Biomphalaria Glabrata Research Articles

Laboratory and field validation of the recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) for snail xenomonitoring for schistosomiasis

Improvements in diagnostics for schistosomiasis in both humans and snail hosts are priorities to be able to reach the World Health Organization (WHO) goal of eliminating the disease as a public health problem by 2030. In this context, molecular isothermal amplification tests, such as Recombinase Polymerase Amplification (RPA), are promising for use in endemic areas at the point-of-need for their accuracy, robustness, simplicity, and time-effectiveness. The developed recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) was used to detect S. mansoni DNA from both laboratory and field Biomphalaria snails. Laboratory snails were experimentally infected and used at one, seven, and 28 days post-exposure (dpe) to 10 S. mansoni miracidia to provide samples in the early pre-patent infection stage. Field samples of Biomphalaria spp. were collected from the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil, which are endemic for S. mansoni. The sensitivity and specificity of the SmMIT-RPA assay were analysed and compared with existing loop-mediated isothermal amplification (LAMP), PCR-based methods, parasitological examination of the snails, and nucleotide sequencing. The SmMIT-RPA assay was able to detect S. mansoni DNA in the experimentally infected Biomphalaria glabrata as early as one dpe to 10 miracidia. It also detected S. mansoni infections (55.5% prevalence) in the field samples with the highest accuracy (100% sensitivity and specificity) compared with the other molecular tests used as the reference. Results from this study indicate that the SmMIT-RPA assay is a good alternative test to be used for snail xenomonitoring of S. mansoni due to its high sensitivity, accuracy, and the possibility of detecting early pre-patent infection. Its simplicity and portability also make it a suitable methodology in low-resource settings.

Yolk proteins of the schistosomiasis vector snail Biomphalaria glabrata revealed by multi-omics analysis

Vitellogenesis is the most important process in animal reproduction, in which yolk proteins play a vital role. Among multiple yolk protein precursors, vitellogenin (Vtg) is a well-known major yolk protein (MYP) in most oviparous animals. However, the nature of MYP in the freshwater gastropod snail Biomphalaria glabrata remains elusive. In the current study, we applied bioinformatics, tissue-specific transcriptomics, ovotestis-targeted proteomics, and phylogenetics to investigate the large lipid transfer protein (LLTP) superfamily and ferritin-like family in B. glabrata. Four members of LLTP superfamily (BgVtg1, BgVtg2, BgApo1, and BgApo2), one yolk ferritin (Bg yolk ferritin), and four soma ferritins (Bg ferritin 1, 2, 3, and 4) were identified in B. glabrata genome. The proteomic analysis demonstrated that, among the putative yolk proteins, BgVtg1 was the yolk protein appearing in the highest amount in the ovotestis, followed by Bg yolk ferritin. RNAseq profile showed that the leading synthesis sites of BgVtg1 and Bg yolk ferritin are in the ovotestis (presumably follicle cells) and digestive gland, respectively. Phylogenetic analysis indicated that BgVtg1 is well clustered with Vtgs of other vertebrates and invertebrates. We conclude that, vitellogenin (BgVtg1), not yolk ferritin (Bg yolk ferritin), is the major yolk protein precursor in the schistosomiasis vector snail B. glabrata.

Species distribution modeling for disease ecology: A multi-scale case study for schistosomiasis host snails in Brazil.

