Characterization of the bacterial microbiota of Biomphalaria glabrata (Say, 1818) (Mollusca: Gastropoda) from Brazil

Roughly 200 000 000 people in 74 countries infected with schistosomes all share the fact that they came in contact freshwater harbouring infected snails. The aim of the study is to characterize the microbiota of wild and laboratory‐reared snails of Biomphalaria glabrata from Pernambuco, Brazil. The microbiota of these molluscs was identified biochemically by the VITEK 2 automated microbiological system. Antimicrobial susceptibility testing was carried out by the disc diffusion method with ß‐lactam antibiotics, aminoglycosides, quinolones, folate pathway inhibitors, fenicols and tetracyclines. The results showed that all bacteria identified were gram‐negative, including 11 bacterial genera: Aeromonas, Citrobacter, Enterobacter, Cupriavidus, Rhizobium, Stenotrophomonas, Pseudomonas, Klebsiella, Acinetobacter, Vibrio and Sphingomonas. Regarding the antimicrobial susceptibility, all the isolates exhibited resistance to amoxicillin and sensitivity to meropenem (beta‐lactam antimicrobials). The microbiota of the wild snails consisted predominantly of Enterobacter cloacae, while the laboratory‐reared snails predominantly showed Citrobacter freundii and Aeromonas sobria.

Significance and Impact of the Study

Biomphalaria glabrata is a Brazilian freshwater Planorbidae of great medical relevance as an intermediate host of Schistosoma mansoni. About a month after being infected by one or more miracidia larvae of a compatible schistosome, B. glabrata sheds thousands of cercariae into the water where they seek human skin and, if successful, penetrate to establish infection, eventually taking up residence and maturing in blood vessels of the small intestine. Results obtained from this study aim at targeting novel biological control strategies for schistosomiasis such as paratransgenesis. This is the first study on the microbiota of B. glabrata from Brazil.     

Observation of some key stages of the embryonic development of Biomphalaria straminea (Dunker, 1848) (Molluska,Planorbidae)

Summary

Biomphalaria straminea is a freshwater snail and one of the intermediate hosts of the trematode parasite which causes schistosomiasis in Brazil. The main stages of embryonic development were analyzed with confocal laser scanning microscopy (CLSM), using the fluorescent probe DiOC and Nile Red as a vital stain in in vivo preparations. In fixed preparations nuclei were stained by the Feulgen reaction or the fluorescent DNA probe, Höechst 33342. Results obtained from the analysis of embryos at the stages of early cleavage, morula, blastula, gastrula, early trocophore and veliger showed that these stages are similar to those described for Biomphalaria glabrata and Biomphalaria tenagophila. CLSM optical sections of early trochophore showed important morphological structures such as the blastopore, stomodeum and shell gland; and in early veliger, internal organs such as the esophagus, stomach and male and female ducts were also clearly identified.     

Tissue responses exhibited by Biomphalaria alexandrina snails from different Egyptian localities following Schistosoma mansoni exposure

Snails’ susceptibilities to infection with Schistosoma mansoni were determined through observation of infection rates, total cercarial production and tissue responses of the first generation (F1) of Biomphalaria alexandrina snails, originally collected from different Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) and responses were compared between groups. The emergence of cercariae for a 3-month period and the calculation of survival and infection rates, in control (Schistosome Biological Supply Center; SBSC) and infected snails were evaluated. SBSC and Giza snails showed greater susceptibilities to infection and lower mortality rates. In addition, at 6 and 72 h post-exposure to miracidia all the snail groups showed no difference in the anatomical locations of sporocysts. The larvae were found in the head-foot, the mantle collar and the tentacles of the snails. Sporocysts showed normal development with low tissue reactions in SBSC and Giza snail groups infected with S. mansoni miracidia (SBSC). However, in Fayoum, Kafr El-Sheikh, Ismailia and Damietta snail groups, variable tissue responses were observed in which numerous hemocytes made direct contact with S. mansoni larvae forming capsules. The results suggested that, different responses of B. alexandrina snail’s hemocytes towards S. mansoni are related to the degree of susceptibility of these snails. So this is important in planning the strategy of schistosomiasis control.     

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.     

Small angle x-ray scattering of the hemoglobin from Biomphalaria glabrata

The hemoglobin from Biomphalaria glabrata is an extracellular respiratory protein of high molecular mass composed by subunits of 360 kDa, each one containing two 180 kDa chains linked by disulfide bridges. In this work, small angle x-ray scattering (SAXS) measurements were performed with the hemoglobin at pH 5.0 and 7.5. Radii of gyration of 98.6 +/- 0.5 and 101.8 +/- 0.2 A and maximum diameters of 300 +/- 10 and 305 +/- 10 A, respectively, were obtained from Guinier plot extrapolation and analytical curve fitting. The pair distance distribution functions p(r) corresponded to globular particles with a somewhat anisotropic shape for both preparations. Computer analysis of the low angle part of the scattering curve led to the determination of the low resolution envelope of the protein, revealing a P(222) symmetry. Shape reconstruction from ab initio calculations using the complete scattering curve furnished a compact prolate three-dimensional (3D) bead model for the protein. Hydrodynamic parameters were obtained from experiments and theoretical calculations using the 3D model. The results of the structural and biochemical studies reported herein indicate that the multisubunit structure of this hemoglobin is compatible with a tetrameric arrangement.     

