Re-assessing thermal response of schistosomiasis transmission risk: Evidence for a higher thermal optimum than previously predicted.

The geographical range of schistosomiasis is affected by the ecology of schistosome parasites and their obligate host snails, including their response to temperature. Previous models predicted schistosomiasis' thermal optimum at 21.7°C, which is not compatible with the temperature in sub-Saharan Africa (SSA) regions where schistosomiasis is hyperendemic. We performed an extensive literature search for empirical data on the effect of temperature on physiological and epidemiological parameters regulating the free-living stages of S. mansoni and S. haematobium and their obligate host snails, i.e., Biomphalaria spp. and Bulinus spp., respectively. We derived nonlinear thermal responses fitted on these data to parameterize a mechanistic, process-based model of schistosomiasis. We then re-cast the basic reproduction number and the prevalence of schistosome infection as functions of temperature. We found that the thermal optima for transmission of S. mansoni and S. haematobium range between 23.1-27.3°C and 23.6-27.9°C (95% CI) respectively. We also found that the thermal optimum shifts toward higher temperatures as the human water contact rate increases with temperature. Our findings align with an extensive dataset of schistosomiasis prevalence in SSA. The refined nonlinear thermal-response model developed here suggests a more suitable current climate and a greater risk of increased transmission with future warming for more than half of the schistosomiasis suitable regions with mean annual temperature below the thermal optimum.

Climate and urbanization drive changes in the habitat suitability of Schistosoma mansoni competent snails in Brazil.

Schistosomiasis is a neglected tropical disease caused by Schistosoma parasites. Schistosoma are obligate parasites of freshwater Biomphalaria and Bulinus snails, thus controlling snail populations is critical to reducing transmission risk. As snails are sensitive to environmental conditions, we expect their distribution is significantly impacted by global change. Here, we used machine learning, remote sensing, and 30 years of snail occurrence records to map the historical and current distribution of forward-transmitting Biomphalaria hosts throughout Brazil. We identified key features influencing the distribution of suitable habitat and determined how Biomphalaria habitat has changed with climate and urbanization over the last three decades. Our models show that climate change has driven broad shifts in snail host range, whereas expansion of urban and peri-urban areas has driven localized increases in habitat suitability. Elucidating change in Biomphalaria distribution-while accounting for non-linearities that are difficult to detect from local case studies-can help inform schistosomiasis control strategies.

Re-assessing thermal response of schistosomiasis transmission risk: evidence for a higher thermal optimum than previously predicted.

The geographical range of schistosomiasis is affected by the ecology of schistosome parasites and their obligate host snails, including their response to temperature. Previous models predicted schistosomiasis' thermal optimum at 21.7 °C, which is not compatible with the temperature in sub-Saharan Africa (SSA) regions where schistosomiasis is hyperendemic. We performed an extensive literature search for empirical data on the effect of temperature on physiological and epidemiological parameters regulating the free-living stages of S. mansoni and S. haematobium and their obligate host snails, i.e., Biomphalaria spp. and Bulinus spp., respectively. We derived nonlinear thermal responses fitted on these data to parameterize a mechanistic, process-based model of schistosomiasis. We then re-cast the basic reproduction number and the prevalence of schistosome infection as functions of temperature. We found that the thermal optima for transmission of S. mansoni and S. haematobium range between 23.1-27.3 °C and 23.6-27.9 °C (95 % CI) respectively. We also found that the thermal optimum shifts toward higher temperatures as the human water contact rate increases with temperature. Our findings align with an extensive dataset of schistosomiasis prevalence in SSA. The refined nonlinear thermal-response model developed here suggests a more suitable current climate and a greater risk of increased transmission with future warming for more than half of the schistosomiasis suitable regions with mean annual temperature below the thermal optimum.

Climate and urbanization drive changes in the habitat suitability of Schistosoma mansoni competent snails in Brazil.

Schistosomiasis is a neglected tropical disease caused by Schistosoma parasites. Schistosoma are obligate parasites of freshwater Biomphalaria snails, so controlling snail populations is critical to reducing transmission risk. As snails are sensitive to environmental conditions, we expect their distribution is significantly impacted by global change. Here, we leveraged machine learning, remote sensing, and 30 years of snail occurrence records to map the historical and current distribution of competent Biomphalaria throughout Brazil. We identified key features influencing the distribution of suitable habitat and determined how Biomphalaria habitat has changed with climate and urbanization over the last three decades. Our models show that climate change has driven broad shifts in snail host range, whereas expansion of urban and peri-urban areas has driven localized increases in habitat suitability. Elucidating change in Biomphalaria distribution - while accounting for non-linearities that are difficult to detect from local case studies - can help inform schistosomiasis control strategies.

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.

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.

Modelling the present and future distribution of Biomphalaria species along the watershed of the Middle Paranapanema region, São Paulo, Brazil.