Species distribution models (SDMs) are increasingly popular tools for profiling disease risk in ecology, particularly for infectious diseases of public health importance that include an obligate non-human host in their transmission cycle. SDMs can create high-resolution maps of host distribution across geographical scales, reflecting baseline risk of disease. However, as SDM computational methods have rapidly expanded, there are many outstanding methodological questions. Here we address key questions about SDM application, using schistosomiasis risk in Brazil as a case study. Schistosomiasis is transmitted to humans through contact with the free-living infectious stage of Schistosoma spp. parasites released from freshwater snails, the parasite's obligate intermediate hosts. In this study, we compared snail SDM performance across machine learning (ML) approaches (MaxEnt, Random Forest, and Boosted Regression Trees), geographic extents (national, regional, and state), types of presence data (expert-collected and publicly-available), and snail species (Biomphalaria glabrata, B. straminea, and B. tenagophila). We used high-resolution (1km) climate, hydrology, land-use/land-cover (LULC), and soil property data to describe the snails' ecological niche and evaluated models on multiple criteria. Although all ML approaches produced comparable spatially cross-validated performance metrics, their suitability maps showed major qualitative differences that required validation based on local expert knowledge. Additionally, our findings revealed varying importance of LULC and bioclimatic variables for different snail species at different spatial scales. Finally, we found that models using publicly-available data predicted snail distribution with comparable AUC values to models using expert-collected data. This work serves as an instructional guide to SDM methods that can be applied to a range of vector-borne and zoonotic diseases. In addition, it advances our understanding of the relevant environment and bioclimatic determinants of schistosomiasis risk in Brazil.

Gastrópodes potenciais hospedeiros intermediários de parasitos na região do Parque Nacional dos Lençóis Maranhenses/Brasil, um destino turístico internacional

Abstract Several aquatic ecosystems present favorable conditions for the establishment of freshwater gastropod populations in Maranhão (Brazil). However, research on these snails in this Brazilian state is more focused on Baixada Maranhense, an endemic area for schistosomiasis in Maranhão. Considering the need to expand information on the occurrence of limnic gastropods in Maranhão, a qualitative study was carried out in the Microregion of Lençóis Maranhenses. The capture of freshwater snails was carried out between June 2016 and November 2019. Were obtained 3,244 specimens of freshwater gastropods: 2,372 specimens of Biomphalaria straminea (Dunker, 1848); 17 specimens of Biomphalaria schrammi (Crosse, 1864); 19 specimens of Drepanotrema lucidum (Pfeiffer, 1939); 115 specimens of (Hydrobiidae; Rissooidea); 24 specimens of Physa marmorata Guilding, 1828; 73 specimens of Pomacea maculata (Perry, 1810); 613 specimens of Pomacea sp. (Perry, 1810). Were also found 11 shells of Biomphalaria glabrata (Say, 1818). The greatest richness of snails was observed in Tutóia. The most abundant species during the study was B. straminea (73,45%). Pomacea sp. was the most representative species with constant frequency (57,15%) in the Lençóis Maranhenses Microregion, present in lentic and lotic ecosystems. The forecast maps indicate the possibility of occurrence and establishment of freshwater gastropods species throughout the investigated region. The presence of gastropods that are hosts of parasites represents a risk to human health, especially in the Lençóis Maranhenses area, which is one of Brazil's tourist destinations. Studies on the occurrence and distribution of limnic gastropods are of great relevance for understanding the conservation status of existing species. Furthermore, they are necessary to monitor snails that act as parasite hosts.

Hemocytes from neonates of Biomphalaria glabrata are functionally less mature than those from adult snails

AbstractGastropod molluscs, which serve as obligatory intermediate hosts for digenetic trematodes, possess an internal defense system (IDS) consisting of phagocytic hemocytes and plasma factors. This IDS is responsible for resistance to infection with larval trematodes, which are encapsulated and killed by hemocytes in incompatible snails. Like other physiological systems, the IDS probably undergoes maturation during early stages of life, and the relatively undeveloped state of the IDS in young snails has been hypothesized to be a factor in their increased susceptibility to infection with larval trematodes. In this study, hemocytes were examined in the BS‐90 laboratory strain of Biomphalaria glabrata that is resistant to infection with Schistosoma mansoni as adults but susceptible to infection as neonates. Compared with hemocytes from adults, hemocytes from neonates had a smaller perimeter and lower intrinsic directional motility on glass microscope slides. Additionally, in vitro assays showed a lower association with fucoidan‐linked polystyrene beads and less ability to produce superoxide anion in hemocytes from neonates compared to hemocytes from adults. These results support the hypothesis that the gastropod IDS undergoes maturation during growth. However, whether the observed differences between hemocytes of neonatal and adult BS‐90 snails play a role in the susceptibility of the former and resistance of the latter to infection with S. mansoni is not known.

