Aspectos morfológicos e histológicos do complexo peniano e vagina de biomphalaria tenagophila tenagophila (Mollusca: planorbidae) / Morphological and histological aspects of the penile and vagina complex of biomphalaria tenagophila tenagophila (Mollusca: planorbidae)

O gênero Biomphalaria (Preston, 1910), da família Planorbidae, tem importância médica no Brasil, pois inclui três espécies suscetíveis ao trematódeo Schistosoma mansoni Sambon, 1907: Biomphalaria glabrata (Say, 1818), Biomphalaria tenagophila tenagophila (d'Orbigny, 1935) e Biomphalaria straminea (Dunker, 1848). A correta determinação destas espécies depende da observação de caracteres taxonômicos confiáveis. Entretanto, as semelhanças existentes entre determinados táxons levaram alguns especialistas a formarem complexos de espécies. No Brasil, existem dois complexos de espécies no gênero Biomphalaria; o complexo Biomphalaria tenagophila proposto por Spatz e colaboradores (1999) e o complexo Biomphalaria straminea proposto por Paraense (1988), cujos táxons, pertencentes a cada um deles, foram assim agrupados devido às semelhanças morfológicas e genéticas. Dos três táxons que integram o complexo Biomphalaria tenagophila, B. t. tenagophila é a única espécie suscetível ao S. mansoni, tendo grande importância epidemiológica nas regiões sul e sudeste do Brasil (MINISTÉRIO DA SAÚDE, 2014). As semelhanças morfológicas entre os três táxons do complexo Biomphalaria tenagophila podem levar a erros de identificação específica, dificultando o reconhecimento de áreas vulneráveis para transmissão da esquistossomose nas regiões sul e sudeste do país. As estruturas morfológicas de maior valor taxonômico em Biomphalaria estão relacionadas ao sistema reprodutor. No presente estudo, propomos estudar os aspectos morfológicos e histológicos do complexo peniano e região da vagina de B. t. tenagophila para fornecer novos subsídios que auxiliem na sistemática de Planorbidae. Foram coletados moluscos B(AU)

Geographic Information System-based association between the sewage network, geographical location of intermediate hosts, and autochthonous cases for the estimation of risk areas of schistosomiasis infection in Ourinhos, São Paulo, Brazil

Abstract INTRODUCTION: Ourinhos is a municipality located between the Pardo and Paranapanema rivers, and it has been characterized by the endemic transmission of schistosomiasis since 1952. We used geospatial analysis to identify areas prone to human schistosomiasis infections in Ourinhos. We studied the association between the sewage network, co-occurrence of Biomphalaria snails (identified as intermediate hosts [IHs] of Schistosoma mansoni), and autochthonous cases. METHODS: Gi spatial statistics, Ripley's K12-function, and kernel density estimation were used to evaluate the association between schistosomiasis data reported during 2007-2016 and the occurrence of IHs during 2015-2017. These data were superimposed on the municipality sewage network data. RESULTS: We used 20 points with reported IH; they were colonized predominantly by Biomphalaria glabrata, followed by B. tenagophila and B. straminea. Based on Gi statistics, a significant cluster of autochthonous cases was superimposed on the Christoni and Água da Veada water bodies, with distances of approximately 300 m and 2200 m from the points where B. glabrata and B. straminea were present, respectively. CONCLUSIONS: The residence geographical location of autochthonous cases allied with the spatial analysis of IHs and the coverage of the sewage network provide important information for the detection of human-infection areas. Our results demonstrated that the tools used for direct surveillance, control, and elimination of schistosomiasis are appropriate.

A preliminary study on the distribution of breeding sites of BIOMPHALARIA GLABRATA in the municipality of peri mirim, a low endemicity area for schistosomiasis in northeast Brazil

Information on areas colonized by snails that transmit Schistosoma mansoni is essential for planning schistosomiasis control measures. Therefore, the purpose of this study was to map the natural breeding sites of Biomphalaria spp. in the municipality of Peri Mirim, Maranhão, Brazil. The snails were manually collected and the breeding sites were geo-referenced (seven in the urban area and five in flooded fields), from November 2017 to March 2018. In the laboratory, the snails were examined for the presence of larval stages of S. mansoni identified by morphology and internal anatomy analysis. While no snails were found in the urban area, sixteen B. glabrata were collected in the flooded fields. No S. mansoni or other trematode infections were detected. Wild rodents, most likely Holochilus sciureus that could act as S. mansoni reservoirs, were also found in the flooded fields. These data indicate possible risk areas for further outbreaks of schistosomiasis transmission in the municipality of Peri Mirim.

Interaction of Biomphalaria cells in primary cultures with Schistosoma mansoni sporocysts

Abstract INTRODUCTION: Biomphalaria snails may display varying levels of susceptibility to Schistosoma mansoni infection. We have been developing an in vitro model to study the interaction between the snail and the parasite, using tissue-derived cell cultures from Biomphalaria. METHODS: The digestive gland- and kidney-derived cells from primary cultures of resistant (B. tenagophila Taim) and susceptible (B. tenagophila HM and B. glabrata BH) strains of Biomphalaria were exposed to S. mansoni sporocysts. RESULTS: S. mansoni sporocysts were surrounded and encapsulated exclusively by cells derived from the digestive gland (DG) of B. tenagophila Taim. The process was followed by a marked decrease in the number of free sporocysts in the culture medium. The morphological characteristics of DG-derived cells in culture have been described. CONCLUSIONS: Cells derived from DG (but not SK) primary cultures of B. tenagophila Taim may participate in S. mansoni sporocyst control.

