Laboratory Snails 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.

Genotypic-specific heat shock response of vector susceptibility to Schistosoma mansoni.

Living organisms are vulnerable to thermal stress which causes a diversity of physiological outcomes. Previous work has shown that the snail vectors (Biomphalaria glabrata) of an important human pathogen, Schistosoma mansoni, revert from resistant to susceptible after short exposure to a heat stress as low as 31oC; however, due to lack of replicability among labs and genetic lines of snails, it has been hypothesized that this effect is genotype dependent. We examined the effects of heat shock on resistance of two species of snail vectors including B. glabrata and B. sudanica. We used 3 different inbred laboratory snail lines in addition to the F1 generation of field collected snails from Lake Victoria, Kenya, an area with high levels of schistosomiasis transmission. Our results showed marginal effects of heat shock on prevalence of infection in B. glabrata, and that this response was genotype specific. We found no evidence of a heat shock effect on prevalence of infection in B. sudanica or on intensity of infection (number of infectious stages shed) in either snail species. Such environmentally influenced defense responses stress the importance of considering this unique interaction between snail and parasite genotypes in determining infection dynamics under climate changes.

Harmful and Beneficial Effects of the Land Snail Monacha Sp. التأثیرات الضارة و المفیدة للقوقع الأرضى من نوع الموناکا

The harmful and beneficial effects of the land snail Monacha sp . were studied in Sharkiah Governorate.Harmful effects were brightly observed in microbiological examination of gastropod and mucus in laboratory, and evaluating its damage on cabbage plant in laboratory and field. Microbial examination revealed that the gastropod was contaminated with (Klepcella) a harmful bacteria of man and animals and (Burkhelderia ceptacia) a non-useful bacteria in mucus. Few data were dealing with contamination of Monacha land snail in Egypt so, this study aims to highlight in this respect for taking into a sustainable infection when contact and thoroughly washing vegetables and fruits that are favorable food for Monacha land snails.Our results showed noticeable damage of cabbage leaves by Monacha snails in laboratory (26. 93%) and field (39. 23%) as a general mean value of cabbage consumption. Beneficial effects were revealed through biochemical analysis of both gastropod and shell components. Analysis revealed that Monacha sp.rich in protean (17.9%) and minerals such as calcium, phosphorus, magnesium and potassium. Calcium determined the highest (Ca, 13%), followed by phosphorus (Ph., 0.60%), magnesium (Mg, 0.12%) and potassium (K, 0.094%) so, the second aim of our study is the revealing of the economic feature of Monacha land snails as an addition to the fodder of farm animals and in traditional medicine.

Urban soil phytomanagement for Zn and Cd in situ removal, greening, and Zn-rich biomass production taking care of snail exposure.

The phytoextraction potential of Arabidopsis halleri (L.) O'Kane & Al Shehbaz and Salix viminalis L. to partially remove Zn and Cd in soil was investigated. In an urban field site, a very short rotation coppice of willows was implemented, and growth parameters were monitored for 3 years. A. halleri was cultivated in the same site with or without fertilizer to improve biomass yield and/or Zn and Cd aerial part concentrations. Effects of harvest and co-cultivation on these two parameters were measured. To determine if willows and A. halleri leaves were risky in case of consumption by a herbivorous invertebrate like the landsnail Cantareus aspersus, metal concentrations of snails fed with Zn- and Cd-enriched and low enriched leaves were compared. Willows and A. halleri grew well on the metal-contaminated soil (1.7 and 616 mg kg-1 Cd and Zn, respectively). The A. halleri Zn foliar concentration reached the Zn hyperaccumulation threshold (> 10,000 mg kg-1 DW) in the presence of NPK fertilizer and although the soil was alkaline (pH > 8.2). Cd concentration increased with harvest and fertilizer. Cd and Zn foliar concentrations of willows were far above baseline values. Laboratory snails exposure revealed that willow leaves ingestion caused a moderate increase of Cd, Pb, and Zn bioaccumulation in snails compared to the one caused by A. halleri ingestion. The soil and plant metal concentrations were reflected by field snail biomonitoring. This study confirmed the interest of selecting A. halleri and willows to partially remove Zn and Cd in the soil and emphasized their potential usefulness in greening urban contaminated area and producing raw materials for green chemistry while paying attention to the environmental pollutant transfer.

