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.

ABSTRACT Approximately 240 million people worldwide are infected by Schistosoma. In Brazil, one of the main intermediate hosts of this parasite is Biomphalaria glabrata snails. The early detection of larval stages in intermediate hosts is an important challenge to public health, but it also represents an opportunity as a new alternative to indicate earlier natural infections before cercariae differentiation and emergence. In this context, we demonstrated that PCR amplification of a 28S gene fragment from the parasite does demonstrate S. mansoni infection in snails 14 days post infection. This conventional polymerase chain reaction amplified clear bands and was able to detect parasitic infection in the intermediate host B. glabrata under experimental conditions. However, we reinforce that this approach requires deeper investigations and further comparisons to confirm its specificity and sensitivity in earlier time points after miracidia infection. This approach has relevant potential as an effective molecular-based strategy for the monitoring of schistosomiasis transmission.

Resumo: Foi realizado estudo morfológico e molecular de Biomphalaria occidentalis, B. oligoza, B. peregrina, B. schrammi, B. straminea e B. tenagophila coletados em 55 pontos situados ao longo da bacia hidrográfica do Alto Rio Tietê a sudoeste do Brasil. A análise morfológica levou em consideração aspectos da concha, manto, órgãos excretores e sistema reprodutor. Os dados moleculares incluíram 122 sequências do gene mitocondrial Citocromo C Oxidase I (COI). Nossos resultados mostram que alguns caracteres da concha, bem como outros relacionados ao manto e ao sistema reprodutor foram aqui considerados fundamentais na determinação das seis Biomphalaria tratadas neste estudo. A utilização do DNA Barcode simultaneamente com a taxonomia morfológica gerou resultados mais fidedignos, demonstrando ser uma abordagem bastante útil, inclusive, para os serviços de vigilância malacológica.

Abstract: Morphological and molecular identifications were carried out for Biomphalaria occidentalis, Biomphalaria oligoza, Biomphalaria peregrina, Biomphalaria schrammi, Biomphalaria straminea and Biomphalaria tenagophila collected from 55 sites located along the upper basin of Tietê River in the Southeast Region of Brazil. Morphological analysis considered aspects of the shell, mantle, excretory organs and reproductive system. Molecular data included 122 sequences of Cytochrome C Oxidase I gene (COI). Our results showed that some shell characters, as well as other characters related to the mantle and the reproductive system, are fundamental for the identification of the six Biomphalaria species included in this study. The use of DNA barcoding together with morphological taxonomy generated more reliable results, proving to be a very useful approach, even for malacological surveillance services.

Resumo: A região do Médio Rio Paranapanema, em São Paulo, Brasil abriga uma diversidade significativa das espécies de Biomphalaria. É também uma região vulnerável a impactos ambientais e de saúde, como a esquistossomose. Este estudo atualiza dados sobre a distribuição de caramujos de água doce em ecossistemas de uma porção da Bacia do Médio Rio Paranapanema, com ênfase no gênero Biomphalaria. Os caramujos foram coletados de 114 corpos distintos de água doce, entre 2015 e 2018. Exemplares pertencentes ao gênero Biomphalaria foram identificados de acordo com características morfológicas e moleculares, enquanto animais de outros gêneros (Drepanotrema, Lymnaea, Melanoides, Physa e Pomacea) foram identificados somente de acordo com características da concha. Ferramentas de análise geoespaciais foram utilizadas para atualizar os sítios de colonização dos caramujos e, consequentemente, auxiliar na identificação de possíveis pontos críticos para hospedeiros intermediários da esquistossomose. As sequências do gene COI relacionadas ao DNA barcode foram testadas quanto à similaridade com sequências encontradas no GenBank, por análise filogenética sob maxima verossimilhança, e analisadas em ABDG, bPTP e GMYC para a delimitação de espécies putativas. Dos 10.722 moluscos coletados, 86,7% pertenciam a família Planorbidae (75,5% Biomphalaria e 11,2% Drepanotrema) e 13,3% a Lymnaea spp., Melanoides spp., Physa spp. e Pomacea spp. A comparação das sequências taxonômicas de COI com o banco de dados de nucleotídeos do NCBI, e a análise filogenética usada para testar a monofilia dos grupos, resultaram em delimitações taxonômicas comparáveis à delimitação morfológica. As espécies B. glabrata e B. tenagophila estão heterogeneamente distribuídas ao longo da área de estudo. B. glabrata foi identificada em apenas cinco coleções de água doce, quatro delas em Ourinhos, enquanto B. tenagophila predominou em Ipaussu. Por outro lado, B. straminea, B. occidentalis e B. peregrina estão distribuídas uniformemente na área de estudo.