New epidemiological profile of schistosomiasis from an area of low prevalence in Brazil

Abstract INTRODUCTION: Schistosomiasis, caused by infection from Schistosoma mansoni, is a disease that represents an important public health problem for Brazil, especially for states in the Northeast region. Thus, the aim of this study is to present a new epidemiological profile for the disease in a municipality with low prevalence in the state of Alagoas, Brazil. METHODS: A cross-sectional study was conducted through a coproparasitological and malacological survey. A structured questionnaire was applied to the study participants to survey possible risk factors and a spatial analysis (kernel density) was used to measure the risk of infection. RESULTS: Of the 347 participants, 106 (30.5%) were infected by Schistosoma mansoni, most of them from the urban area of the municipality (68.9%; 73/106). A 3-fold risk of infection was found for individuals living in the urban area and a risk of 2.15 times for self-declared farmers. Biomphalaria glabrata and B. straminea were the species found in the municipality, but no animals were diagnosed as infected by the parasite. Spatial analysis showed a random distribution of vectors and human cases of the disease, and the formation of two clusters of human cases in the urban area was seen. CONCLUSIONS: A new epidemiological profile for schistosomiasis from S. mansoni infection was presented in a municipality of low endemicity: a high proportion of positive individuals in the urban area; presence of snails without positive diagnosis for S. mansoni infection; random distribution of vectors and human cases; and absence of association between classical risk factors and human infection.

Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata

BACKGROUND Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5' end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.

Ocotea pulchellaas an alternative against schistosomiasis: chemical analysis, development of nanoemulsion and biological control activity / Ocotea pulchellacomo una alternativa ante esquistosomiasis: análisis químico, desarrollo de una nanoemulsión y actividad en control biológico

The aim of this work was to evaluate the potential of the essential oil (EO) from Ocotea pulchella leaves as an alternative in the control of schistosomiasis. It was tested O. pulchella EO nanoformulation to assess its activity against adult Biomphalaria glabrata, their spawning and Schistossoma mansoni cercariae. Additionally, the EO chemical composition was investigated by gas-chromatography. Nanoemulsion were elaborated by the low energy method. The adult mollusks, their spawning and cercariae were placed in contact with nanoemulsion to calculate lethal concentrations. Myristicin, bicyclogermacrene and α-Pinene were the main substances in the EO. Nanoemulsion caused mortality of adult B. glabrata, its egg embryos and S. mansoni. These results suggest the use of this nanoemulsion as an alternative in the control of the schistosomiasis cycle.

El objetivo de este trabajo fue evaluar el potencial de los aceites esenciales (AE) de las hojas de Ocotea pulchellacomo una alternativa en el control de esquistosomiasis. Se probó una nanoformulación de AE de O. pulchellapara evaluar su actividad ante adultos de Biomphalaria glabrata, sus huevos y cercarías de Schistossoma mansoni. La nanoemulsión fue elaborada por el método de baja energía. Los moluscos adultos, sus huevos y cercarías se colocaron en contacto con la nanoemulsión para calcular concentraciones letales. Los compuestos mayoritarios en el AE fueron miristicina, biciclogermacreno y α-pineno. La nanoemulsión causó mortalidad en adultos de B. glabrata, sus huevos y a S. mansoni. Los resultados sugieren el uso de esta nanoemulsión como una alternativa en el control del ciclo de esquistosomiasis.

Influence of food type and calcium supplementation on growth, oviposition and survival parameters of Biomphalaria glabrata and Biomphalaria straminea

Schistosomiasis is a parasitic disease caused by Schistosoma mansoni whose intermediate host is the snail of the genus Biomphalaria. This snail is geographically widespread, making the disease a serious public health problem. The purpose of this study was to analyze the growth, reproductive rates and mortality of B. glabrata and B. straminea in different calcium concentrations and food types. Freshly hatched snails stored in aquariums under different dietary and calcium supplementation programs were studied. Under these conditions, all planorbids survived, so there was no mortality rate and 79,839 eggs of B. straminea and 62,558 eggs of B. glabrata were obtained during the 2 months of oviposition. The following conditions: lettuce + fish food and lettuce + fish food + powdered milk resulted in the highest reproductive rates. In addition, supplementation with calcium carbonate and calcium sulfide in three different concentrations did not significantly influenced the amount of eggs or ovigerous masses. Thus, this study shows that changes in diet are crucial for the survival/oviposition of these planorbids, being an important study tool for population control. Calcium is also a key factor in these conditions, but more work is necessary to better assess its effect on snail survival.