Inhibition of Fasciola hepatica infection in Galba truncatula snails by application of monosaccharides to the aquatic environment

Fasciola hepatica is one of the etiological agents of fasciolosis, a widespread disease in domestic animals and occasionally in humans. Fasciolosis may be reduced by blocking the parasite transmission through its intermediate hosts. In the present work, different monosaccharides have been tested for their ability to impact on ligand/receptor interactions at the interface between the parasite and the intermediate host. Laboratory snails were subjected to miracidia in the presence of methyl-α-D-mannopyranoside (MetMan), α-D-glucose (Glc), N-acetyl-D-glucosamine (GlcNAc), D-(+)-galactose (Gal), N-acetyl-D-galactosamine (GalNAc) or L-(−)-fucose (Fuc), in 10 mM concentration. The snail survival rates and the prevalence of infection were determined after 50 days. Survived snails in the study groups varied from 78% to 97%. A remarkable reduction in the number of parasite-infected snails was observed in groups subjected to MetMan, Glc, or GlcNAc – 36.9%, 10.9%, and 11.9%, respectively, compared to 92.7% in the control group. Other tested monosaccharides had a low impact on snail infection. The results point to the implication that surface carbohydrate/receptor interactions are among the determining factors concerning the transmission of F. hepatica by the specific vector Galba truncatula. Biological recognition between the two organisms can be interfered with appropriate monosaccharides and this can be used to develop an alternative method for control of fasciolosis at the intermediate host level.

Transcriptomic responses of the freshwater snail (Parafossarulus striatulus) following dietary exposure to cyanobacteria

Freshwater snails are promising bioindicators that can be used in ecotoxicological testing and ecological risk assessments. To screen molecular responses following mollusk exposure to algal blooms, whole transcriptome sequencing was performed with the freshwater snail (Parafossarulus striatulus) fed with blue algae (Microcystis aeruginosa). A total of 86,848 unigenes were assembled, and 10,413 unigenes were annotated in the TrEMBL, Pfam, KEGG, and SwissProt databases. In snails fed with both green and blue algae, a total of 276 differentially expressed unigenes were identified, though there were limited differences in snails fed with only green algae. In addition, ten randomly selected differentially expressed unigenes were analyzed in snails collected from Taihu Lake, China. The expression of four unigenes exhibited a trend consistent with that observed in transcriptome profiling of laboratory snails. The results of this study provide an invaluable resource for enhancing our understanding of ecotoxicology following the occurrence of algal blooms in lakes.

Molluscicidal effect of suspension concentrate of niclosamide ethanolamine salt

Objective To evaluate the molluscicidal effect of suspension concentrate of niclosamide ethanolamine salt (SCNE) against Oncomelania hupensis snails in laboratory and field. Methods The experiment of SCNE against the snails by using the immersing and spraying methods was performed in laboratory and field, with control groups of wettable powder of niclosamide ethanolamine salt (WPN). Results In the laboratory, LC50(s) of SCNE for 24, 48 h and 72 h by using the immersion method were 0.092 6, 0.062 9 mg/L and 0.054 9 mg/L, respectively. The mortality rates of snails for 24, 48 h and 72 h by using the immersion method were all 100% with the concentrations of 0.25 mg/L. The mortality rates of snails were all 100% while spraying SCNE for 3 d in the laboratory with the concentrations of 0.25 g/m2. In Jiangling County, except 0.5 g/m3 SCNE immersing the snails for 24 h, the mortality rates of snails by using SCNE with the immersing method were all 100%. While the concentration of SCNE was 0.5 g/m3 or above, the mortality rates were all 100% after the use of it with the immersion method for 2 d in Gong'an County. In Jiangling County, the mortality rates of snails by using SCNE 0.5 g/m3 for 1 d, 3 d, and 7 d with the spraying method were 87.5%, 92.82% and 97.40% respectively. While the concentration of SCNE was 0.5 g/m3, the mortality rates were 85.94%, 86.78% and 94.21% respectively after the use of it with the spraying method for 1 d, 3 d, 7 d in Gong'an County, and the molluscicidal effect of SCNE (1.0 g/m2) was higher than that of WPN. Conclusion SCNE has a high molluscicidal effect in the laboratory and field, and it is a novel and simple formulation of niclosamide.