Abstract: The Middle Paranapanema River region of São Paulo, Brazil is home to significant diversity of Biomphalaria species and is very vulnerable to health and environmental impacts such as schistosomiasis. This study updates freshwater malacological surveys for ecosystems in one portion of the Middle Paranapanema River Basin, with emphasis on the genus Biomphalaria. Snails were collected from 114 distinct bodies of water between 2015 and 2018. Biomphalaria specimens were identified according to morphological and molecular characteristics, while animals in other genera (Drepanotrema, Lymnaea, Melanoides, Physa and Pomacea) were identified solely according to shell characteristics. A geographic information system was used to update intermediate host colonization sites and consequently assist in identifying probable hotspots for intermediate hosts of schistosomiasis. The sequences of the COI gene relating to the DNA barcode stretch were tested for similarity against sequences found in GenBank, for monophyly through Maximum Likelihood phylogenetic inference, and analyzed in ABDG, bPTP and GMYC for the delimitation of putative species. Of the 10,722 snails collected, 86.7% were in the Planorbidae family (75.5% Biomphalaria and 11.2% Drepanotrema) and 13.3% were other non-Planorbidae species (Lymnaea, Melanoides, Physa and Pomacea). The taxonomic COI reference sequences in the NCBI nucleotide database used for DNA sequence comparison, and phylogenetic analysis used to test the monophyly of the groups, resulted in more reliable taxonomic units than delimitation of the COI sequences in MOTUs using statistical taxonomic models. Analysis of the species distribution shows that B. glabrata and B. tenagophila are heterogeneously distributed in the study area. B. glabrata colonizes only five water bodies, in the study area, most of them in Ourinhos, while B. tenagophila predominates in water bodies in Ipaussu. Contrasting with this, B. straminea, B. occidentalis and B. peregrina are evenly distributed throughout the study area.

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.

Neste trabalho analisamos a distribuição atual de caramujos do gênero Biomphalaria nos setores norte e sul da planície fluvial da costa litorânea de São Paulo. A região está inserida numa extensa planície costeira inundável. Os dados de 29 sítios de coletas confirmam que a coleção de água doce em ambos os setores é colonizada por Biomphalaria tenagophila e Biompalaria straminea. Ambas as espécies são hospedeiras intermediárias naturais do Schistosoma mansoni. A abundânciade B. straminea em sítios de coleta em que foram registrados recentemente apenas B. tenagophila, indica o potencial de expansão de B. straminea na região. A análise quantitativa de espécimes/espécie demonstra que o crescimento local das populações de B. tenagophila ocorre em períodos de baixa pluviosidade. Há maior risco de dispersão das populações de B. tenagophila entre diferentes coleções de água em períodos úmidos, quando a alta pluviosidade promove a cheia dos principais rios e inundações decorrentes do escoamento de água da Serra do Mar para a planície costeira. O aumento da temperatura causada por alterações climáticas globais, e consequentemente o aumento de cheias e inundações, deve interferir na distribuição dos caramujos na planície fluvial do litoral, com risco de alteração na transmissão autóctone da esquistossomose nesta região.

We analyze the current distribution of snails from genus Biomphalaria in the north and south of the coastal fluvial plain in the state of São Paulo, which is part of a large coastal floodplain. Data from twenty-nine collection sites confirmed that the freshwater body in both regions is colonized by Biomphalaria tenagophila and Biomphalaria straminea, which are natural intermediate hosts of Schistosoma mansoni. The abundance of B. straminea in collection sites where only B. tenagophila had previously been recorded indicates the potential for B. straminea to expand in the region. While quantitative analysis of the number of specimens per species showed that local growth of B. tenagophila populations occurs during periods with little rainfall, there is a greater risk of B. tenagophila populations spreading between different water bodies during rainy periods, when heavy rainfall results in water levels rising in the main rivers and flooding caused by water flowing from the Serra do Mar mountain range to the coastal plain. The temperature increase caused by global climate changes and the consequent increasing frequency of high-water levels and floods can be expected to affect the distribution of these snails on the coastal fluvial plain, leading to the risk of a change in the autochthonous transmission pattern of schistosomiasis in the region.