Ocorrência de Biomphalaria straminea em corpos hídricos do município de Picos, Piauí: avaliação do risco de transmissão da esquistossomose mansoni / Occurrence of Biomphalaria straminea in water bodies in the municipality of Picos, Piauí: risk assessment of transmission of schistosomiasis mansoni

A esquistossomose é uma parasitose de veiculação hídrica causada por trematódeos do gênero Schistosoma, transmitida por caramujos planorbídeos do gênero Biomphalaria. O presente estudo teve por objetivo identificar e analisar os caramujos do gênero Biomphalaria encontrados em coleções hídricas do perímetro urbano de Picos quanto à infecção por S. mansoni. O inquérito foi realizado em sete coleções hídricas durante os meses de julho, agosto e setembro de 2019. Os caramujos coletados foram encaminhados ao laboratório e separados em número de até 10 indivíduos em frascos identificados. Para detectar a infecção por S. mansoni foram realizados os testes de exposição à luz, nested-PCR e SmMIT-LAMP. Foram coletados 1.214 espécimes de Biomphalaria straminea nas sete coleções hídricas, com a seguinte distribuição: São José, n = 667 (54,94%); Centro, n = 273 (22,49%); Canto da Várzea, n = 108 (n = 8,90%); Passagem das Pedras, n = 102 (8,40%); Malvas, n = 32 (2,64%); Ipueiras, n = 12 (0,98%) e Catavento, n = 20 (1,65%). Não foram encontrados caramujos infectados por S. mansoni pelas técnicas de exposição à luz e pela nested-PCR. Através da técnica de SmMIT-LAMP houve positividade em uma amostra de B. straminea analisada em conjunto, sendo possível caracterizar a Estação São José como um foco potencial de transmissão da esquistossomose mansoni. Em relação à abundância de moluscos vetores entre os meses de coletas, o mês de setembro foi o que se destacou pelo número de exemplares coletados, com 449 indivíduos (40%). Pela técnica de exposição à luz foi possível identificar quatro tipos cercarianos diferentes: dois tipos de distoma brevifurcada faringeada, um tipo de distoma longifurcada faringeada e um de distoma brevifurcada afaringeada. Demonstramos que a combinação entre o Teste de Eliminação Clássica e métodos moleculares fornecem informações mais fidedignas acerca da positividade por S. mansoni em caramujos Biomphalaria, contribuindo assim para a detecção de focos de transmissão de esquistossomose.

Schistosomiasis is a waterborne parasitic disease caused by trematodes of the genus Schistosoma, transmitted by planorbid snails of the genus Biomphalaria. The present study aimed to identify and analyze Biomphalaria snails found in water collections in the urban area of Picos, state of Piauí, for infection by S. mansoni. The survey was carried out in seven collecting sites from July to September 2019. The collected snails were sent to the laboratory and separated in a number of up to 10 individuals in identified jars. To detect S. mansoni infection, light exposure, nested-PCR and SmMIT-LAMP tests were performed. In all 1,214 specimens of Biomphalaria straminea were collected as the following: São José, n = 667 (54.94%); Centro, n = 273 (22.49%); Canto da Várzea, n = 108 (n = 8.90%); Passagem das Pedras, n = 102 (8.40%); Malvas, n = 32 (2.64%); Ipueiras, n = 12 (0.98%) and Catavento, n = 20 (1.65%). No snails infected by S. mansoni were found by light exposure and nested-PCR techniques. However, SmMIT-LAMP detected positivity in a sample of B. straminea specimens, making it possible to characterize the São José colleting site as a potential focus of schistosomiasis mansoni transmission. In relation to the abundance of vector mollusks between the months of collection, September was the one that stood out for the number of samples collected, with 449 individuals (40%). The light exposure technique, made possible to identify four different cercarian types: two types of distoma pharyngeal brevifurcated, one type of distoma pharyngeal longifurcated and one of distoma pharyngeal brevifurcated. We highlight that the combination of Classical Elimination Test and molecular methods provide more reliable information about positivity for S. mansoni in Biomphalaria snails, contributing to the characterization of the foci of schistosomiasis transmission.

Efeitos das nanopartículas de Óxido de Zinco (NPs ZnO) em alguns aspectos da sobrevivência, reprodução e desenvolvimento de Biomphalaria glabrata (Say, 1818) (Gastropoda: Pulmonata) / Effects of Zinc Oxide Nanoparticles (NPs ZnO) on some aspects of the survival, reproduction and development of Biomphalaria glabrata (Say, 1818) (Gastropoda: Pulmonata)

A nanotecnologia representa uma tendência chave na ciência e tecnologia do século XXI. As nanopartículas de óxido de zinco (NPs de ZnO) estão entre as mais utilizadas devido a sua capacidade de refletir, absrover e dispersar a radiação ultravioleta. A partir da sua larga utilização em fotoprotetores, estas nanopartículas podem ser liberadas em efluentes e águas residuais e os ecossistemas aquáticos se tornam alvos diretos da contaminação por estes xenobióticos. A avaliação do impacto biológico das NPs de ZnO se torna de grande importância na área de Ecotoxicologia. Neste trabalho, foram analisados os efeitos das NPs de ZnO na sobrevivência, reprodução, sistema imune e nas alterações bioquímicas por estresse oxidativo em Biomphalaria glabrata. As nanopartículas foram caracterizadas quanto ao tamanho, índice de polidispersão e potencial zeta. O tamanho médio nas NPs de ZnO utilizadas neste estudo foi de 34,2 nm. Foram realizados testes agudos, 48h de exposição para determinar as concentrações letais e subletais (CL10, CL30, CL50, CL90). Para avaliar os efeitos das NPs de ZnO na atividade reprodutiva, caramujos B. glabrata, foram expostos a concentrações subletais em diferentes períodos (7 e 21 dias). Após a exposição, a fecundidade e fertilidade de B. glabrata foi afetada de forma significativa em relação ao grupo controle.