DIVERSITY AND COMPARATIVE STUDIES ON BULINUS SNAILS COLLECTED FROM TWO LOCALITIES IN EGYPT

A comparative study of the shell structure, seasonal temperature and Calcium content of Bulinus snails from two areas (Damietta and Giza) in Egypt was done and compared with laboratory snails from Schistosome Biological Supply Center (SBSC). The shells of collected snails identified as Bulinus truncatus, showed a wide variation in shape. The results showed a significant differences were detected between the populations from SBSC and Damietta (p<0.05) for mean of measured shell width, aperture length, length of spire and number of whorls. The populations from Giza and Damietta governorates showed significant differences (P<0.05) in mean of measured length of diagonal, length of body whorl above aperture, length of spire and number of whorls. There were no statically significant differences between the populations from SBSC and Giza. The seasonal temperature affected on susceptibility of snails to infection with Schistosoma haematobium. The. mean prepatent period was short in summer and long in winter. The shells of S. haematobium - infected B. truncatus snails showed hypocalcifiction from all localities.

Diversity and Comparative Studies on Bulinus Snails Collected from Two Localities in Egypt

A comparative study of the shell structure, seasonal temperature and Calcium content of Bulinus snails from two areas (Damietta and Giza) in Egypt was done and compared with laboratory snails from Schistosome Biological Supply Center (SBSC). The shells of collected snails identified as Bulinus truncatus, showed a wide variation in shape. The results showed a significant differences were detected between the populations from SBSC and Damietta (p<0.05) for mean of measured shell width, aperture length, length of spire and number of whorls. The populations from Giza and Damietta governorates showed significant differences (P<0.05) in mean of measured length of diagonal, length of body whorl above aperture, length of spire and number of whorls. There were no statically significant differences between the populations from SBSC and Giza. The seasonal temperature affected on susceptibility of snails to infection with Schistosoma haematobium. The. mean prepatent period was short in summer and long in winter. The shells of S. haematobium - infected B. truncatus snails showed hypocalcifiction from all localities.

Effects of trematode parasitism on the shell morphology of snails from flow and nonflow environments.

The primary function of the gastropod shell is protection. However, shells that function well in one environment may be maladaptive in another. Upon infection, the snail shell protects internal parasites and it is to the parasite's advantage to optimize, or not interfere with, shell functionality. However, parasites, particularly trematodes, are often pathogenic and it is not clear if parasitism will induce environment-dependent or -independent changes to gastropod shells. We conducted a field study and a complementary laboratory experiment to examine the effects of trematode parasitism on shell characteristics (shape, size, and crush resistance) of Physa acuta snails in flow and nonflow environments using geometric morphometrics and crush assays. Field results indicate wetland (nonflow) snails had large, crush resistant shells with narrow apertures and tall spires. In contrast, stream (flow) snails had small, weak shells with wide apertures and short spires. Parasitism had no apparent effect on the crush resistance of wetland snails but significantly reduced the crush resistance of stream snails. Parasitism had no significant effect on overall shell shape in stream or wetland snails. Similar to the results of our field study, nonflow tank snails had significantly more crush resistant shells than flow tank snails. Additionally, the shapes of flow and nonflow tank snails significantly differed where nonflow tank snails resembled wetland snails and flow tank snails resembled stream snails. For laboratory snails, parasitism reduced crush resistance regardless of flow/nonflow treatment. Our results demonstrate that habitat and/or flow treatment was the primary factor affecting P. acuta shell morphology and that trematode parasitism played a secondary role.

