Biological and histological changes in reproductive patterns of Biomphalaria glabrata experimentally co-infected by Echinostoma paraensei and Angiostrongylus cantonensis.

Investigation of co-infection by different helminths in snails can provide essential information about the biology of parasites co-existence under natural conditions. This study aimed to investigate the reproductive and histological changes of Biomphalaria glabrata experimentally co-infected with Echinostoma paraensei and Angiostrongylus cantonensis. Five groups of snails i.e. uninfected, with single and double infections, either E. paraensei first (E + A) or A. cantonensis first (A + E) were followed; three times a week during four weeks the numbers of egg masses, eggs and hatched snails were counted. Histological analysis of the ovotestis and albumen gland was performed after four weeks. The number of egg masses/snail, eggs/snail and hatched snails showed significative differences comparing the control group to all infected groups, especially in group E + A, with the majority of values of parameters analyzed lower than 50% of those observed for control snails. In addition, the mortality was higher in the group E + A than to control group. The histological analysis showed that presence of both parasites in the reproductive organs was associated with tissue damages.

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

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

Preliminary studies investigating the occurrence of Biomphalaria cousini in Brazil.

Specific genetic profiles of Brazilian Biomphalaria species were previously standardized by molecular taxonomy through the analysis of restriction fragments, which were generated by digesting the internal transcribed spacer region of rDNA with the DdeI endonuclease. Biomphalaria amazonica displayed three distinct profiles. To investigate these distinct profiles, the same molecular technique, polymerase chain reaction and restriction fragment length polymorphism, was used with different endonucleases. In addition, morphological data were also used to compare B. amazonica specimens that were collected from Brazil, Colombia and Bolivia. The morphological characters of Bolivian molluscs were similar to B. amazonica, displayed a molecular profile of five restriction fragments and morphological data, whereas the Colombian mollusc population showed morphological characters similar to Biomphalaria cousini and a molecular profile of three restriction fragments, similar to B. cousini. The Brazilian specimens showed the B. amazonica and B. cousini molecular profiles as well as a third profile, which resembled a combination of the Colombian and Bolivian molecular profiles.

Toxicological assessment of lambda-cyhalothrin and acetamiprid insecticides formulated mixture on hatchability rate, histological aspects, and protein electrophoretic pattern of Biomphalaria alexandrina (Ehrenberg, 1831) snails.

Several formulated mixtures of pesticides are widely used in modern agriculture. Nevertheless, the agriculture runoff causes a serious damage to the aquatic ecosystem. Therefore, the present study aims to use B. alexandrina snails as bioindicators for 30 g/l lambda-cyhalothrin and 17 g/l acetamiprid as a formulated mixture insecticide. Results showed that it has a molluscicidal activity against snails at LC50 7.9 mg/l. The hatchability percent of both treated 1-day-aged and/or 3-day-aged groups were less than that of the control group. The sublethal concentrations of the tested insecticide caused a remarkable abnormal necrosis in male and female gametogenic cells, besides a severe damage in both secretory and digestive cells. The results of SDS-PAGE protein profiles of treated snails showed that the least number of protein bands was noticed in snail groups that subjected to LC10 (6.6 mg/l) and LC25 (7.2 mg/l) concentrations when compared to control protein fractions.

Distribution and current infection status of Biomphalaria straminea in Hong Kong.