Os resultados demonstraram um efeito tempodependente e concentração-dependente. A partir destes resultados, fez-se importante a realização de um teste crônico, 4 semanas de exposição, para avaliar o potencial de reversibilidade da atividade reprodutiva de caramujos expostos as concentrações de 1 e 2 mg L-1 . Os caramujos só apresentaram recuperação da atividade reprodutiva após a exposição a menor concentração, 1 mg L-1 . Testes agudos também foram realizados a fim de avaliar a viabilidade celular e quantificar espécies reativas de oxigênio (Peróxido de Hidrogênio) envolvidas no estresse oxidativo em diferentes tecidos (massa cefalopediosa e visceral, hemolinfa e glândula digestiva) de B. glabrata. A exposição aguda às NPs de ZnO alterou o perfil de hemócitos dos caramujos, com uma tendência ao aumento na população de granulócitos. Concentrações subletais (1, 2 e 6 mg L-1 ) foram capazes de induzir um aumento no nível de peróxido de hidrogênio em amostras de massa cefalopediosa e visceral (partes moles), glândula digestiva e hemolinfa. Este trabalho também avaliou o efeito das NPs de ZnO em diferentes estágios de vida de B. glabrata. Neonatos e massas ovígeras foram expostos a concentrações subletais e foram realizados testes agudos e crônicos. Os resultados indicaram que os estágios iniciais de vida apresentam uma sensibilidade maior do que os caramujos adultos.

A alta taxa de mortalidade dos embriões e neonatos pode siginifcar uma alteração na população desta espécie e consequentemente, implicações ecológicas. Os dados obtidos evidenciam a necessidade da utilização de endpoints mais sensíveis que a mortalidade de organismos adultos. A alteração de processos fisiológicos primordiais como a atividade reprodutiva, não representam efeitos apenas para a população de moluscos B. glabrata, mas podem representar um efeito em todo o ecossistema. Isso porque os moluscos estão associados a diferentes níveis tróficos e representam um elo na cadeia trófica. Alem disso, são responsáveis pela ciclagem de nutrientes, sendo considerados detritívoros-herbivoros. O protocolo básico deste trabalho poderá vir a ser empregado como ferramenta importante, já que as avaliações ecotoxicológicas dispõem de metodologias eficientes para análise da saúde ambiental e dos ecossistemas aquáticos. (AU)

Schistosomiasis in the middle paranapanema river region, state of São Paulo, Brazil: does it matter today for public health?

Abstract INTRODUCTION: The Middle Paranapanema watershed is known for the transmission of schistosomiasis, and there have been autochthonous cases since 1952. This study aimed to describe this disease in space and time and evaluate its current importance as a public health problem. METHODS: Thematic maps showing the risk areas for transmission of schistosomiasis, using scan statistics, and flow maps were created in the period 1978-2016. Incidence was calculated, and the existence of spatial dependence between autochthonous and imported cases was evaluated using Ripley's K12-function. Species of snails were identified in high-risk clusters. RESULTS: A total of 1,511 autochthonous cases were reported in eight of the 25 municipalities in the study area, of which 92.8% occurred in Ourinhos. A total of 2,189 imported cases were reported (27% in Ourinhos and 20% in Assis), mainly originating in the states of Paraná and Minas Gerais. Clusters of autochthonous and imported cases with higher risk were identified in Ourinhos, Assis and Ipaussu. However, over the years, the cases began to occur in low density in Ourinhos and no longer in other municipalities in the region. The cluster detected in the period 2007-2016 in Ourinhos still has risk for the transmission of schistosomiasis. K12-function analysis indicated positive spatial dependence between autochthonous and imported cases. CONCLUSIONS: The study showed that, currently, schistosomiasis as a public health problem in Middle Paranapanema is restricted to Ourinhos. This fact may be related to the presence of Biomphalaria glabrata at a specific point and low coverage of basic sanitation.

Prevalence of infection of biomphalaria glabrata by schistosoma mansoni and the risk of urban schistosomiasis mansoni in Salvador, Bahia, Brazil

