material and Methods: Giemsa stained hemocytes were characterized using light microscopy. Total anddifferential hemocyte counts (THC and DHC) were calculated in the hemolymph of two groups composedof 60 susceptible and 60 resistant snails. Each group was further subdivided as 12 control pre-exposuresnails (PE) and 48 post-exposure snails (PO) to S. mansoni at different time points (6 h, 1, 3 and 7 days).THC and DHC counts were recorded by a snail hemogram.Results: Results revealed that granulocytes constituted the most common population all through theexperiment with the large dense-granulated granulocytes subpopulation being the largest-sized cellsdetected. The highly reactive subpopulations that increased in number upon exposure to S. mansoni werethe few-granulated and the large-granulated granulocytes, suggesting their possible participation in earlyparasite destruction.Conclusion: The resulting hemograms helped determine the participation of hemocyte populations andsubpopulations in the defense against S. mansoni, aiding in understanding snail compatibility patterns.Further studies to propagate transgenic B. alexandrina snails abundant in large granular granulocytesutilizing (gene editing) CRISPR-Cas9 technique are recommended. This would be required to spreadschistosome resistance traits in snail populations, thus, contributing to reduced schistosomiasistransmission in the long run.

Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba

Pseudosuccinea columella snails transmit the trematode Fasciola hepatica, but in Cuba, six naturally occurring populations successfully resist parasite infection. Here, we present an updated distribution of P. columella in Cuba; 68 positive sites with the earliest records more abundant in west-central Cuba and with east-central populations generally corresponding to the newest samples. No records were found farther east. The IPA site reported 10.5% prevalence of F. hepatica-infected snails. Population genetics, studied through microsatellites, showed low allelic and multilocus genotypic richness (MLGT), mainly in susceptible populations, strong deviations from panmixia and high self-fertilization rates. Susceptible individuals were grouped in one major cluster containing the majority of MLGT, and two independent clusters grouped the MLGT of resistant individuals from western and central populations, respectively. From these, we propose that several introductions of P. columella occurred in Cuba, primarily in the west, with the early arrivals deriving on the resistant populations. A more recent introduction of susceptible P. columella carrying MLGT T and Y may have occurred, where the latter spread quickly through the island and possibly increase the risk of parasite transmission in Cuba since all snails naturally infected with F. hepatica were carriers of the MLGT Y. Interestingly, even though resistant populations are highly diverse and are likely the oldest within Cuba, they are only found in six localities characterized by soft (total hardness, TH = 6.3 ± 1.03°d) and slightly acidic (pH = 6.2 ± 0.12) waters with low richness in snail species (3.2 ± 1.02). This tendency was also observed in a two-year follow-up ecological study that was conducted on a farm where both phenotypes occurred in sympatry; colonization events by resistant over susceptible snails coincided with a reduction in the pH and TH of the water. A comparison of life traits in susceptible and resistant isolates reared at two different pH/TH conditions (5.9/4°d or 7.8/14°d) showed that low pH/TH negatively affects P. columella, irrespective of the phenotype. However, evidence of higher tolerance (higher survival, life expectancy, egg viability) to such conditions was observed in resistant isolates. Finally, we speculate that the limited distribution of resistant populations might be related to a better exploitation of sites that are less suitable to snails (thus, with lower competition), rather than to a differential ecological restriction to specific environmental conditions from susceptible P. columella.

The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: Hemocytes in the spotlight

The Fasciola hepatica/Pseudosuccinea columella interaction in Cuba involves a unique pattern of phenotypes; while most snails are susceptible, some field populations are naturally resistant to infection and parasites are encapsulated by snail hemocytes. Thus, we investigated the hemocytes of resistant (R) and susceptible (S) P. columella, in particular morphology, abundance, proliferation and in vitro encapsulation activity following exposure to F. hepatica. Compared to susceptible P. columella, hemocytes from exposed resistant snails showed increased levels of spreading and aggregation (large adherent cells), proliferation of circulating blast-like cells and encapsulation activity of the hemocytes, along with a higher expression of the cytokine granulin. By contrast, there was evidence of a putative F. hepatica-driven inhibition of host immunity, only in susceptible snails. Additionally, (pre-)incubation of naïve hemocytes from P. columella (R and S) with different monosaccharides was associated with lower encapsulation activity of F. hepatica larvae. This suggests the involvement in this host-parasite interaction of lectins and lectins receptors (particularly related to mannose and fucose sensing) in association with hemocyte activation and/or binding to F. hepatica.