BACKGROUND: Schistosomiasis, also generally known as snail fever, is a parasitic disease caused by trematode flatworms of the genus Schistosoma. In Hong Kong and mainland China, the freshwater snail Biomphalaria straminea has been introduced and has the potential to transmit intestinal schistosomiasis caused by S. mansoni, a parasite of man which has a wide distribution in Africa and parts of the New World, especially Brazil. The first identification of B. straminea in Hong Kong dates back to the 1970s, and its geographical distribution, phylogenetic relationships, and infection status have not been updated for more than 30 years. Thus, this study aims to reveal the distribution and current infection status of B. straminea in contemporary Hong Kong. METHODS: Snails were collected from different parts of Hong Kong from July 2016 to January 2017. Both anatomical and molecular methods were applied to identify B. straminea. Cytochrome c oxidase subunit 1 (cox1), internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and 16S ribosomal DNA (rDNA) were sequenced from individual snails and analyzed. To detect the presence of S. mansoni, both biopsy and PCR analyses were carried out. RESULTS: Using both anatomical and molecular analyses, this study demonstrated the existence of black- and red-coloured shell B. straminea in different districts in the New Territories in Hong Kong, including places close to the mainland China border. None of the B. straminea (n = 87) investigated were found to be infected with S. mansoni when tested by biopsy and PCR. The Hong Kong B. straminea are genetically indistinguishable, based on the chosen molecular markers (cox1, ITS1-5.8S-ITS2, and 16S rDNA), and are similar to those obtained in mainland China and South America. CONCLUSION: Biomphalaria straminea is now well established in freshwater habitats in Hong Kong. No evidence of infection with S. mansoni has been found. Surveillance should be continued to monitor and better understand this schistosomiasis intermediate host in mainland China and Hong Kong.

Pathogen-associated molecular patterns activate expression of genes involved in cell proliferation, immunity and detoxification in the amebocyte-producing organ of the snail Biomphalaria glabrata.

The anterior pericardial wall of the snail Biomphalaria glabrata has been identified as a site of hemocyte production, hence has been named the amebocyte-producing organ (APO). A number of studies have shown that exogenous abiotic and biotic substances, including pathogen associated molecular patterns (PAMPs), are able to stimulate APO mitotic activity and/or enlarge its size, implying a role for the APO in innate immunity. The molecular mechanisms underlying such responses have not yet been explored, in part due to the difficulty in obtaining sufficient APO tissue for gene expression studies. By using a modified RNA extraction technique and microarray technology, we investigated transcriptomic responses of APOs dissected from snails at 24 h post-injection with two bacterial PAMPs, lipopolysaccharide (LPS) and peptidoglycan (PGN), or with fucoidan (FCN), which may mimic fucosyl-rich glycan PAMPs on sporocysts of Schistosoma mansoni. Based upon the number of genes differentially expressed, LPS exhibited the strongest activity, relative to saline-injected controls. A concurrent activation of genes involved in cell proliferation, immune response and detoxification metabolism was observed. A gene encoding checkpoint 1 kinase, a key regulator of mitosis, was highly expressed after stimulation by LPS. Also, seven different aminoacyl-tRNA synthetases that play an essential role in protein synthesis were found to be highly expressed. In addition to stimulating genes involved in cell proliferation, the injected substances, especially LPS, also induced expression of a number of immune-related genes including arginase, peptidoglycan recognition protein short form, tumor necrosis factor receptor, ficolin, calmodulin, bacterial permeability increasing proteins and E3 ubiquitin-protein ligase. Importantly, significant up-regulation was observed in four GiMAP (GTPase of immunity-associated protein) genes, a result which provides the first evidence suggesting an immune role of GiMAP in protostome animals. Moreover, altered expression of genes encoding cytochrome P450, glutathione-S-transferase, multiple drug resistance protein as well as a large number of genes encoding enzymes associated with degradation and detoxification metabolism was elicited in response to the injected substances.

A molecular survey of biomphalaria in Egypt: is B. glabrata present?

Two species of Biomphalaria are reported from Egypt, the indigenous Biomphalaria alexandrina and Biomphalaria glabrata, the latter believed to be introduced during the past few decades. Both are known to be excellent hosts of Schistosoma mansoni, the human-infecting blood fluke common in Egypt. Given the concerns regarding the spread of the exotic B. glabrata, this study was carried out to get a more current picture of the status of Biomphalaria in Egypt. Snail collections were undertaken during 2002-2003 from regions between Alexandria and Ismailia in the north of the Nile Delta, to as far south as Abu Simbel at Lake Nasser. Biomphalaria snails were found in 37 out of 76 sampled localities and were widely distributed in the Nile Delta and along the Nile as far south as Aswan. According to the results of species-specific polymerase chain reaction assays that sampled both nuclear and mitochondrial genomes, and according to DNA sequence data, all Biomphalaria collected during this survey were B. alexandrina. There was no evidence of the presence of B. glabrata or of hybridization of B. alexandrina with B. glabrata in the examined sites. The results were surprising given that some field-collected snails strongly resembled B. glabrata in both size and conchology and that previous survey work suggested B. glabrata had established in Egypt. Continued scrutiny to ascertain the possible presence of B. glabrata in Egypt is warranted. Also, the planorbid Helisoma duryi was detected in the Delta and as far south as Aswan, so it is important for Egyptian schistosomiasis workers to accurately distinguish this non-schistosome-transmitting snail from Biomphalaria.