Abstract INTRODUCTION: Biomphalaria glabrata is considered to be responsible for the incidence of schistosomiasis in Brazil. Therefore, surveillance of areas where schistosomiasis is prevalent is fundamental for public health planning. This study was aimed to evaluate B. glabrata populations in water bodies of the city of Salvador, determine their distribution, estimate the prevalence of Schistosoma mansoni infections, characterize shed cercariae, and identify transmission foci. METHODS: Malacological surveys were carried out in 17 water collections from Salvador. Snail species were identified based on shell and mantle characteristics. Snails were evaluated for S. mansoni infection by exposure to light and via real time polymerase chain reaction (qPCR) using S. mansoni-18S rRNA subunit specific primers. RESULTS: 1,403 B. glabrata were collected. Classical cercarial shedding indicated that 5 snails (0.4%) were positive for S. mansoni. A higher prevalence of infections was found in Horta de Saramandaia (5.5%) and Lagoa do IAT (1.9%). Non-Schistosoma larvae, such as Xiphidiocercaria, Strigeidae, Spirorchiidae and Clinostomidae, were observed in 3.2% of the snails. S. mansoni DNA was detected in 6.2% snails via qPCR. CONCLUSIONS: B. glabrata is widely distributed in Salvador, as indicated by 7 water collections associated with a risk of schistosomiasis transmission. To our knowledge, this is the first study to identify B. glabrata eliminating cercariae of Clinostomidae, Strigeidae, and Spirorchiidae in Salvador. We propose that qPCR may be employed in combination with classical cercarial shedding. Estimating S. mansoni prevalence in snails by only considering the results of light exposure method classical into account may underestimate the problem.

Nanoemulsion containing essential oil from Xylopia ochrantha Mart. produces molluscicidal effects against different species of Biomphalaria (Schistosoma hosts)

BACKGROUND This work describes a chemical study of the essential oil from leaves of Xylopia ochrantha, an endemic Annonaceae species from Brazil, and its activity against Biomphalaria species. Considering its poor solubility in aqueous medium, the essential oil was nanoemulsified to evaluate its action on controlling some mollusc species of genus Biomphalaria, snail hosts of Schistosoma mansoni that causes schistosomiasis, which mainly affects tropical and subtropical countries. OBJECTIVES The main aims of this work were to analyse the chemical composition of essential oil from X. ochrantha, and to evaluate the effect of its nanoemulsion on molluscs of genus Biomphalaria and their oviposition. METHODS Chemical analysis was performed by gas chromatography coupled to mass spectrometry. Nanoemulsions were prepared by a low energy method and characterised by particle size and polydispersity index. Biological assays evaluating the mortality of adult species of B. glabrata, B. straminea and B. tenagophila and their ovipositions upon contact with the most stable nanoemulsion during 24 and 48 h were performed. FINDINGS Chemical analysis by mass spectrometry revealed the majority presence of bicyclogermacrene and germacrene D in the essential oil. The formulation with a hydrophilic-lipophilic balance (HLB) of 9.26 was the most suitable for the oil delivery system. This nanoemulsion caused the mortality in B. tenagophila, B. straminea and B. glabarata of different sizes at levels ranging from 50 to 100% in 48 h. Additionally, the formulation could inhibit the development of deposited eggs. CONCLUSION Thus, these results suggest the use of nanoemulsified essential oil from X. ochrantha as a possible alternative in controlling some Biomphalaria species involved in the schistosomiasis cycle.

Padrão de distribuição da diversidade genética molecular e espacial de Biomphalaria spp. e sua relação com a ocorrência da esquistossomose na região do Médio Paranapanema, estado de São Paulo / Distribution pattern of the molecular and spatial genetic diversity of Biomphalaria and its relation with the occurrence of schistosomiasis, region of the Middle Paranapanema, State of São Paulo

A UGRHI-17 da bacia hidrográfica do Médio Paranapanema (São Paulo, Brasil) é reconhecidamente uma área de alta biodiversidade de espécies de Biomphalaria e possui grande vulnerabilidade a acometimentos ambientais e em saúde, no caso da esquistossomose. O objetivo do estudo foi identificar áreas de maior risco para a ocorrência da esquistossomose utilizando dados de transmissão da esquistossomose e de diversidade genética molecular, associando-os às ferramentas geoespaciais, e com isso estabelecer áreas potenciais para a vigilância malacológica e da infecção em coleções de água doce na região do Médio Paranapanema. Os moluscos do gênero de Biomphalaria foram identificados por meio de características morfológicas e moleculares; enquanto os outros grupos taxonômicos (Drepanotrema, Lymnaea, Melanoides, Physa e Pomacea) foram identificados por características conchiológicas ou morfológicas. A análise filogenética das espécies de Biomphalaria foi realizada por meio da análise de sequências dos genes mitocondriais COI, 16S rRNA e COI+16S. As sequências do gene COI referentes ao trecho DNA Barcode foram testadas quanto à similaridade com sequências depositadas no GenBank e analisadas em ABDG, bPTP e GMYC para delimitação de espécies putativas. Na análise espacial foram utilizadas as estatísticas de varredura, Gi e de fluxo. Foram utilizadas as fichas notificação e investigação dos casos de esquistossomose na região de estudo entre 1978-2016. Foi calculado as taxas de incidência e foi avaliado a dependência espacial entre os casos autóctones e importados com a função K12 de Ripley. Foram gerados mapas da distribuição espacial dos caramujos do gênero Biomphalaria; dos casos de esquistossomose ocorridos na região de estudo; e da diversidade genética em haplótipos do gene 16S. Além disso, foi realizada modelagem de nicho para estimar cenários futuros de alteração na distribuição dos caramujos, utilizado o algoritmo de máxima entropia. Foram utilizados os dados de variáveis climáticas e topográficas obtidas no WorldClim, HydroSHEDS e TOPODATA e o modelo climático regional HadGEM2-ES do período de 2041-2060, considerando dois cenários de mudança climática possíveis: RCP2.6 e RCP8.5. Foram identificados aglomerados de alto risco para ocorrência de esquistossomose em Ourinhos, Assis e Ipaussu. Entretanto, ao longo dos anos, os casos passaram a ocorrer em baixa densidade em Ourinhos e deixaram de ocorrer nos demais municípios da região. Dos caramujos coletados, 75.5% eram Biomphalaria, 11.2% Drepanotrema e 13.3% de outros gêneros não planorbídeos. O modelo de máxima entropia mostrou que há probabilidade futura da espécie B. glabrata permanecer nos municípios de Ourinhos e Assis, e uma probabilidade em torno de 50% de a espécie expandir sua colonização a corpos de água doce de outros municípios da região de estudo, isto em função das mudanças climáticas. Os resultados para B. straminea mostram que esta espécie tem maior probabilidade de expansão de colonização no futuro, especialmente nos municípios próximos a Ourinhos. A análise filogenética mostrou árvores com cinco ramos monofiléticos com alto suporte estatístico. A diversidade de haplótipos está distribuída de forma diferente em cada um dos cinco taxa analisados. Conclui-se, em um dos resultados deste trabalho, que atualmente a esquistossomose, como problema de saúde pública no Médio Paranapanema, está restrita a Ourinhos. Tal fato pode estar relacionado à presença de B. glabrata em pontos específicos e à cobertura deficiente do saneamento básico. Desta forma, o estudo contribuiu para eleger áreas prioritárias para o combate aos caramujos e à doença para evitar ou reduzir transmissões futuras nesta região