Molecular approach for detecting early prepatent Schistosoma mansoni infection in Biomphalaria alexandrina snail host

The present study aimed to evaluate a polymerase chain reaction (PCR) assay used for detection of Schistosoma mansoni infection in Biomphalaria alexandrina snails in early prepatent period and to compare between it and the ordinary detection methods (shedding and crushing). Biomphalaria alexandrina snails are best known for their role as intermediate hosts of S. mansoni. DNA was extracted from infected snails in addition to non-infected "negative control" (to optimized the efficiency of PCR reaction) and subjected to PCR using primers specific to a partial sequence of S. mansoni fructose-1,6-bus phosphate aldolase (SMALDO). SMALDO gene was detected in the infected laboratory snails with 70, 85, and 100% positivity at the 1st, 3rd, and 7th day of infection, respectively. In contrast, the ordinary method was not sensitive enough in detection of early prepatent infection even after 7days of infection which showed only 25% positivity. By comparing the sensitivity of the three methods, it was found that the average sensitivity of shedding method compared to PCR was 23.8% and the average sensitivity of crushing method compared to PCR was 46.4% while the sensitivity of PCR was 100%. We conclude that PCR is superior to the conventional methods and can detect positive cases that were negative when examined by shedding or crushing methods. This can help in detection of the areas and times of high transmission which in turn will be very beneficial in planning of the exact timing of the proper control strategy.

Morphological and molecular analyses of larval and adult stages of Echinoparyphium recurvatum von Linstow 1873 (Digenea: Echinostomatidae) from central Mexico.

In central Mexico, populations of the freshwater snail Physella cubensis were infected with metacercariae from a species of Echinoparyphium (Digenea: Echinostomatidae). In the current study, we describe both larval and adult stages of this species obtained from experimental and natural infections. A total 180 snails were collected from Patzcuaro Lake, Michoacan state in central Mexico in July 2012. In the laboratory snails were placed in individual vials and exposed to light with the aim of observing emergence of cercariae. To obtain metacercariae, uninfected snails (P. cubensis) were exposed to cercariae. Chicks were infected with metacercariae to obtain adults. Nine days post-infection, eggs were recovered and incubated in tap water at room temperature to observe miracidia. Adults obtained from natural and experimentally infected hosts possess a head collar with 45 spines in two alternating rows, confirming the identification as Echinoparyphium recurvatum von Linstow 1873. To test the conspecificity of all stages, sequences of nuclear internal transcribed spacer 1 (ITS1), 5.8S and ITS2 rDNA were obtained from two adult worms recovered from chicks and also a natural avian host, the shoveler duck Anas clypeata, together with five cercarial and four metacercarial isolates from nine snails. The genetic divergence estimated among the 13 isolates was very low, ranging from 0 to 0.6%. Phylogenetic analyses inferred by maximum likelihood and Bayesian methods showed that all isolates of E. recurvatum form a single clade with strong support. The presence of E. recurvatum in P. cubensis and A. clypeata from central Mexico represents new host reports, and extends the distribution range in the Americas.

Tissue invasion of laboratory-reared Biomphalaria glabrata by a harpacticoid copepod

Copepods were observed in the tissues of 3 of 23 Biomphalaria glabrata snails examined histologically. All were heavily encapsulated by hemocytes and were dead. The copepods are most likely members of the order Harpacticoida, based on external morphology. This tissue invasion appears to be accidental rather than symbiotic or predatory, but could be a cause of observed mortality in laboratory snail colonies.