The Middle Paranapanema region in the state of São Paulo, Brazil, is an area with high diversity for Biomphalaria species, with municipalities historically marked by cases of schistosomiasis transmission. The objectives of the study were to evaluate the current distribuition and predict the future distribution of habitats of Biomphalaria species at a high spatial resolution along 114 freshwater sites in the Middle Paranapanema watershed. The modelling encompassed 55 municipalities of the Middle Paranapanema region, which were analyzed through the maximum entropy algorithm. All geographic coordinates of the Biomphalaria species collected from 2015-2018 and environmental data were obtained through WorldClim, HydroSHEDS, TOPODATA and Secretaria do Meio Ambiente for the 1970-2017 period. For the 2041-2060 period we used the HadGEM2-ES climate model. Due to climate change, MaxEnt showed that there was a high probability for the maintenance of B. glabrata habitats near Ourinhos and Assis, an expansion of scattered spots, and a 50% probability that the species will spread throughout new suitable areas. The results showed that the geographical range of B. straminea will most likely expand in the future along the Middle Paranapanema hydrographic basin, especially in the municipalities near Ourinhos. For B. glabrata and B. straminea, the geographic expansion was related to the predicted increase in the annual temperature range. The habitats suitable for B. tenagophila and B. peregrina seemed to slightly expand around the west border of the Middle Paranapanema region. Biomphalaria occidentalis may have a small reduction in its distribution due to climate change. The variables that contributed the most to the future modelling for these three species were precipitation and temperature. Identifying the sites with intermediate hosts for schistosomiasis may guide public health measures to avoid or reduce future transmissions in this region.

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.

Molecular detection of prepatent Schistosoma mansoni infection in Biomphalaria glabrata snail vectors.

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.

Schistosomiasis in the Middle Paranapanema river region, state of São Paulo, Brazil: Does it matter today for public health?

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.

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.

Diversity of Biomphalaria spp. freshwater snails and associated mollusks in areas with schistosomiasis risk, using molecular and spatial analysis tools / Diversidade de caramujos de água doce Biomphalaria spp. e outros moluscos associados, em áreas de risco da esquistossomose, empregando ferramentas moleculares e espaciais

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.

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.

Molecular and morphological identification of Biomphalaria species from the state of São Paulo, Brazil.

DNA barcoding and morphological characters were used to identify adult snails belonging to the genus Biomphalaria from 17 municipalities in the state of São Paulo, Brazil. The DNA barcode analysis also included twenty-nine sequences retrieved from GenBank. The final data set of 104 sequences of the mitochondrial cytochrome oxidase I (COI) gene was analyzed for K2P intraspecific and interspecific divergences, through tree-reconstruction methods (Neighbor-Joining, Maximum Likelihood and Bayesian inference), and by applying different models (ABGD, bPTP, GMYC) to partition the sequences according to the pattern of genetic variation. Twenty-seven morphological parameters of internal organs were used to identify specimens. The molecular taxonomy of Biomphalaria agreed with the morphological identification of specimens from the same collection locality. DNA barcoding may therefore be a useful supporting tool for identifying Biomphalaria snails in areas at risk for schistosomiasis.

The current distribution pattern of Biomphalaria tenagophila and Biomphalaria straminea in the northern and southern regions of the coastal fluvial plain in the state of São Paulo

We analyze the current distribution of snails from genus Biomphalaria in the north and south of the coastal fluvial plain in the state of Sa˜o 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

Distribuição e diversidade de espécies do gênero Biomphalaria em microrregiões localizadas no Médio Paranapanema, São Paulo, SP, Brasil / Diversity and distribution of the Biomphalaria species in the middle reaches of the Paranapanema River, São Paulo, SP, Brazil

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.

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.

ITS2 variability of Biomphalaria (Mollusca, Planorbidae) species from the Paranapanema Valley (São Paulo State, Brazil): diversity patterns, population structure, and phylogenetic relationships

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.

Genetic markers from Biomphalaria tenagophila (Gastropoda: Pulmonata: Planorbidae) obtained by the double stringency polymerase chain reaction technique

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

Biologia de Biomphalaria tenagophila (Pulmonata, Planorbídae) em relação ao uso do moluscicida químico Bayluscide (Bayer): implicações para o controle / Biology of Biompharia tenagophila (Pulmonata, planorbidae) in relation to the use of chemical moluscicide Bayluscide (Bayer); Implications for the control

A Biomphalaria tenagóphila é um molusco pulmonado, hermafrodita simultâneo, com preferência pela fecundação cruzada. Esta espécie é hospedeira intermediária do parasita causador da esquistossomose no homem e, no Estado de São Paulo a espécie é muito freqüente. O Controle da abundância dos caramujos no campo é feito através da aplicação nas coleções d'água do moluscicida Bayluscide da Bayer, uma niclosamida muito toxica aos caramujos. Nosso objetivo foi medir os efeitos deste produto químico sobre a biologia de Biomphalaria tenagophila em laboratório e no campo. Calculamos a CL 50 para uma linhagem de laboratório, e os sobreviventes aos ensaios biológicos foram observados quanto a sobrevivência e eficiência reprodutiva em várias doses. A sobrevivência foi afetada em animais adultos, houve um atraso na maturação sexual dos jovens e aumento do número de ovos no indivíduo sobrevivente a uma dose alta do moluscicida. Pode ter havido seleção deste indivíduo com maior capacidade reprodutiva feminina, esta vantagem adaptativa pode ser mantida por autofecundação. O Bayluscide não alterou a relação do número de caramujos infestados pelo parasita mesmo após mais de dez anos de aplicação do moluscicida, provavelmente porque a relação parasita/hospedeiro seja estável evolutivamente. Os dados de campo mostram que houve alteração da densidade de caramujos, que pode ser resultado das extremas flutuações populacionais devido a mortalidade dos animais pela ação tóxica da droga, alterando a estrutura das populações naturais de caramujos.

Spermatogenesis and desiccation in Biomphalaria tenagophila (orbigny, 1985) (Gastropoda, Planorbidae)

Os resultados mostram o efeito de períodos prolongados de dessecaçäo sobre a espermatogênese em Biomphalaria tenagophila. Para tanto adaptou-se ao ovotestis, a técnica de suspensäo celular, o que possibilitou a obtençäo de ótimas preparaçöes cromossômicas