The UGRHI-17 of the Middle Paranapanema watershed (São Paulo, Brazil) is recognized as an area of high biodiversity of Biomphalaria species and great vulnerability to environmental and health impacts for schistosomiasis. The objective of the study is to identify areas of greatest risk for the occurrence of schistosomiasis using transmission data from schistosomiasis and molecular genetic diversity, associating them with the geospatial tools, and thereby establishing potential areas for malacological surveillance and infection in collections of freshwaters in the region of Middle Paranapanema. Molluscs of the genus Biomphalaria were identified by morphological and molecular characteristics; while the other taxonomical groups (Drepanotrema, Lymnaea, Melanoides, Physa and Pomacea) were identified through conchiological or morphological characteristics. Molecular genetic analysis of the species was done through sequence analysis of the mitochondrial genes COI, rRNA16S and COI+16S. The COI gene sequences related to DNA Barcode portions were tested for similarity to sequences deposited in GenBank and analyzed ABDG, BPTP and GMYC for delimiting putative species. In the spatial analysis we used the scan statistics, Gi and flow maps. Reporting and investigation records of cases schistosomiasis in the study regions between 1978 and 2016 were used. Incidence were calculated and the existence of spatial dependence between autochthonous and imported cases was evaluated using Ripley's K12-function. Maps of the spatial distribution of snails of the genus Biomphalaria; cases of schistosomiasis occurred in the study region; and the genetic diversity in haplotypes of the 16S gene were generated. In addition, the ecological niche modeling to estimate future scenarios of alteration in the distribution of snails, used the maximum entropy algorithm in MaxEnt software. Climate and altitude data obtained from WorldClim, HydroSHEDS and TOPODATA and the regional climate model HadGEM2-ES from the period of 2041-2060 were used, considering two possible scenarios of climate change: Representative Concentration Pathways - RCP2.6 and RCP8.5. High-risk clusters were identified for the occurrence of schistosomiasis in Ourinhos, Assis and Ipaussu. However, over the years, cases occurred in low density in Ourinhos and ceased to occur in other municipalities in the region. Of the snails collected, 75.5% were Biomphalaria, 11.2% Drepanotrema and 13.3% of other non-planorbid genera. The maximum entropy model showed that B. glabrata is a future likely to remain in the municipality of Ourinhos and Assis and a probability around 50% of species to expand their colonization to freshwater bodies of other municipality of the study region, due to the climatic changes. The results for B. straminea showed that this is the species most likely to expand colonization in the future, especially in the municipalities near Ourinhos. The phylogenetic analysis showed trees with five monophyletic branches with high statistical support. The diversity of haplotypes is distributed differently at each of the five taxa analyzed. As one of the results of this work it was concluded that, currently, schistosomiasis as a public health problem in the Middle Paranapanema is restricted to Ourinhos. This may be related to the presence of B. glabrata at specific point and poor coverage of basic sanitation. In this way, the study contributed to the selection of priority areas for combating snails and disease in order to avoid or reduce future transmissions in this region

Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata

BACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.