Foram estudadas a diversidade e a abundância de espécies do gênero Biomphalaria em córregos próximos aos Rios Paranapanema e Pardo (São Paulo, SP, Brasil), em locais antigamente associados à transmissão do Schistosoma mansoni, sujeitos ainda a drásticas variações na disponibilidade de água. Os dados confirmam a predominância de Biomphalaria glabrata (Say, 1818) em córregos do município de Ourinhos, localizados nas margens do Rio Pardo e do Rio Paranapanema. Em Ipauçu, distante 30 km de Ourinhos, a predominância de Biomphalaria tenagophila (Orbigny, 1835) é acompanhada da ausência de B. glabrata. Foram estimados os índices de Diversidade e Dominância de Simpson, que evidenciam uma distribuição variada, provavelmente associada com o substrato aquático onde vivem os caramujos.

The predominant landscape in the studied region, composed by the Paranapanema and Pardo rivers, has been significantly affected by human interference in order to create extensive artificial irrigation ditches for agriculture. These environments are subject to drastic variations in water supply whereby draughts are created what bears a drastic populational fluctuation. The composition of schistosomiasis-associated planorbid fauna in Ourinhos and Ipauçu herein presented corroborate previous malacological surveys, which incriminates Biomphalaria. glabrata (Say, 1818) as the predominant species mainly at the Ourinhos micro-regions. At this site the snail distribution might be linked to the composition of deposited sediments in the hydrographic basin. Data from Ipauçu, a collection site that is only 30 km from Ourinhos, indicates absence of B. glabrata, and predominance of B. tenagophila (Orbigny, 1835). Despite closeness, the micro-regions show important differences in values of diversity index, indicating a heterogeneous distribution of Biomphalaria species.

The ribosomal DNA internal transcribed spacer 2 (ITS2) has been shown to be a useful genetic marker for species identification and phylogenetic reconstruction in the genus of freshwater snails Biomphalaria (Preston 1910). Additionally, ITS2 studies in Biomphalaria have uncovered significant intra-specific genetic variability suggesting the presence of cryptic species complexes. We obtained ITS2 sequences for the Biomphalaria species B. glabrata, B. tenagophila, B. occidentalis and B. peregrina from the Paranapanema Valley (São Paulo State, Brazil) and compared them with a comprehensive set of published Biomphalaria ITS2 sequences using Bayesian inference of phylogeny. Analysis of the resulting trees showed that the newly obtained B. glabrata sequences did not cluster with those from other Brazilian localities and that sub-structuring occurred among Brazilian B. tenagophila populations. Moreover, although ITS2 sequences seem to indicate clear genetic differentiation within both B. glabrata and B. tenagophila, evidence in support of the occurrence of cryptic species is more compelling for the latter. We discuss the significance and implications of the detected patterns of ITS2 variability for taxonomic studies in Biomphalaria.

Biomphalaria tenagophila, one of the intermediate hosts of the trematoda Schistosoma mansoni, is a simultaneous hermafrodite snail species. In order to analyse the genetic structure of these populations, we performed a double-stringency PCR technique to obtain genetic markers with microsatellites and arbitrary primers in a single reaction.

A Biomphalaria tenagophila é um caramujo hermafrodita simultâneo com potencial para a autofecundação e fecundação cruzada. É portanto, um excelente objeto de estudo sobre o modo de reprodução como estratégia evolutiva. Neste trabalho comparamos a quarta geração produzida sucessivamente por autofecundação a animais cruzados em relação ao tamanho da concha na maturidade sexual, à fecundidade e à fertilidade dos ovos. Os parâmetros reprodutivos analisados apresentaram semelhança nos dois grupos. No entanto, o tamanho da concha na maturidade sexual foi significativamente maior na quarta geração produzida por sucessivas autofecundações.

Biomphalaria tenagophila is a simultaneous hermaphrodite freshwater snail, which can breed by self and cross-fertilization. It is, therefore, well suited for studying reproduction as an evolutive strategy. Several characteristics (shell size and age at sexual maturity, egg fecundity and fertility) were analyzed in four consecutive self-fertilized generations and compared to cross-fertilized individuals. The reproductive parameters were similar in the two groups. Shell size was significantly greater in the fourth self-fertilized generation than in the cross-fertilized individuals.