A neotropical snail host of Schistosoma mansoni introduced into Africa and consequences for the schistosomiasis transmission Biomphalaria tenagophila in Kinshasa (Democratic Republic of Congo).

Malacological surveys carried out in the early 1970s in water bodies of the Kinshasa area, Lower Zaire (Democratic Republic of Congo), showed the appearance of a Biomphalaria species which was identified as Biomphalaria camerunensis. In 1976, other surveys confirmed the presence of the species in several sites and showed numerous infected snails with Schistosoma mansoni, demonstrating for the first time an active transmission of the parasite responsible of the intestinal schistosomiasis in this area. The most recent malacological sampling was carried out by one of us in 1994 in Mangungu River and revealed the presence of apparently the same snail species. However, conchological, anatomical and molecular studies showed that this snail may be considered as an introduced neotropical species, B. tenagophila. To our knowledge, this is the second example of the introduction of a neotropical snail host of schistosomes into Africa.

Effect of temperature on the reproduction of the snail Biomphalaria glabrata.

1. The reproduction of the snail Biomphalaria glabrata, one of the intermediate hosts of Schistosoma mansoni, was evaluated under laboratory conditions by measuring egg production, number of ovipositions, number of eggs per oviposition, and hatching rate using a Latin square design with five different temperature treatments. This permitted the elimination of occasional variation in oviposition due to manipulation or a priori group. 2. Egg production and spawning rates were higher at temperatures between 20.0 and 27.5 degrees C than at 17.5 degrees C. Number of eggs per spawning and hatching rate did not vary with temperature. 3. During a 3-day adaptation phase, a short-term thermal effect appeared in which some indices of reproductive rates differed significantly from those of the experimental phase.

The effect of size of M line Biomphalaria glabrata on the course of development of Echinostoma paraensei.

M line Biomphalaria glabrata snails of 4-, 6-, 8-, 10-, 12-, or 20-mm shell diameter were individually exposed to 10 miracidia each of Echinostoma paraensei. Snails 10 mm in size or larger were found to be significantly less likely to harbor intraventricular sporocysts than snails in smaller size categories. The percentage of snails with intraventricular sporocysts that also developed hemocyte encapsulation responses generally increased with snail size, whereas the number of snails that ultimately became heavily parasitized with large numbers of daughter rediae decreased significantly with snail size. However, at least some snails in each size category developed such disseminated infections. Comparative histological study of 6- and 12-mm snails revealed that parasites readily penetrated both groups of snails, but were more likely to be encapsulated and destroyed in larger snails. Encapsulation reactions were noted from 1 to 15 days postexposure (dpe) in 12-mm snails, indicating that unlike other commonly studied models of trematode-gastropod interactions, snail resistance is not always manifested during the first few days following exposure. Upon infection with E. paraensei, both 6- and 12-mm snails showed significant increases in the number of circulating hemocytes/mm3 of hemolymph. In 6-mm snails, such increases occurred concurrently with successful parasite development. Hemocyte counts in 6-mm snails were significantly elevated from 4 to 15 dpe whereas in 12-mm snails they were significantly elevated from 2 to 30 dpe. A significant degree of resistance to E. paraensei develops as B. glabrata grows and attains sexual maturity. A mechanistic understanding of this phenomenon awaits further investigation.

Superinfection of Biomphalaria glabrata with Schistosoma mansoni.

