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

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

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.

Characterization of adenosine deaminase (ADA) in Hemolymph of Biomphalaria glabrata

A adenosina é uma molécula sinalizadora de muitos eventos celulares. A adenosina desaminase (ADA) é enzima chave para o controle dos níveis intra e extra celulares de adenosina. A atividade da ADA foi detectada em hemolinfa de B. glabrata e suas condições ótimas de ensaio foram determinadas experimentalmente. A variação do pH de 6,2 até 7,8 não causou mudança significativa na atividade. O Km aparente foi de 734 µmoles L-1, usando adenosina como substrato. A maior atividade foi encontrada usando 37ºC como temperatura de incubação. As condições de ensaio padrão foram então estabelecidas como sendo 15 minutos de tempo de incubação, 0,4 µL de hemolinfa por ensaio, pH 6.8 e 37ºC de temperatura de incubação. A enzima apresentou atividades de 834 ± 67 µmols.min-1.L-1 (25ºC) e 2029 ± 74 µmols.min-1.L-1 (37ºC), em torno de 40 e 100 vezes maiores que os níveis encontrados em soro de humanos sadios. Em temperaturas superiores, essa atividade cai 20% a 43ºC e 70% a 50ºC, em 15 minutos. A ADA perde 26 a 78% de sua atividade quando a hemolinfa é pré-incubada a 50ºC de 2 a 15 minutos, respectivamente. Considerando os altos níveis de ADA encontrados pode-se inferir que, em animais sadios e alimentados, a adenosina é mantida em baixas concentrações na hemolinfa. Tendo a atividade da enzima permanecido constante frente à larga faixa de pH testada, sugere-se que a ADA pode atuar com eficiência mesmo em situações adversas que determinem variações no pH da hemolinfa.

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath: deferens vans and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This techniques is based on the amplification of the internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mn I, Hae III, RSA I, Hpa II and AluI. The restriction patterns obtained with Dde I presented the best profile for separation of the four species of Biomphalaria. The profiles obtained will all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Partial purification of an endoglucanase from Biomphalaria glabrata

Previous studies have shown that Biomphalaria glabrata contains a complete cellulolytic system which includes an endoglucanase, an exoglucanase and a ß-glucosidase. In the present report, a scheme for the purification of the endoglucanase from this invertebrate is proposed. Two major problems were encountered during the study: 1) the presence of a green-brownish pigment, which could not be eliminated by thermal shock or ammonium sulfate precipitation and 2) relative instability of enzymatic activity. Various alternatives were tested and the best sequence of steps was: 1) a sample of the crude extract, obtained by homogenization of the digestive glands in 50 mM Tris-HCl buffer, pH 8.4, and ultracentrifugation, was applied to a Q-Sepharose FPLC column (50 mM Tris-HCl buffer, pH 8.4; 10 mm x 22.2 cm column; flow rate 1.5 ml/min; 0.1 to 0.5 M NaCl gradient); 2) the eluate peak containing activity was dialyzed, lyophilized and eluted from a Superdex-75 gel filtration FPLC column (50 mM ammonium acetate buffer, pH 4.8; 16 mm x 60 cm column; flow rate 1.0 ml/min). A low degree of purification (about 36-fold) and recovery (about 12 percent) were observed, probably due to enzyme instability. SDS-electrophoresis of the active fraction showed a major peak of 30 kDa. In order to improve the purification scheme, further studies are required to stabilize this enzyme during purification and storage