Detection of Schistosoma mansoni and Schistosoma haematobium DNA by Loop-Mediated Isothermal Amplification: Identification of Infected Snails from Early Prepatency

Monitoring post-control transmission of schistosomes by examining humans becomes less effective as infection rates among humans decrease. Molecular monitoring of prepatent schistosome infection in snails by the polymerase chain reaction (PCR) has been used for studying human-to-snail transmission, and snail prepatent infection rates were found to correspond to infection prevalence and average intensity in human populations contacting the sites studied. We have now developed loop-mediated isothermal amplification (LAMP) assays for identifying Schistosoma mansoni and S. haematobium to facilitate large-scale evaluation of post-intervention transmission potential. LAMP primers were designed based on the Sm1-7 and DraI repeated sequences of the corresponding schistosomes, and amplification by LAMP of these 121-basepair highly abundant sequences provided a detection sensitivity of 0.1 fg of genomic DNA. When these LAMP assays were applied for examining infected laboratory snails, it was possible to identify infection from the first day after exposure to miracidia. The potential advantages of these assays are discussed.

Épidémiologie des bilharzioses humaines en Mauritanie. L’exemple de la rive droite du fleuve Sénégal

A study was performed to determine the transmission and prevalence of human schistosomiasis in the Mauritanian side of the Senegal River basin. Parasitological investigations by Kato-Katz and urine filtration conducted on 1,259 school children indicated a mean prevalence of S. haematobium--29.0%, 25.9% and 34.3%, respectively, in the children of the lower, middle and high valley. Only the school children of the lower delta valley were infected by S. mansoni with a mean prevalence rate of 21.5%. The malacological investigations carried out in the water points of each visited village highlighted the presence of B. pfeifferi, B. senegalensis, B. globosus, B. umbilicatus, B. truncatus and B. forskalii. The last three species are announced for the first time in the Mauritanian side of the Senegal River. The laboratory snail infection experiments indicate that B. senegalensis and B. globosus are the most important intermediate hosts for S. haematobium in the Mauritanian side of the Senegal River basin. However, an incompatibility between the oasis strains of S. haematobium and the snails of the lower valley was noted. In the middle valley and high valley, the infection of the school children takes place during the rainy season, because of the creation of the temporary water points, in the lower valley; the transmission seems to be continuous.

Development of an Intelligent Robot for an Agricultural Production Ecosystem (VI) : Counting of Snails in Laboratory and Paddy by using Developed Programs of Image Processing

An image processing analysis to segment and count the golden apple snails in paddy field was done in order to develop an intelligent robot for a rice production ecosystem. The Simulink models for counting snails were constructed for three different condition of the paddy and for the case they are mating. It was found useful to use the color of the reflection of the sunshine on the shells of the snails in the daytime and the color of the snail itself after raining at night in order to count the number of snails in paddy field by image processing. These models enable the robot to count the number of snails in paddy.

Detection of Ornithobilharzia turkestanikum cercaria (trematoda) by nested-PCR in intermediate host snail, Lymnaea gedrosiana

Trematodes are important in economic and public health. Ornithobilharzia turkestanicum (O. turkestanicum) is one of the important economic trematodes in domestic animals. Ornithobilharzia infection in intermediate host (Lymnaea gedrosiana ) can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites. The current available diagnostic methods are inefficient for identification of prepatent infections and/or after dead of snails. For the above difficulties we adapted a nested polymerase chain reaction (Nested-PCR) assay for sensitive detection of O. turkestanicum in clinical samples and its cercaria in snails. The life cycle of parasite was maintained in sheep and snails in laboratory in Razi Institute. Adult worms were isolated from sheep and DNA was extracted from worms by a procedure using DNA extraction solution developed in NIGEB. PCR and nested-PCR primers were designed based on 28s ribosomal RNA gene of O. turkestanicum and the DNA was amplified by PCR assay. PCR product was purified and cloned in pTZ57R/T and sequenced. The comparison of the obtained sequences with the GenBank using blast program was showed in NCBI Sequence viewer just 682 bp. PCR amplicon was submitted in GenBank and can be assessed using AY862391 accession number. DNA was extracted from the infected and non-infected snails 2-5 days post-infection. The infected snails could be rapidly detected with Nested-PCR. Results indicate that this assay is specific for detecting O. turkestanicum. The high sensitivity of the test enabled identification of single infected snail even when its DNA was pooled with uninfected snails. Thus demonstrating the possibility of mass diagnosis in pools of snails, therefore, the assay has the potential for large-scale demonstration of prepatent infection prevalence in snails and offers a new diagnostic tool for evaluation of bilharziosis trasnsmission and for control of infection as discussed.