Biomphalaria glabrata 2 to 12 mm in diameter were exposed to 1 miracidium of Schistosoma mansoni and after 15, 30, or 60 days were reexposed to the homologous strain. The criterion of superinfection was the subsequent escape of cercariae of both sexes. Opposite sex superinfection was acquired by 45 of 106 snails of various sizes, all of which were in the group reexposed at 30 days. The observations confirm those of Kagan and Geiger and suggest that in the schistosomes a maximizing of the number of intermediate hosts with larval populations of both sexes may be an adaptation having a high species survival value.

Schistosoma mansoni: changes in the albumen gland of Biomphalaria glabrata snails selected for nonsusceptibility to the parasite.

The LAC-line strain of the snail Biomphalaria glabrata has very low susceptibility to the parasite Schistosoma mansoni and a very low reproductive potential. Upon examination of the reproductive tract of these snails, light and electron microscopy revealed obvious abnormalities in the albumen gland. Secretory cells that are normally cuboidal in susceptible NMRI (F0) snails were squamous in LAC-line snails. These LAC-line cells contained small secretory granules and negligible rough endoplasmic reticulum and Golgi, compared to large granules and an extensive array of both organelles in F0 albumen gland cells. Comparative analyses of soluble protein extracts of F0 and LAC-line albumen glands showed several qualitative differences. Among the most prominent was an 18-kDa protein in F0 snails that was remarkably reduced in the soluble protein extracts of LAC-line snails. Also, metabolic incorporation of [35S]-methionine was impaired in LAC-line albumen glands. Whether these albumen gland changes are caused by decreased susceptibility to parasitism is yet to determined.

Contribution to the histology of Biomphalaria glabrata.

A combination of histological techniques applied to the study of Biomphalaria glabrata yielded some interesting new data about the histology of this snail, a major intermediate host of Schistosoma mansoni in Brazil. Three kinds of pigments were identified: a dark pigment which bleached following oxidation with potassium permanganate; a lipofuchsin-like, diastase-resistant PAS-positive pigment and an iron-containing pigment, probably related to hemosiderin. Calcium was detected in small deposits within the connective tissue and forming a dense core inside the chitinous radular teeth. The presence of fibrils, staining with Sirius-red and birefringence under polarized light strongly suggest primitive collagen tissue. The radular apparatus appeared as a storing site for glycogen, while abundant Alcian-blue positive material (proteoglycans) was extremely concentrated in the radular sac.

Comparative studies for development of Schistosoma mansoni sporocysts in Puerto Rican and Brazilian Biomphalaria glabrata.

The development of sporocysts of Schistosoma mansoni was monitored in pigmented and albino Biomphalaria glabrata from Puerto Rico and Brazil. The snails were exposed individually to 20 miracidia, and sporocysts were allowed to develop for 3 to 12 weeks. Most of the immature sporocysts were found in the seminal receptacle sac and vas deferens during development. In contrast, mature daughter sporocysts were detected everywhere except in the foot at 12 weeks after exposure to the miracidia. It was found that mature daughter sporocysts formed more rapidly in the pigmented than in the albino snails, but no difference was observed in the formative time between the same types of Puerto Rican and Brazilian snails. It seems likely that there is a correlation between the infection rate and the time required for formation of mature daughter sporocysts in B. glabrata.

Biomphalaria species in Alexandria water channels.

Of the several species of Biomphalaria snails worldwide that serve as the intermediate host for Schistosoma mansoni, Biomphalaria alexandrina is a species that is indigenous to Egypt. Recently, there has been much debate concerning the presence of Biomphalaria glabrata and the hybrid of the species with Biomphalaria alexandrina. Due to this debate, the absence of a clear explanation for the presence of B. glabrata in Egyptian water channels and the probability that they may be reintroduced, we conducted this field study to identify Biomphalaria species present in Alexandria water channels. Laboratory-adapted susceptible snails to Schistosoma mansoni of the following species were used as a reference; Biomphalaria alexandrina, Biomphalaria glabrata and their hybrid. These snails were used to perpetuate the Schistosoma life cycle at the Theodor Bilharz Research Institute (TBRI), Cairo, Egypt. Morphological and molecular studies were conducted on these reference snails as well as on the first generation of Biomphalaria snails from two areas in the Alexandria governorate. The morphological study included both external shell morphology and internal anatomy of the renal ridge. The molecular study used a species-specific PCR technique. The results demonstrated that there was an absence of Biomphalaria glabrata and the hybrid from Alexandria water channels. Moreover, the susceptibility patterns of these reference snails were studied by measuring the different parasitological parameters. It was found that Biomphalaria glabrata and the hybrid were significantly more susceptible than Biomphalaria alexandrina to the Egyptian strain of Schistosoma mansoni. The results demonstrated that if Biomphalaria glabrata was reintroduced and adapted to the local environment in Egypt, it would have important epidemiologic impacts that would have a serious effect on the health of Egyptian people.