Covariation between microeukaryotes and bacteria associated with Planorbidae snails.

Microbial communities associated with macroorganisms might affect host physiology and homeostasis. Bacteria are well studied in this context, but the diversity of microeukaryotes, as well as covariations with bacterial communities, remains almost unknown. To study microeukaryotic communities associated with Planorbidae snails, we developed a blocking primer to reduce amplification of host DNA during metabarcoding analyses. Analyses of alpha and beta diversities were computed to describe microeukaryotes and bacteria using metabarcoding of 18S and 16S rRNA genes, respectively. Only three phyla (Amoebozoa, Opisthokonta and Alveolata) were dominant for microeukaryotes. Bacteria were more diverse with five dominant phyla (Proteobacteria, Bacteroidetes, Tenericutes, Planctomycetes and Actinobacteria). The composition of microeukaryotes and bacteria were correlated for the Biomphalaria glabrata species, but not for Planorbarius metidjensis. Network analysis highlighted clusters of covarying taxa. Among them, several links might reflect top-down control of bacterial populations by microeukaryotes, but also possible competition between microeukaryotes having opposite distributions (Lobosa and Ichthyosporea). The role of these taxa remains unknown, but we believe that the blocking primer developed herein offers new possibilities to study the hidden diversity of microeukaryotes within snail microbiota, and to shed light on their underestimated interactions with bacteria and hosts.

Fluorescent non transgenic schistosoma to decipher host-parasite phenotype compatibility.

Schistosomiasis is considered as a significant public health problem, imposing a deeper understanding of the intricate interplay between parasites and their hosts. Unfortunately, current invasive methodologies employed to study the compatibility and the parasite development impose limitations on exploring diverse strains under various environmental conditions, thereby impeding progress in the field. In this study, we demonstrate the usefulness for the trematode parasite Schistosma mansoni, leveranging a fluorescence-imaging-based approach that employs fluorescein 5-chloromethylfluorescein diacetate (CMFDA) and 5-chloromethylfluorescein diacetate (CMAC) as organism tracker for intramolluscan studies involving the host snail Biomphalaria glabrata. These probes represent key tools for qualitatively assessing snail infections with unmatched accuracy and precision. By monitoring the fluorescence of parasites within the snail vector, our method exposes an unprecedented glimpse into the host-parasite compatibility landscape. The simplicity and sensitivity of our approach render it an ideal choice for evolutionary studies, as it sheds light on the intricate mechanisms governing host-parasite interactions. Fluorescent probe-based methods play a pivotal role in characterizing factors influencing parasite development and phenotype of compatibility, paving the way for innovative, effective, and sustainable solutions to enhance our understanding host-parasite immunobiological interaction and compatibility.

Viability Analysis of Biomphalaria glabrata Hemocytes During Schistosoma mansoni Infection And Glyphosate-Based Herbicide Exposure

Pesticides are chemical agents that have a range of harmful effects on human health and the environment. They can cause drastic changes in natural communities, such as macroinvertebrates, plankton and fish. Snails, including Biomphalaria glabrata, are often present in aquatic communities and have multiple roles in limnic ecosystems. B. glabrata is an intermediate host of several species of helminths of medical and veterinary importance, such as Schistosoma mansoni, the etiological agent of schistosomiasis. The original Roundup® herbicide can affect snails and directly affects S. mansoni hemocytes, cells that act in the snails’ immune defense. Here we analyzed the effect of herbicide exposure on B. glabrata hemocytes divided into four groups: control group, infected-only group, treated-only group and infected+treated group. For this, flow cytometry and Neubauer-chamber counting were used to determine the morphology, viability and lectin expression profiles of hemocytes. We observed that the group infected by S. mansoni and treated with herbicide had a higher concentration of dead hemocytes in relation to the other groups. The treated group showed similar results to the control group, suggesting that the herbicide (Roundup™) alone does not interfere with the snails’ immune system. Regarding cellular-morphological-characterization analysis, hyalinocytes were the cells most commonly found in all groups studied. These findings suggest that S. mansoni infection and exposure to pesticides directly the immune system of the snails, stimulating the production of hemocytes, especially hyalinocytes, which have a high phagocytic power to quell the infection, but with toxic effects on the snail.

