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.

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.

Prevalence and distribution of angiostrongylus cantonensis (nematoda, angiostrongylidae) in achatina fulica (mollusca, gastropoda) in baixada santista, São Paulo, Brazil

ABSTRACT INTRODUCTION Angiostrongylus cantonensis is causes eosinophilic meningoencephalitis in humans. Worldwide expansion of this nematode is linked to the dispersion of their hosts. This study aimed to determine the prevalence of A. cantonensis infection in Achatina fulica in the nine municipalities that make up Baixada Santista, São Paulo, Brazil. METHODS Angiostrongylus cantonensis larvae were analyzed using optical microscopy. We performed polymerase chain reaction and restriction fragment length polymorphism using restriction endonuclease ClaI, directed to the internal transcribed spacer region 2 of A. cantonensis larval DNA. RESULTS Of the 540 snails analyzed, 117 (21.7%) were infected by A. cantonensis. For morphological and morphometric analyses, 60 larvae were used. Second-stage larvae were, on average, 358.2µm long and 26.4µm wide, while third-stage larvae were, on average, 450µm long and 21.12µm wide. The tails of the larvae ended in a fine tip. CONCLUSIONS All municipalities comprising Baixada Santista had A. fulica that were naturally infected with A. cantonensis. All of the observed characteristics were typical of the species.

SPATIAL DISTRIBUTION OF LYMNAEIDAE (MOLLUSCA, BASOMMATOPHORA), INTERMEDIATE HOST OF Fasciola hepatica LINNAEUS, 1758 (TREMATODA, DIGENEA) IN BRAZIL / Distribuição espacial de Lymnaeidae (Mollusca, Basommatophora), hospedeiros intermediários de Fasciola hepatica Linnaeus, 1758 (Trematoda, Digenea) no Brasil

Snails of the family Lymnaeidae act as intermediate hosts in the biological cycle of Fasciola hepatica, which is a biological agent of fasciolosis, a parasitic disease of medical importance for humans and animals. The present work aimed to update and map the spatial distribution of the intermediate host snails of F. hepatica in Brazil. Data on the distribution of lymnaeids species were compiled from the Collection of Medical Malacology (Fiocruz-CMM, CPqRR), Collection of Malacology (MZUSP), “SpeciesLink” (CRIA) network and through systematic surveys in the literature. Our maps of the distribution of lymnaeids show that Pseudosuccinea columella is the most common species and it is widespread in the South and Southeast with few records in the Midwest, North and Northeast regions. The distribution of the Galba viatrix, G. cubensis and G. truncatula showed a few records in the South and Southeast regions, they were not reported for the Midwest, North and Northeast. In addition, in the South region there are a few records for G. viatrix and one occurrence of Lymnaea rupestris. Our findings resulted in the first map of the spatial distribution of Lymnaeidae species in Brazil which might be useful to better understand the fasciolosis distribution and delineate priority areas for control interventions.

Moluscos da família Lymnaeidae são hospedeiros intermediários no ciclo biológico de Fasciola hepatica, agente etiológico da fasciolose, doença parasitária de importância médica para humanos e animais. O presente trabalho teve como objetivo datar e mapear a distribuição espacial dos hospedeiros intermediários de F. hepatica no Brasil. Os dados de distribuição das espécies de limneídeos foram obtidos das Coleções de Malacologia Medica (Fiocruz-CMM, CPqRR) e de Malacologia do Museu de Zoologia da Universidade de São Paulo (MZUSP), rede “SpeciesLink” (CRIA) e através de pesquisas sistemáticas na literatura. Os mapas de distribuição dos limneideos mostram que Pseudosuccinea columella é a espécie mais comum e distribuída nas regiões Sul e Sudeste com poucos registros nas regiões Centro-Oeste, Norte e Nordeste. Além disso, na região Sul os registros para G. viatrix são escassos e existe apenas um único relato de ocorrência para L. rupestris. Os dados obtidos resultaram no primeiro mapa de distribuição espacial das espécies de Lymnaeidae no Brasil, informação básica e relevante para estruturação de políticas de controle da fasciolose humana e animal.

ocorrência de moluscos do gênero Biomphalaria em parques da cidade de Belo Horizonte, Minas Gerais, Brasil / Occurrence of mollusks, genus Biomphalaria, in parks of the city of Belo Horizonte, Minas Gerais, Brazil

Até pouco tempo a esquistossomose era considerada a mais prevalente endemia entre a população rural de baixa renda, especialmente nos estados do Nordeste e Sudeste do Brasil, sendo por issoclassificada como endemia rural. Estudos recentes têm demonstrado que o perfil epidemiológico desta doença está mudando, uma vez que vem sendo transmitida nas periferias e mesmo dentro de grandes centros urbanos. A cidade de Belo Horizonte-MG tem 71 parques, dos quais 55 são abertos àvisitação pública. Em 31 (43,6por cento) deles há uma ou mais coleções hídricas. O objetivo deste trabalhofoi investigar a presença de moluscos de importância médica nas coleções hídricas destes 31 parques da capital mineira. Foram coletados, em 11 parques, 551 exemplares de Biomphalaria, hospedeiraintermediária do Schistosoma mansoni. Foram também coletados exemplares dos gêneros Drepanotrema sp, Pomacea sp, Melanoides sp, Physa sp e Lymnaea sp, este último hospedeirointermediário de outro parasito causador da fasciolose. Todos os exemplares se mostraram negativos para cercárias de S. mansoni. No entanto, considerando a possível contaminação do meio ambiente com fezes humanas infectadas com ovos de S. mansoni e a presença dos hospedeirosintermediários, estes achados servem de alerta para uma possível instalação do ciclo de transmissão da esquistossomose em parques municipais da cidade de Belo Horizonte.