Development of a natural molluscicide prototype kit (MoluSchall) for the control of schistosomiasis mansoni transmission

Abstract INTRODUCTION In Brazil, Biomphalaria glabrata, B. tenagophila, and B. straminea are intermediate hosts of Schistosoma mansoni, the etiological agent of schistosomiasis mansoni. Molluscicide use is recommended by the WHO for controlling the transmission of this parasite. Euphorbia milii latex has shown promising results as an alternative molluscicide. Thus, a natural molluscicide prototype kit based on freeze-dried E. milii latex was developed and evaluated against Biomphalaria spp. METHODS E. milii latex was collected, processed, and lyophilized. Two diluents were defined for freeze-dried latex rehydration, and a prototype kit, called MoluSchall, was produced. A stability test was conducted using prototype kits stored at different temperatures, and a toxicity assay was performed using Danio rerio. Additionally, MoluSchall was tested against B. glabrata under semi-natural conditions according to defined conditions in the laboratory. RESULTS MoluSchall was lethal to three Brazilian snail species while exhibiting low toxicity to D. rerio. Regardless of storage temperature, MoluSchall was stable for 24 months and was effective against B. glabrata under semi-natural conditions, with the same LD100 as observed under laboratory conditions. CONCLUSIONS MoluSchall is a natural, effective, and inexpensive molluscicide with lower environmental toxicity than existing molluscicides. Its production offers a possible alternative strategy for controlling S. mansoni transmission.

Accumulation of oocysts of Cryptosporidium parvum in Biomphalaria glabrata (Pulmonata: Planorbidae) under experimental conditions

Abstract INTRODUCTION: Cryptosporidium oocysts are easily transported to various aquatic environments. The objective of this study was to evaluate B. glabrata mollusks exposed to food containing C. parvum oocysts. METHODS: Six experimental groups were used with B. glabrata either exposed or not to C. parvum oocysts. Microscopic and molecular diagnostics were conducted in water samples and tissues of B. glabrata. RESULTS: By light microscopy, C. parvum oocysts were identified in the water of the exposed groups. C. parvum DNA was not detected in water but was detected in tissue samples. CONCLUSIONS: Further studies should be conducted under natural conditions.

Biological screening of extracts from leaf and stem bark of Croton floribundus Spreng. (Euphorbiaceae) / Triagem biológica de extratos das folhas e caules de Croton floribundus Spreng. (Euphorbiaceae)

Abstract This work describes the preliminary evaluation of cytotoxic, antimicrobial, molluscicidal, antioxidant and anticholinesterase activities from leaf (LECF) and stem bark alcoholic extracts (BECF) of the species Croton floribundus Spreng. (Euphorbiaceae), popularly known as capixingui or tapixingui. BECF presented significant toxicity (LC50 = 89.6 μg/ml) in the Artemia salina Leach, 1819 (Crustacea: Branchiopoda) bioassay, whereas LECF did not show activity (LC50 > 1000 μg/ml). From DPPH method, the values of IC50 for the LECF and BECF were 61.2 μg/ml and 62.2 μg/ml, respectively, showing that C. floribundus has an expressive antioxidant activity. Antimicrobial susceptibility was evaluated by microdilution technique and only BECF was active against Staphylococcus aureus (MIC = 39.6 μg/ml). The extracts did not present molluscicidal activity against snail Biomphalaria glabrata Say, 1818 (Gastropoda: Planorbidae). Both extracts revealed the presence of several components with an inhibiting capacity of acetylcholinesterase enzyme on the bioautographic assay. C. floribundus showed to be a promising species considering that it exhibited good biological activity in the most assays performed.

Resumo Este trabalho descreve a avaliação preliminar das atividades citotóxica, antimicrobiana, moluscicida, antioxidante e anticolinesterásica de extratos alcoólicos das folhas (LECF) e das cascas do caule (BECF) da espécie Croton floribundus Spreng. (Euphorbiaceae), popularmente conhecida como capixingui ou tapixingui. No bioensaio com Artemia salina Leach, 1819 (Crustacea: Branchiopoda), BECF apresentou toxicidade significante (LC50 = 89,6 µg/ml), enquanto que LECF não apresentou atividade (LC50 > 1000 µg/ml). A partir do método de DPPH, os valores de IC50 para o LECF e BECF foram 61,2 µg/ml e 62,2 µg/ml, respectivamente, evidenciando que C. floribundus tem uma atividade antioxidante expressiva. A susceptibilidade antimicrobiana foi avaliada pela técnica de microdiluição e apenas BECF foi ativo contra Staphylococcus aureus (MIC = 39,6 mg/ml). Os extratos não apresentaram atividade moluscicida contra o caramujo Biomphalaria glabrata Say, 1818 (Gastropoda: Planorbidae). Ambos os extratos revelaram a presença de componentes com capacidade inibidora da enzima acetilcolinesterase no ensaio bioautográfico. C. floribundus mostrou ser uma espécie promissora considerando que exibiu boa atividade biológica na maioria dos ensaios testados.