Comparison of Nmri Schistosoma mansoni Infection in Three Susceptible Laboratory Albino Strains of Biomphalaria glabrata

Albino strains of Biomphalaria glabrata that are compatible with Schistosoma mansoni are commonly used to investigate snail-schistosome interactions, but whether they are all equally compatible is not known. In this study, compatibility with the Naval Medical Research Institute (NMRI) strain of S. mansoni was compared among 3 widely used albino strains: NMRI (the normal laboratory host for NMRI S. mansoni), M line, and University of Massachusetts Lowell (UML). Compatibility was assessed on the basis of infection prevalence following exposure to miracidia, the histological fate of sporocysts, and mitotic response in the snail amebocyte-producing organ (APO), a component of the internal defense system (IDS). Infection prevalence in UML was nearly identical to that in NMRI but was significantly lower in M line. Although the APO of UML showed no response to infection over the course of 9 days, mitotic activity was elevated in the APO of NMRI and M line, with that in M line being higher and more prolonged than in the APO of resistant BS-90 snails. Finally, hemocyte responses against some small primary sporocysts occurred at 1 and 3 days post-exposure (DPE) in all 3 strains, but in 2 of 5 M line a response also occurred against large primary sporocysts at 6 DPE. Thus, based on infection prevalence and tissue responses, compatibility with NMRI S. mansoni is lowest in M line, whereas UML and NMRI show the same degree of compatibility, despite decades of maintenance of this parasite strain in NMRI snails. The elevated mitotic response in the APO of M line and NMRI snails suggests that a response of the IDS can occur even in a compatible host-parasite relationship.

Natural resistance to Fasciola hepatica (Trematoda) in Pseudosuccinea columella snails: A review from literature and insights from comparative “omic” analyses

The snail Pseudosuccinea columella is one of the main vectors of the medically-important trematode Fasciola hepatica. In Cuba, the existence of natural P. columella populations that are either susceptible or resistant to F. hepatica infection offers a unique snail-parasite for study of parasite-host compatibility and immune function in gastropods. Here, we review all previous literature on this system and present new “omic” data that provide a molecular baseline of both P. columella phenotypes from naïve snails. Comparison of whole snail transcriptomes (RNAseq) and the proteomes of the albumen gland (2D-electrophoresis, MS) revealed that resistant and susceptible strains differed mainly in an enrichment of particular biological processes/functions and a greater abundance of proteins/transcripts associated with immune defense/stress response in resistant snails. These results indicate a differential allocation of molecular resources to self-maintenance and survival in resistant P. columella that may cause enhanced responsiveness to stressors (i.e. F. hepatica infection or tolerance to variations in environmental pH/total water hardness), possibly as trade-off against reproduction and the ecological cost of resistance previously suggested in resistant populations of P. columella.

Proteomic Analysis of Biomphalaria glabrata Hemocytes During in vitro Encapsulation of Schistosoma mansoni Sporocysts

Circulating hemocytes of the snail Biomphalaria glabrata, a major intermediate host for the blood fluke Schistosoma mansoni, represent the primary immune effector cells comprising the host's internal defense system. Within hours of miracidial entry into resistant B. glabrata strains, hemocytes infiltrate around developing sporocysts forming multi-layered cellular capsules that results in larval death, typically within 24–48 h post-infection. Using an in vitro model of hemocyte-sporocyst encapsulation that recapitulates in vivo events, we conducted a comparative proteomic analysis on the responses of hemocytes from inbred B. glabrata strains during the encapsulation of S. mansoni primary sporocysts. This was accomplished by a combination of Laser-capture microdissection (LCM) to isolate sections of hemocyte capsules both in the presence and absence of sporocysts, in conjunction with mass spectrometric analyses to establish protein expression profiles. Comparison of susceptible NMRI snail hemocytes in the presence and absence of sporocysts revealed a dramatic downregulation of proteins in during larval encapsulation, especially those involved in protein/CHO metabolism, immune-related, redox and signaling pathways. One of 4 upregulated proteins was arginase, competitor of nitric oxide synthetase and inhibitor of larval-killing NO production. By contrast, when compared to control capsules, sporocyst-encapsulating hemocytes of resistant BS-90 B. glabrata exhibited a more balanced profile with enhanced expression of shared proteins involved in protein synthesis/processing, immunity, and redox, and unique expression of anti-microbial/anti-parasite proteins. A final comparison of NMRI and BS-90 host hemocyte responses to co-cultured sporocysts demonstrated a decrease or downregulation of 77% of shared proteins by NMRI cells during encapsulation compared to those of the BS-90 strain, including lipopolysaccharide-binding protein, thioredoxin reductase 1 and hemoglobins 1 and 2. Overall, using this in vitro model, results of our proteomic analyses demonstrate striking differences in proteins expressed by susceptible NMRI and resistant BS-90 snail hemocytes to S. mansoni sporocysts during active encapsulation, with NMRI hemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed immune-relevant proteins (e.g., FREP2) compared to BS-90. Our data suggest that snail strain differences in hemocyte protein expression during the encapsulation process account for observed differences in their cytotoxic capacity to interact with and kill sporocysts.