Food and habitat partitioning in grazing snails (Turbo smaragdus), Northern New Zealand

Gut content analyses and fatty acid profiles were used to identity food consumption and assimilation in a dominant grazing snail (Turbo smaragdus) in rocky shore and estuarine habitats, at Waiwera Estuary, northern New Zealand. Gut contents of freshly collected individuals indicated that snails utilize a wide range of food sources within their habitats, including microalgae and foliose-corticated macrophytes (rocky intertidal), and mangrove tissue and filamentous algae (mangrove stand). Laboratory feeding experiments revealed thatT. smaragdus prefers microalgae and possibly filamentous epiphytes, regardless of snail size or habitat of origination. The fatty acid profiles of snails fed different diets (brown algae [Hormosira banksii], diatoms, mangrove pneumatophores, and filamentous green algae [Chaetomorpha sp.]) confirmed the assimilation of the given foods, except for the mangrove treatment, which resulted in a low, long chain, fatty acid (LCFA) signature. Incongruities between the gut content analyses (high number of mangrove particles) and fatty acid profiles (low LCFA signature) of field and laboratory snails exposed to a mangrove diet suggest that snails target microalgal and filamentous epiphyte food sources on the pneumatophores and inadvertently ingest mangrove particles. Snails within all food treatments, except diatoms, had a relatively strong bacterial signature (18∶1n–7, Σ15+Σ17). Bacteria may have readily accumulated in the experimental tanks and been selected by snails as an alternative food source. In the field, snails and other grazers may ingest detritus and biofilms to access dense populations of bacteria. This research highlights the need for a comprehensive and multianalytical approach to elucidate the role of grazers in algal-plant dominated ecosystems.

Identification of an avian schistosome recovered from Aythya novaeseelandia and infectivity of its miracidia to Lymnaea tomentosa snails.

A partial life cycle involving miracidia hatched from the eggs of Trichobilharzia sp. recovered from New Zealand scaup (Aythya novaeseelandia) to the release of furcocercariae by laboratory snails (Lymnaea tomentosa) was accomplished. Challenges with five and ten miracidia per snail were lethal. Challenge with three miracidia resulted in development to the daughter sporocyst stage and death in five, development to furcocercarial stage and death in one, and shedding of furcocercariae in one of seven snails. Observed lethality of schistosome miracidia to L. tomentosa may explain the low infection prevalence observed in the wild. Future work should plan challenge exposures of three or fewer miracidia to ensure snail survival and successful recovery of furcocercariae. The Trichobilharzia sp. found in the New Zealand scaup does not key morphologically to the literature. It may be a new species and further work is needed.

Endemic echinostome infections of candidate hosts

Wild Lymnaea tomentosa snails, recovered from Lake Wanaka, New Zealand, were established in the laboratory. Wild snails, naturally infected with echinostomes, provided metacercariae for infection of laboratory maintained snails. Metacercarial cysts from wild and laboratory snails were then used to attempt infection of definitive host candidates. Laboratory snails provided convenient packaging of known numbers of cysts. Metacercariae excysted in the small intestines of ducklings to mature in 6 days. Worms were expelled as they became gravid. Attempts to establish infections in experimental hosts other than ducklings were not successful. No worms were recovered from mice, white rats, guinea pigs, hamsters or immunosuppressed white rats.