Angiostrongylus cantonensis (Nematode: Metastrongyloidea) in molluscs from harbour areas in Brazil

Angiostrongylus cantonensis is the most common aetiological agent of human eosinophilic meningoencephalitis. Following a report indicating the presence of this parasite in Brazil in 2007, the present study was undertaken to investigate the presence of A. cantonensis in the surrounding Brazilian port areas. In total, 30 ports were investigated and the following molluscs were identified: Achatina fulica, Belocaulus sp., Bradybaena similaris sp., Cyclodontina sp., Helix sp., Leptinaria sp., Melampus sp., Melanoides tuberculata, Phyllocaulis sp., Pomacea sp., Pseudoxychona sp., Rhinus sp., Sarasinula marginata, Streptaxis sp., Subulina octona, Succinea sp., Tomigerus sp., Wayampia sp. and specimens belonging to Limacidae and Orthalicinae. Digestion and sedimentation processes were performed and the sediments were examined. DNA was extracted from the obtained larvae and the internal transcribed spacer region 2 was analysed by polymerase chain reaction-restriction fragment length polymorphism after digestion with the endonuclease ClaI. Of the 30 ports investigated in this study, 11 contained molluscs infected with A. cantonensis larvae. The set of infected species consisted of S. octona, S. marginata, A. fulica and B. similaris. A total of 36.6% of the investigated ports were positive for A. cantonensis, indicating a wide distribution of this worm. It remains uncertain when and how A. cantonensis was introduced into South America.

Hybridism between Biomphalaria cousini and Biomphalaria amazonica and its susceptibility to Schistosoma mansoni

Molecular techniques can aid in the classification of Biomphalaria species because morphological differentiation between these species is difficult. Previous studies using phylogeny, morphological and molecular taxonomy showed that some populations studied were Biomphalaria cousini instead of Biomphalaria amazonica. Three different molecular profiles were observed that enabled the separation of B. amazonica from B. cousini. The third profile showed an association between the two and suggested the possibility of hybrids between them. Therefore, the aim of this work was to investigate the hybridism between B. cousini and B. amazonica and to verify if the hybrids are susceptible to Schistosoma mansoni. Crosses using the albinism factor as a genetic marker were performed, with pigmented B. cousini and albino B. amazonica snails identified by polymerase chain reaction-restriction fragment length polymorphism. This procedure was conducted using B. cousini and B. amazonica of the type locality accordingly to Paraense, 1966. In addition, susceptibility studies were performed using snails obtained from the crosses (hybrids) and three S. mansoni strains (LE, SJ, AL). The crosses between B. amazonica and B. cousini confirmed the occurrence of hybrids. Moreover, hybrids can be considered potential hosts of S. mansoni because they are susceptible to LE, SJ and AL strains (4.4 percent, 5.6 percent and 2.2 percent, respectively). These results indicate that there is a risk of introducing schistosomiasis mansoni into new areas.

Preliminary studies investigating the occurrence of Biomphalaria cousini in Brazil

Specific genetic profiles of Brazilian Biomphalaria species were previously standardized by molecular taxonomy through the analysis of restriction fragments, which were generated by digesting the internal transcribed spacer region of rDNA with the DdeI endonuclease. Biomphalaria amazonica displayed three distinct profiles. To investigate these distinct profiles, the same molecular technique, polymerase chain reaction and restriction fragment length polymorphism, was used with different endonucleases. In addition, morphological data were also used to compare B. amazonica specimens that were collected from Brazil, Colombia and Bolivia. The morphological characters of Bolivian molluscs were similar to B. amazonica, displayed a molecular profile of five restriction fragments and morphological data, whereas the Colombian mollusc population showed morphological characters similar to Biomphalaria cousini and a molecular profile of three restriction fragments, similar to B. cousini. The Brazilian specimens showed the B. amazonica and B. cousini molecular profiles as well as a third profile, which resembled a combination of the Colombian and Bolivian molecular profiles.

Biomphalaria molluscs (Gastropoda: Planorbidae) in Rio Grande do Sul, Brazil

The present study was aimed at characterising Biomphalaria species using both morphological and molecular (PCR-RFLP) approaches. The specimens were collected in 15 localities in 12 municipalities of the southern region of the state of Rio Grande do Sul, Brazil. The following species were found and identified: Biomphalaria tenagophila guaibensis, Biomphalaria oligoza and Biomphalaria peregrina. Specimens of the latter species were experimentally challenged with the LE Schistosoma mansoni strain, which showed to be refractory to infection.