Malacological survey and geographical distribution of vector snails for schistosomiasis within informal settlements of Kisumu City, western Kenya.

BACKGROUND: Although schistosomiasis is generally considered a rural phenomenon, infections have been reported within urban settings. Based on observations of high prevalence of Schistosoma mansoni infection in schools within the informal settlements of Kisumu City, a follow-up malacological survey incorporating 81 sites within 6 informal settlements of the City was conducted to determine the presence of intermediate host snails and ascertain whether active transmission was occurring within these areas. METHODS: Surveyed sites were mapped using a geographical information system. Cercaria shedding was determined from snails and species of snails identified based on shell morphology. Vegetation cover and presence of algal mass at the sites was recorded, and the physico-chemical characteristics of the water including pH and temperature were determined using a pH meter with a glass electrode and a temperature probe. RESULTS: Out of 1,059 snails collected, 407 (38.4%) were putatively identified as Biomphalaria sudanica, 425 (40.1%) as Biomphalaria pfeifferi and 227 (21.5%) as Bulinus globosus. The spatial distribution of snails was clustered, with few sites accounting for most of the snails. The highest snail abundance was recorded in Nyamasaria (543 snails) followed by Nyalenda B (313 snails). As expected, the mean snail abundance was higher along the lakeshore (18 ± 12 snails) compared to inland sites (dams, rivers and springs) (11 ± 32 snails) (F(1, 79) = 38.8, P < 0.0001). Overall, 19 (1.8%) of the snails collected shed schistosome cercariae. Interestingly, the proportion of infected Biomphalaria snails was higher in the inland (2.7%) compared to the lakeshore sites (0.3%) (P = 0.0109). B. sudanica was more abundant in sites along the lakeshore whereas B. pfeifferi and B. globosus were more abundant in the inland sites. Biomphalaria and Bulinus snails were found at 16 and 11 out of the 56 inland sites, respectively. CONCLUSIONS: The high abundance of Biomphalaria and Bulinus spp. as well as observation of field-caught snails shedding cercariae confirmed that besides Lake Victoria, the local risk for schistosomiasis transmission exists within the informal settlements of Kisumu City. Prospective control interventions in these areas need to incorporate focal snail control to complement chemotherapy in reducing transmission.

Experimental evidence of size/age-biased infection of Biomphalaria glabrata (Pulmonata: Planorbidae) by an incompatible parasite species: consequences for biological control.

Because the digenetic trematode Plagiorchis elegans can elicit a rapid, severe and permanent suppression of the reproductive output in the snail Biomphalaria glabrata, it is considered as a potential biological control agent of human schistosomiasis. This assumption however is derived from laboratory experiments that are poor approximations of what occurs in a natural ecosystem. In order to recreate conditions that resemble those found in nature, we exposed B. glabrata as individual populations composed of a young, juvenile and adult snails to various concentrations of P. elegans eggs to assess the probability of encountering the parasite eggs by the different snail sizes/age groups. We demonstrated that within populations composed of different size/age classes, larger/older snails displayed the negative effects typical of exposure to P. elegans, whereas smaller individuals appeared relatively unaffected, particularly at lower levels of exposure. These findings coupled with the difficulty of producing large quantities of parasite eggs suggest that P. elegans has limited efficiency as a biological control agent of human schistosomiasis.