Histopathological and ultrastructural studies on biomphalaria alexandrina snails infected with schistosoma mansoni miracidia and treated with plant extracts

Background: Biomphalaria alexandrina snails are the intermediate host of Schistosoma mansoni in Egypt

Aim of the work: this study aimed to evaluate the molluscicidal activity of the methanol extract of the plants Anagallis arvensis and Viburnum tinus against B. alexandrina [Normal and S. mansoni infected]

Results: the present results proved high activity for both plant extracts [LC[50] and LC[90] which reached 45and 60 ppm and 38 and 59 ppm for A. arvensis and V. tinus, respectively]. The effect of sub-lethal concentration, 1/2 LC[5], of the two plant extracts [26 and 11 ppm, respectively] affected B. alexandrina survival rate to be in the following order, control > V. tinus treated > A. arvensis treated > infected > infected-A. arvensis treated > infected- V. tinus treated. On the other hand, exposure to those sub-lethal doses caused considerable reduction in the infection percentages. In addition, the histopathological effects of the examined sub-lethal concentrations on hepatopancreatic tubules of the treated snails showed cells vacuolation, presence of hyaline substances filled the lumens of the tubules and necrotic focal areas in case of A. arvnsis and vacuolar degeneration with the necrotic changes in case of V. tinus. While, alterations in the hermaphrodite glands of the treated snails included: degeneration and necrotic changes in the acini. The severity of lesions was progressed with infection as a result of invading of snail tissue by developmental stages of the S. mansoni cercariae. The ultrastructural micrographs were used to explain and confirm the recorded histopathological alterations in the hermaphrodite glands of the infected-treated snails. In comparison with the control and infected snail groups, infected-treated snails showed degeneration with severe deformation and destruction in their reproductive units, degeneration in developmental stages tissues of S. mansoni cercariae and accumulation of the toxic agents

Conclusion: the two examined plants, A. arvensis and V. tinus plant extracts showed high activity against B. alexandrina and provide a considerable scope in exploiting local indigenous resources for snail's molluscicidal agents. The sub-letal concentrations, 1/2 LC[5], of the two plant extracts caused a considerable reduction in survival rate and infection rate among S. mansoni infected snails. Histopathological changes in the digestive glands showed cells vacuolation, hyaline substance filled lumens of the tubules and necrotic focal areas in the digestive glands. Histopathological effects explained and confirmed by TEM images showed degeneration with severe deformation and destruction in the reproductive units

Distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni nos estados do Paraná, Minas Gerais, Bahia, Pernambuco e Rio Grande do Norte, 2012-2014 / Distribución geográfica de los huéspedes intermedios de Schistosoma mansoni en los estados de Paraná, Minas Gerais, Bahia, Pernambuco y Rio Grande do Norte, Brasil, 2012-2014 / Geographical distribution of intermediate hosts of Schistosoma mansoni in the states of Paraná, Minas Gerais, Bahia, Pernambuco and Rio Grande do Norte, Brazil, 2012-2014

Objetivo: descrever a distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni em cinco estados brasileiros. Métodos: estudo transversal; foram selecionados municípios dos estados do Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) e Rio Grande do Norte (98), nos anos de 2012 a 2014; esses municípios foram escolhidos por não possuírem registros atualizados da presença de caramujos hospedeiros intermediários de S. mansoni; moluscos foram capturados, taxonomicamente identificados e examinados para verificação de cercárias de S. mansoni. Resultados: os trabalhos foram realizados em 427 municípios (99,5% dos 429 selecionados); foi registrada presença de moluscos em 300 (70,2%) municípios e a ocorrência de Biomphalaria glabrata em 62 (21%) municípios, B. straminea em 181 (60%), B. tenagophila em três (1%); associação de B. glabrata/B. straminea foi encontrada em 53 (18%), e de B. glabrata/B. tenagophila em um (0,3%). Conclusão: os registros de B. glabrata, B. straminea e B. tenagophila estão de acordo com a distribuição conhecida.

Objetivo: describir la distribución geográfica de los hospedadores intermediarios de Schistosoma mansoni en cinco estados brasileños. Métodos: estudio epidemiológico transversal; el estudio fue realizado en municipios de los estados de Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) y Rio Grande do Norte (98), entre 2012 y 2014; estos municipios fueron elegidos por no poseer registros actualizados de la presencia de caracoles vectores de S. mansoni; los moluscos fueron capturados, taxonómicamente identificados y examinados para la verificación de cercarias de S. mansoni. Resultados: los trabajos fueron realizados en 427 municipios (99,5% de 429 municipios seleccionados); fue registrada presencia de moluscos en 300 (70,2%) municípios; la presencia de Biomphalaria glabrata fue registrada en 62 (21%) municipios, B. straminea en 181 (60%) y B. tenagophila en três (1%); se observó asociación de B. glabrata con B. straminea en 53 (18%) y de B. glabrata con B. tenagophila en uno (0,3%). Conclusión: los registros de Biomphalaria están de acuerdo con la distribución conocida.

Objective: to describe the geographical distribution of intermediate hosts of Schistosoma mansoni in five Brazilian states. Methods: this was a descriptive cross-sectional study; municipalities were selected in the states of Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) , and Rio Grande do Norte (98), for the period 2012 to 2014; these municipalities were chosen because they did not have current records of the presence of snails vectores de S. mansoni. The molluscs were captured and taxonomically identified and examined for S. mansoni cercariae. Results: the work was carried out in 427 municipalities (99.5% of the 429 selected); the presence of mollusks was registered in 300 (70.2%) municipalities; Biomphalaria glabrata were found in 62 (21%) municipalities, B. straminea in 181 (60%), B. tenagophila in three (1%); B. glabrata/B. straminea association was found in 53 municipalities (18%) and B. glabrata/B. tenagophila association in one (0.3%) municipality. Conclusion: B. glabrata, B. straminea and B. tenagophila distribution records obtained in this study are consistent with previously known distribution.