Hemocyte-like cells in larvae of the freshwater snail Biomphalaria glabrata (Mollusca: Panpulmonata)

Despite undergoing development within a germfree egg capsule, embryos and larvae of the freshwater snail Biomphalaria glabrata possess passive immune protection in the form of parentally-derived antimicrobial proteins in the perivitelline fluid. However, the point at which larvae begin to form their own internal defense system (IDS), which consists of both plasma proteins and hemocytes, is not known. In this study, hemocyte-like cells were observed in mechanically-disrupted late trochophores and veligers of the BS-90 strain of B. glabrata. These cells showed the properties of glass adherence, spreading, motility, and binding and phagocytosing polystyrene microspheres. No hemocyte-like cells were recovered from the early trochophore stage, and therefore their formation first occurs during subsequent maturation. Numbers of hemocyte-like cells increased during larval development. Although the functional significance of these cells is not known, they may represent the initial cellular component of the IDS.

Temperature affects predation of schistosome-competent snails by a novel invader, the marbled crayfish Procambarus virginalis.

The human burden of environmentally transmitted infectious diseases can depend strongly on ecological factors, including the presence or absence of natural enemies. The marbled crayfish (Procambarus virginalis) is a novel invasive species that can tolerate a wide range of ecological conditions and colonize diverse habitats. Marbled crayfish first appeared in Madagascar in 2005 and quickly spread across the country, overlapping with the distribution of freshwater snails that serve as the intermediate host of schistosomiasis-a parasitic disease of poverty with human prevalence ranging up to 94% in Madagascar. It has been hypothesized that the marbled crayfish may serve as a predator of schistosome-competent snails in areas where native predators cannot and yet no systematic study to date has been conducted to estimate its predation rate on snails. Here, we experimentally assessed marbled crayfish consumption of uninfected and infected schistosome-competent snails (Biomphalaria glabrata and Bulinus truncatus) across a range of temperatures, reflective of the habitat range of the marbled crayfish in Madagascar. We found that the relationship between crayfish consumption and temperature is unimodal with a peak at ~27.5°C. Per-capita consumption increased with body size and was not affected either by snail species or their infectious status. We detected a possible satiation effect, i.e., a small but significant reduction in per-capita consumption rate over the 72-hour duration of the predation experiment. Our results suggest that ecological parameters, such as temperature and crayfish weight, influence rates of consumption and, in turn, the potential impact of the marbled crayfish invasion on snail host populations.

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.

Chemical composition, larvicidal and molluscicidal activity of the essential oil Alpinia zerumbet

In this present study, we describe the chemical composition, larvicidal and molluscicidal activity of essential oil distilled from Alpinia zerumbet leaves against third-stage larvae of Ae.aegypti and the snail Biomphalaria glabrata, respectively. For this, we distilled the oil by hydrodistillation, identified its components by gas chromatography coupled to mass spectrometry and gas chromatography with flame ionization detection and calculated the lethal concentration of larvicidal and molluscicidal activity using the probit method. From these analyses, we showed that the oil is predominantly composed of monoterpenes, in which p-cymene (34.12%), 1,8-cineole (25.67%) and terpinen-4-ol (21.01%) stood out. In addition, we also proved that the oil has larvicidal and molluscicidal activity with lethal concentrations respectively of 43.74 and 36.52 μg/mL. Therefore, we conclude that the oil is active and has the potential to replace synthetic larvicides and molluscicides.