CHARACTERIZATION OF THE HEMOCYTES OF SUSCEPTIBLE AND RESISTANT BIOMPHALARIA ALEXANDRINA SNAIL

The internal defense system consists of soluble components of hemolymph and circulating cells known as hemocytes. The circulating hemocytes play a central role in innate immunity. This work aimed to study the hemocytes of both susceptible and resistant B. alexandrina snails exposed to S. mansoni infection using light and electron microscopes. Two tested groups were included in the study; 60 susceptible and 60 resistant B. alexandrina snails. Both tested groups were studied as regad the hemocyte count (before and after infection) and the morphological characteristics of both circulating and tissue hemocytes by light and electron microscopes. Before infection, .there was no significant difference between the two groups as regard the hemocyte count, however after infection, there is a significant decrease in the circulating hemocytes of the resistant group. Light microscopy revealed five morphological types of circulating cells of both susceptible and resistant snails. Regarding scannig electron microscopy, hemocytes of susceptible snails appeared rounded with smooth or slightly rough surface. However, that of the resistant snails appeared irregular in shaped with corrugated surface. Furthermore, Light microscopy and the transmission electron microscopy revealed signs of cell activation in the hemocytes of the resistant group. The circulating hemocytes consist of five cell types in both susceptible and resistant B. alexandarina and morphologies of these cells are quite similar, but with more signs of cell activations in the resistant group. More specific studies on the functional activities of the hemocytes and mechanisms that may affect or influence the susceptibility and/or non-susceptibility of molluscs to invade microorganisms is essential and how they can act in the immune response.

Characterization of the Hemocytes of Susceptible and Resistant Biomphalaria Alexandrina Snail

The internal defense system consists of soluble components of hemolymph and circulating cells known as hemocytes. The circulating hemocytes play a central role in innate immunity. This work aimed to study the hemocytes of both susceptible and resistant B. alexandrina snails exposed to S. mansoni infection using light and electron microscopes. Two tested groups were included in the study; 60 susceptible and 60 resistant B. alexandrina snails. Both tested groups were studied as regad the hemocyte count (before and after infection) and the morphological characteristics of both circulating and tissue hemocytes by light and electron microscopes. Before infection, .there was no significant difference between the two groups as regard the hemocyte count, however after infection, there is a significant decrease in the circulating hemocytes of the resistant group. Light microscopy revealed five morphological types of circulating cells of both susceptible and resistant snails. Regarding scannig electron microscopy, hemocytes of susceptible snails appeared rounded with smooth or slightly rough surface. However, that of the resistant snails appeared irregular in shaped with corrugated surface. Furthermore, Light microscopy and the transmission electron microscopy revealed signs of cell activation in the hemocytes of the resistant group. The circulating hemocytes consist of five cell types in both susceptible and resistant B. alexandarina and morphologies of these cells are quite similar, but with more signs of cell activations in the resistant group. More specific studies on the functional activities of the hemocytes and mechanisms that may affect or influence the susceptibility and/or non-susceptibility of molluscs to invade microorganisms is essential and how they can act in the immune response.