Identification of the putative mannose 6-phosphate receptor (MPR 46) protein in the invertebrate mollusc

Mannose 6-phosphate receptor (MPR 300) protein was earlier affinity purified on phosphomannan gel from the membrane extracts of whole animal acetone powder of a mollusc, unio, in the presence of EDTA (Udaya Lakshmi, Y., Radha, Y., Hille-Rehfeld, A., von Figura, K., and Siva Kumar, N. (1999) Biosci. Rep. 19:403–409). In the present study we demonstrate that the unio also contains the putative mannose 6-phosphate receptor (MPR 46) that can be purified on the same gel in presence of divalent metal ions (10 mM each of calcium, manganese, and magnesium), and in the absence of sodium chloride and at pH 6.5. Chicken and Fish cell MPR 46 proteins were purified under these conditions (Siva Kumar, N., Udaya Lakshmi, Y., Hille-Rehfeld, A., and von Figura, K. (1999) Comp. Biochem. & Physiol. 123B:261–265). The authenticity of the receptor is further confirmed by its ability to react with the MSC1 antibody that is specific for MPR 46 protein. Additional evidence for the presence of MPR 46 in molluscs could be obtained by metabolic labeling of mollusc cells Biomphalaria glabrata (Bg cells) with [³⁵S] methionine and cysteine, and passing the labeled membrane extract on phosphomannan gel (at pH 6.5 and 7.0). On elution with mannose 6-phosphate, followed by immunoprecipitation of the column fractions, we identified the putative MPR 46 protein in the Bg cells. When Bg cell MPR 46 was deglycosylated along with chicken MPR 46 (control) both species yielded a single polypeptide corresponding to molecular mass of 26 kDa, suggesting that both contain the same receptor protein.     

Profiling Schistosoma mansoni development using serial analysis of gene expression (SAGE)

Despite the widespread use of chemotherapy and other control strategies over the past 50years, transmission rates for schistosomiasis have changed little. Regardless of the approach used, future control efforts will require a more complete understanding of fundamental parasite biology. Schistosomes undergo complex development involving an alteration of parasite generations within a mammalian and freshwater molluscan host in the completion of its lifecycle. Little is known about factors controlling schistosome development, but understanding these processes may facilitate the discovery of new control methods. Therefore, our goal in this study is to determine global developmentally regulated and stage-specific gene expression in Schistosoma mansoni using serial analysis of gene expression (SAGE). We present a preliminary analysis of genes expressed during development and sexual differentiation in the mammalian host and during early larval development in the snail host. A number of novel, differentially expressed genes have been identified, both within and between the different developmental stages found in the mammalian and snail hosts.     

Uptake of Mineral Particles by Biomphalaria glabrata (Say.) (Pulmonata,Mollusca) and its Importance for Growth Rate and Egg Production

The ingestion of mineral particles by Biomphalaria glabrata is proved and the importance of these particles in the diet of the snails is investigated. The number of particles ingested rises with the size of the snails and they are retained in quantity in the alimentary tract when an adequate supply is not provided. The size of grit found in the gut differs widely from the mixture offered. Mineral particles are ingested actively and selectively. Quantitative investigations comparing snails cultured with and without sand showed a slight advantage concerning growth and reproduction for snails cultured with sand.     

Capacity of irradiated echinostome sporocysts to protect Schistosoma mansoni in resistant Biomphalaria glabrata

Three closely related species of Echinostoma flukes each has a distinctive pattern of protection of Schistosoma mansoni in schistosome-resistant Biomphalaria glabrata host snails. Protection of developing S. mansoni by irradiated E. paraensei sporocysts in the schistosome-resistant snail host was strong; protection induced by irradiated E. lindoense and E. liei sporocysts was weak or not measurable. The capacity of irradiated E. paraensei sporocysts to interfere with the host's innate anti-schistosome response also differed between strains of B. glabrata. Protection of S. mansoni strain Lc-1 was greater in B. glabrata strain 10-R2 than it was in strain M-RLc snails. Irradiated E. paraensei sporocysts also induced a different response to the two schistosome strains in a single host strain. Irradiated E. paraensei sporocysts induced in B. glabrata 10-R2 snails a stronger protection of S. mansoni strain PR-1 than of strain Lc-1. Exposure of each snail to the irradiated E. paraensei miracidia usually protected the following challenge schistosome infection better when 30 rather than 10 irradiated echinostome miracidia were used.