Host-bacteriome transplants of the schistosome snail host Biomphalaria glabrata reflect species-specific associations.

Microbial symbionts can affect host phenotypes and, thereby, ecosystem functioning. The microbiome is increasingly being recognized as an important player in the tripartite interaction between parasitic flatworms, snail intermediate hosts, and the snail microbiome. In order to better understand these interactions, transplant experiments are needed, which rely on the development of a reliable and reproducible protocol to obtain microbiome-disturbed snails. Here, we report on the first successful snail bacteriome transplants, which indicate that Biomphalaria glabrata can accrue novel bacterial assemblies depending on the available environmental bacteria obtained from donor snails. Moreover, the phylogenetic relatedness of the donor host significantly affected recipients' survival probability, corroborating the phylosymbiosis pattern in freshwater snails. The transplant technique described here, complemented by field-based studies, could facilitate future research endeavors to investigate the role of specific bacteria or bacterial communities in parasitic flatworm resistance of B. glabrata and might ultimately pave the way for microbiome-mediated control of snail-borne diseases.

Molluscicidal and Cercaricidal Effects of Myrciaria floribunda Essential Oil Nanoemulsion.

Schistosomiasis is a tropical disease transmitted in an aqueous environment by cercariae from the Schistosoma genus. This disease affects 200 million people living in risk areas around the world. The control of schistosomiasis is realized by chemotherapy, wastewater sanitation, health education, and mollusk control using molluscicidal agents. This work evaluates the effects of a nanoemulsion containing essential oil from Myrciaria floribunda leaves as a molluscicidal and cercaricidal agent against Biomphalaria glabrata mollusks and Schistosoma mansoni cercariae. The Myrciaria floribunda essential oil from leaves showed nerolidol, β-selinene, 1,8 cineol, and zonarene as major constituents. The formulation study suggested the F3 formulation as the most promising nanoemulsion with polysorbate 20 and sorbitan monooleate 80 (4:1) with 5% (w/w) essential oil as it showed a smaller droplet size of approximately 100 nm with a PDI lower than 0.3 and prominent bluish reflection. Furthermore, this nanoemulsion showed stability after 200 days under refrigeration. The Myrciaria floribunda nanoemulsion showed LC50 values of 48.11 µg/mL, 29.66 µg/mL, and 47.02 µg/mL in Biomphalaria glabrata embryos, juveniles, and adult mollusks, respectively, after 48 h and 83.88 µg/mL for Schistosoma mansoni cercariae after 2 h. In addition, a survival of 80% was observed in Danio rerio, and the in silico toxicity assay showed lower overall human toxicity potential to the major compounds in the essential oil compared to the reference molluscicide niclosamide. These results suggest that the nanoemulsion of Myrciaria floribunda leaves may be a promising alternative for schistosomiasis control.

The FMRF-NH2 gated sodium channel of Biomphalaria glabrata: Localization and expression following infection by Schistosoma mansoni.

The neglected tropical disease schistosomiasis impacts over 700 million people globally. Schistosoma mansoni, the trematode parasite that causes the most common type of schistosomiasis, requires planorbid pond snails of the genus Biomphalaria to support its larval development and transformation to the cercarial form that can infect humans. A greater understanding of neural signaling systems that are specific to the Biomphalaria intermediate host could lead to novel strategies for parasite or snail control. This study examined a Biomphalaria glabrata neural channel that is gated by the neuropeptide FMRF-NH2. The Biomphalaria glabrata FMRF-NH2 gated sodium channel (Bgl-FaNaC) amino acid sequence was highly conserved with FaNaCs found in related gastropods, especially the planorbid Planorbella trivolvis (91% sequence identity). In common with the P. trivolvis FaNaC, the B. glabrata channel exhibited a low affinity (EC50: 3 x 10-4 M) and high specificity for the FMRF-NH2 agonist. Its expression in the central nervous system, detected with immunohistochemistry and in situ hybridization, was widespread, with the protein localized mainly to neuronal fibers and the mRNA confined to cell bodies. Colocalization of the Bgl-FaNaC message with its FMRF-NH2 agonist precursor occurred in some neurons associated with male mating behavior. At the mRNA level, Bgl-FaNaC expression was decreased at 20 and 35 days post infection (dpi) by S. mansoni. Increased expression of the transcript encoding the FMRF-NH2 agonist at 35 dpi was proposed to reflect a compensatory response to decreased receptor levels. Altered FMRF-NH2 signaling could be vital for parasite proliferation in its intermediate host and may therefore present innovative opportunities for snail control.