Total protein composition of young and adult Biomphalaria alexandrina snails with different compatibilities to Schistosoma mansoni infection

Schistosomiasis remains a disease of major global public health concern since it is a chronic and debilitating illness. The widely distributed Schistosoma mansoni that causes intestinal schistosomiasis represents a great threat. Its world-wide distribution is permitted by the broad geographic range of the susceptible species of its intermediate host, Biomphalaria, which serves as an obligatory host for the larval stage, at which humans get infected. The objectives were to identify the proteins responsible for the snails’ compatibility outcome through differentiation between the total proteins among Biomphalaria alexandrina snails at different ages. The work was conducted on snails that differ in age and genetic backgrounds. Four subgroups (F1) from the progeny of self-reproduced susceptible and resistant snails (F0) were studied. Infection rates of these subgroups (young susceptible, adult susceptible, young resistant and adult resistant) were 90 %, 75 %, 40 % and 0 %, respectively. Using Sodium Dodecyl Sulphate Polyacrylamide Gel electrophoresis (SDS-PAGE), differences in protein expression were evaluated between adult and young snails of different subgroups. Dice similarity coefficient was calculated to determine the percentage of band sharing among the experimental subgroups. The results showed that the combination of similarities between age and compatibility status of the snails, lead to the highest similarity coefficient, followed by the combination of similarities of both genetic origin and age, even though they differ in the compatibility status. On the other hand, the differences in the genetic background, age and compatibility status, lead to the least similarity index. It was also found that the genetic background in young snails plays a major role in the determination of their compatibility, while the internal defense system has the upper hand in determining the level of adult compatibility. In conclusion, the findings of the present work highlight the great impact of the snail age in concomitance with the genetics and the internal defense in the determination of B. alexandrina/S.mansoni compatibility. Future works are recommended, as further characterization of the shared protein bands among the studied subgroups is needed to clarify their role in host-parasite relationship.

A Novel Toll-Like Receptor (TLR) Influences Compatibility between the Gastropod Biomphalaria glabrata, and the Digenean Trematode Schistosoma mansoni.

Schistosomiasis, a devastating disease caused by parasitic flatworms of the genus Schistosoma, affects over 260 million people worldwide especially in tropical and sub-tropical regions. Schistosomes must undergo their larval development within specific species of snail intermediate hosts, a trait that is shared among almost all digenean trematodes. This unique and long-standing host-parasite relationship presents an opportunity to study both the importance of conserved immunological features in novel immunological roles, as well as new immunological adaptations that have arisen to combat a very specific type of immunological challenge. While it is well supported that the snail immune response is important for protecting against schistosome infection, very few specific snail immune factors have been identified and even fewer have been functionally characterized. Here, we provide the first functional report of a snail Toll-like receptor, which we demonstrate as playing an important role in the cellular immune response of the snail Biomphalaria glabrata following challenge with Schistosoma mansoni. This TLR (BgTLR) was identified as part of a peptide screen of snail immune cell surface proteins that differed in abundance between B. glabrata snails that differ in their compatibility phenotype to challenge by S. mansoni. The S. mansoni-resistant strain of B. glabrata (BS-90) displayed higher levels of BgTLR compared to the susceptible (M-line) strain. Transcript expression of BgTLR was found to be very responsive in BS-90 snails when challenged with S. mansoni, increasing 27 fold relative to β-actin (non-immune control gene); whereas expression in susceptible M-line snails was not significantly increased. Knockdown of BgTLR in BS-90 snails via targeted siRNA oligonucleotides was confirmed using a specific anti-BgTLR antibody and resulted in a significant alteration of the resistant phenotype, yielding patent infections in 43% of the normally resistant snails, which shed S. mansoni cercariae 1-week before the susceptible controls. Our results represent the first functional characterization of a gastropod TLR, and demonstrate that BgTLR is an important snail immune receptor that is capable of influencing infection outcome following S. mansoni challenge.

Impact of the age of Biomphalaria alexandrina snails on Schistosoma mansoni transmission: modulation of the genetic outcome and the internal defence system of the snail.