Effects of Commercial Sunscreens on Survival, Reproduction and Embryonic Development of the Aquatic Snail Biomphalaria glabrata (SAY, 1818).

Over the past few years, there has been a significant increase in the use of sunscreens. Consequently, the occurrence in aquatic environments of ultraviolet filters has also increased. The present study aims to evaluate the toxicity of two commercial sunscreens to the aquatic snail Biomphalaria glabrata. Acute assays were performed with adult snails exposed to solutions of the two products in synthetic soft water. Reproduction and development assays were carried out, involving individual adult and egg masses exposure to assess fertility and embryonic development. Sunscreen A showed a LC50-96h of 6.8g/L and reduction in number of eggs and egg masses per individual in the concentration of 0.3g/L. Sunscreen B presented higher malformation rates in 0.4g/L with 63% of malformed embryos. Results indicate that the formulation used in sunscreens is an important factor in aquatic toxicity and needs to be evaluated before the final product is commercialized.

Toxicological evaluation of antiretroviral Tenofovir Disoproxil Fumarate on the mollusk Biomphalaria glabrata and its hemocytes

Tenofovir disoproxil fumarate (TDF) is a drug used in HIV treatment, and several studies have detected its presence in surface water. Furthermore, more information on its environmental impact is needed in the scientific literature. Thus, due to the lack of data on the impact of this drug, and its presence in different waters of the world, this work aimed to evaluate the potential toxicological effects of TDF on the mollusk Biomphalaria glabrata in vivo and in vitro. For in vitro analysis, hemocytes were exposed to different drug concentrations for 1 h and evaluated for feasibility, and phagocytic and metabolic activity. The in vivo analysis consisted of the exposure of groups of five mollusks, in triplicate, at the same drug concentrations for 72 h and 21 days, evaluating mortality, and mollusk and hemolymph behavior. Although the exposure of the mollusk to TDF did not reduce its survival, however it was toxic to its hemocytes. Even if toxicity was identified on the mollusk and its hemocytes initially, further studies should be conducted to understand the effects of this residue on the environment and different life stages of the mollusk because, per the Globally Harmonized System of Classification and Labeling of Chemicals, for aquatic ecosystems, the results obtained were classified as toxic (EC50% 2.65 [1.98; 5.29] mg/L) and could cause unfeasibility in hemocytes at concentrations below 10 mg/l.

Downstream effects: Impact of antibiotic pollution on an aquatic host–parasite interaction

AbstractThe global increase in antibiotic use has led to contamination of freshwater environments. Despite the identified impacts of antibiotics on humans and wildlife, the effect of antibiotics on host–parasite life cycles in freshwater is relatively unexplored. In the current study, we utilize the trematode parasiteSchistosoma mansoni, and its snail intermediate host,Biomphalaria glabrata, to investigate the influence of an ecologically relevant antibiotic concentration on the life history characteristics of both parasite and host. Our results demonstrate that antibiotics not only accelerate parasite development time, but also increase host reproduction and delay parasite‐induced host castration. Using a mathematical model, we suggest that life history alterations associated with antibiotics are likely to increase parasite transmission and disease burden. Our study suggests that antibiotic pollution could impact freshwater ecosystems by influencing host–parasite dynamics and potentially increase the burden of schistosomiasis in endemic regions.