Of the approximately 34 identified Biomphalaria species,Biomphalaria alexandrina represents the intermediate host of Schistosoma mansoni in Egypt. Using parasitological and SOD1 enzyme assay, this study aimed to elucidate the impact of the age of B. alexandrina snails on their genetic variability and internal defence against S. mansoni infection. Susceptible and resistant snails were reared individually for self-reproduction; four subgroups of their progeny were used in experiment. The young susceptible subgroup showed the highest infection rate, the shortest pre-patent period, the highest total cercarial production, the highest mortality rate and the lowest SOD1 activity. Among the young and adult susceptible subgroups, 8% and 26% were found to be resistant, indicating the inheritance of resistance alleles from parents. The adult resistant subgroup, however, contained only resistant snails and showed the highest enzyme activity. The complex interaction between snail age, genetic background and internal defence resulted in great variability in compatibility patterns, with the highest significant difference between young susceptible and adult resistant snails. The results demonstrate that resistance alleles function to a greater degree in adults, with higher SOD1 activity and provide potential implications for Biomphalaria control. The identification of the most susceptible snail age enables determination of the best timing for applying molluscicides. Moreover, adult resistant snails could be beneficial in biological snail control.

Susceptibility of Snails to Infection with Schistosomes is influenced by Temperature and Expression of Heat Shock Proteins.

The freshwater snail, Biomphalaria glabrata is the obligate intermediate host for the transmission of the parasitic trematode, Schistosoma mansoni the causative agent of the chronic debilitating neglected tropical disease, schistosomiasis. We showed previously that in juvenile snails, early and significant induction of stress manifested by the expression of stress proteins, Hsp 70, Hsp 90 and reverse transcriptase (RT) of the non- LTR retrotransposon, nimbus, is a characteristic feature of juvenile susceptible NMRI but not resistant BS-90 snails. These latter, however, could be rendered susceptible after mild heat shock at 32°C, revealing that resistance in the BS-90 resistant snail to schistosomes is a temperature dependent trait. Here we tested the hypothesis that maintenance of BS-90 resistant snails at the permissive temperature for several generations affects the resistance phenotype displayed at the non-permissive temperature of 25°C. The progeny of BS-90 snails bred and maintained through several generations (F1 to F4) at 32°C were susceptible to the schistosome infection when returned to room temperature, shedding cercariae at four weeks post-infection. Moreover, the study of expression levels of the heat shock protein (Hsp) 70 protein by ELISA and western blot analysis, showed that this protein is also differentially expressed between susceptible and resistant snails, with susceptible snails expressing more protein than their resistant counterparts after early exposure to wild-type but not to radiation-attenuated miracidia. These data suggested that in the face of global warming, the ability to sustain a reduction in schistosomiasis by using refractory snails as a strategy to block transmission of the disease might prove challenging since non-lethal elevation in temperature, affects snail susceptibility to S. mansoni.

Efeito moluscicida do óleo essencial de Cymbopogon winterianus Jowitt (Poaceae) sobre Lymnaea columella (Say, 1817) e Biomphalaria tenagophila(D’Orbigny, 1835)

RESUMO O uso das substâncias moluscicidas convencionais no controle de planorbídeos vetores constitui-se uma importante ferramenta no combate da fasciolose hepática e esquistossomose. Sendo, portanto, de extrema relevância para a pecuária e para os serviços de Vigilância Epidemiológica. Por outro lado, a seleção de caramujos resistentes a tais substâncias e sua baixa seletividade estimulam a busca por novas substâncias. Neste sentido, o presente trabalho foi desenvolvido para avaliar o efeito do óleo essencial de Cymbopogon winterianusJowitt sobre Lymnaea columella e Biomphalaria tenagophila, hospedeiros intermediários de Fasciola hepatica e esquistossomose mansônica, respectivamente. O óleo essencial foi extraído a partir de folhas frescas utilizando o sistema Clevenger. A análise qualitativa foi realizada por meio de cromatografia gasosa acoplada a espectrometria de massas (CG/EM) e a quantificação dos constituintes presentes no óleo foi determinada por cromatografia gasosa acoplada ao detector de ionização de chama (CG/DIC). O efeito moluscicida foi avaliado utilizando seis moluscos de cada espécie e o óleo essencial de C. winterianus nas concentrações finais de 10, 20, 30, 40, 60, 80 e 100 ppm. A análise por cromatografia gasosa do óleo essencial possibilitou a identificação dos componentes majoritários geraniol (28,62%), citronelal (23,62%) e citronelol (17,10%). Os valores de DL100 e DL50para os moluscos das espécies L. columella e B. tenagophila foram, respectivamente, 60 e 40 ppm; 80 ppm e 60 ppm. O óleo de Cymbopogon winterianus demonstrou-se uma alternativa promissora para o controle dos moluscos, sendo a espécie L. columella mais sensível ao mesmo.