Comparative analysis of three families of hygrophilid snails shows that the egg mass fluid protein haemocyanin-like 1 (Hcl-1) is unique to planorbids

ABSTRACT The egg mass fluid (EMF) of the freshwater snail Biomphalaria glabrata (Hygrophila: Planorbidae) contains haemocyanin-like 1 (Hcl-1) protein, distinct from respiratory haemocyanins. The distribution of Hcl-1 was investigated among major families of Hygrophila, Physidae and Lymnaeidae, both of which employ respiratory haemocyanins, and Planorbidae, a group that evolved haemoglobin as a respiratory pigment. Immunoblotting detected c. 150 kDa protein (molecular weight of Hcl-1) cross-reactive with anti-keyhole limpet haemocyanin antiserum in the EMF of planorbids Bulinus globosus and Planorbella duryi (from a genus closely related to Biomphalaria), but not Physella acuta (Physidae) and Ladislavella elodes (Lymnaeidae). High throughput sequence data revealed Hcl-1 homologs from Bulinus globosus and Planorbella duryi, representative species that span the range of planorbid phylogeny, but not from Physella acuta (Physidae) and Lymnaea stagnalis (Lymnaeidae). A domain architecture comprising only three functional units (FUs) and predicted secondary structures within the C-terminal FU distinguish planorbid Hcl-1 protein from molluscan respiratory haemocyanins that are natively assembled as functional didecamers. Immunoblotting confirmed a monomeric configuration of native Hcl-1. Molecular clock analysis estimated divergence of Hcl-1 proteins from gastropod respiratory haemocyanins at 267 ± 143 Ma. It is hypothesized that Hcl proteins originated in the ancestor of the planorbid lineage when evolution of respiratory haemoglobin altered selective pressures for maintaining original function, facilitating mutation and refunctionalization of the ancestral respiratory haemocyanin in Planorbidae.

No evidence for schistosome parasite fitness trade-offs in the intermediate and definitive host

BackgroundThe trematode parasite Schistosoma mansoni uses an aquatic snail intermediate and a vertebrate definitive host to complete its life cycle. We previously showed that a key transmission trait—the number of cercariae larvae shed from infected Biomphalaria spp. snails—varies significantly within and between different parasite populations and is genetically controlled by five loci. We investigated the hypothesis that the success of parasite genotypes showing high propagative fitness in the intermediate snail host may be offset by lower reproductive fitness in the definitive vertebrate host.MethodsWe investigated this trade-off hypothesis by selecting parasite progeny producing high or low number of larvae in the snail and then comparing fitness parameters and virulence in the rodent host. We infected inbred BALB/c mice using two Schistosoma mansoni parasite lines [high shedder (HS) and low shedder (LS) lines] isolated from F2 progeny generated by genetic crosses between SmLE (HS parent) and SmBRE (LS parent) parasites. We used the F3 progeny to infect two populations of inbred Biomphalaria glabrata snails. We then compared life history traits and virulence of these two selected parasite lines in the rodent host to understand pleiotropic effects of genes determining cercarial shedding in parasites infecting the definitive host.ResultsHS parasites shed high numbers of cercariae, which had a detrimental impact on snail physiology (measured by laccase-like activity and hemoglobin rate), regardless of the snail genetic background. In contrast, selected LS parasites shed fewer cercariae and had a lower impact on snail physiology. Similarly, HS worms have a higher reproductive fitness and produced more viable F3 miracidia larvae than LS parasites. This increase in transmission is correlated with an increase in virulence toward the rodent host, characterized by stronger hepato-splenomegaly and hepatic fibrosis.ConclusionsThese experiments revealed that schistosome parasite propagative and reproductive fitness was positively correlated in intermediate and definitive host (positive pleiotropy). Therefore, we rejected our trade-off hypothesis. We also showed that our selected schistosome lines exhibited low and high shedding phenotype regardless of the intermediate snail host genetic background. ​Graphical