Esquistossomose em área de ecoturismo do Estado de Minas Gerais, Brasil / Schistosomiasis in an ecotourism area in Minas Gerais State, Brazil

Neste trabalho, são discutidos os modos de transmissão da esquistossomose na localidade de São José da Serra, com 500 habitantes, no Município de Jaboticatubas, Minas Gerais, Brasil. A região ao redor recebe, durante todo o ano, milhares de pessoas para a prática de ecoturismo. A ocorrência de esquistossomose aguda em um casal que viajou para essa área, no carnaval de 2007, motivou este estudo. Exames de fezes foram feitos em 268 moradores, 53,6 por cento da população total. Trinta e cinco (13 por cento) estavam eliminando ovos nas fezes. Comparando os resultados coproscópicos com os de um inquérito feito em 2005, observa-se um aumento da proporção de positivos de 9,6 por cento para 12,5 por cento, entre 56 pessoas que participaram em ambos os estudos. Foram coletados 65 exemplares de Biomphalaria glabrata, sendo 1 (1,5 por cento) positivo. No inquérito malacológico realizado em 2005, em 182 biomphalarias, nenhum exemplar foi encontrado positivo. Esses dados indicam a ocorrência de transmissão ativa de esquistossomose na área, revelando a necessidade de planejamento coordenado no desenvolvimento do setor turístico, incluindo a implantação de programas educativos integrados para a comunidade e, principalmente, para os turistas.

This paper discusses schistosomiasis transmission in São José da Serra, a village with a population of 500 in the county of Jaboticatubas, Minas Gerais State, Brazil. The area receives thousands of visitors a year for ecotourism. The study was motivated by a case of acute schistosomiasis involving a couple that spent the 2007 Carnival (Mardi Gras) holiday in the area. Stool tests from 268 local residents (53.6 percent of the population) showed that 35 (13 percent) were positive for the infection. A comparison with a previous survey (2005) in the same location showed an increase in the schistosomiasis-positive rate from 9.6 percent to 12.5 percent, among the 56 individuals who participated in both surveys. A malacological survey of 65 Biomphalaria glabrata snails showed one specimen (1.5 percent) eliminating cercariae. In a similar survey in 2005, no positive snail specimens were found. The study indicates that active schistosomiasis transmission is occurring in the area, and that integrated educational programs are needed for both the local community and tourists.

First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from São Vicente, state of São Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.

Biomphalaria tenagophila: dynamics of populations of resistant and susceptible strains to Schistosoma mansoni, with or without pressure of the parasite

Resistant (Taim, RS) and susceptible albino (Joinville, SC) Biomphalaria tenagophila populations were kept together, at different proportions, throughout a 18-month-period. Some of the snail groups were submitted to Schistosoma mansoni infection. The targets of this study were (a) to analyze the populational dynamics among resistant and susceptible individuals to S. mansoni; (b) to study the resistance phenotype in descendants of cross-breeding; (c) to observe whether the parasite could exert any kind of selection in those snail populations. Throughout the experiment it could be observed that the susceptible B. tenagophila strain (Joinville) underwent a selective pressure of the parasite that was negative, since the individuals showed a high mortality rate. Although B. tenagophila (Taim) population presented a higher mortality rate without pressure of the parasite, this event was compensated by a reproductive capacity. B. tenagophila Taim was more fecund than B. tenagophila Joinville and was able to transmit the resistance character to their descendants. F1 generation obtained by cross-breeding between resistant and susceptible lineages was completely resistant to S. mansoni infection, irrespective of the Taim proportion. Moreover, less than 5 percent of F2 progeny were susceptible to S. mansoni infection.

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 × coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63 percent AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.

Biomphalaria spp. (Preston, 1910) snails in the municipality of Juiz de Fora, Zona da Mata Mineira mesoregion, ate of Minas Gerais, Brazil

This study focuses on the geographic distribution of the snail of the genus Biomphalaria and evaluates its infectivity by Schistosoma mansoni in 5264 specimens collected in the municipality of Juiz de Fora, Minas Gerais, Brazil. Of the 31 locations studied, 6 were reservoirs, 11 rudimentary holding ponds, 7 irrigation ditches, 5 lakes, 1 ornamental pond, and 1 waterfall. Intermediate hosts were found only in the rudimentary ponds and ditches, which were 100 percent positive. Using morphological and molecular analysis techniques, B. tenagophila, B. peregrina, and B. straminea were identified. This is the first report of B. stramínea in the municipality, and evaluation of its infective potential revealed susceptibility of 25.4 percent. Although we did not find specimens of Biomphalaria infected by S. mansoni, the data obtained indicate the presence of intermediate hosts, especially in the irrigation ditches in Juiz de Fora, and their proximity to contaminated areas.

Dominant character of the molecular marker of a Biomphalaria tenagophila strain (Mollusca: Planorbidae) resistant to Schistosoma mansoni

Biomphalaria tenagophila population from Taim (state of Rio Grande do Sul, Brazil) is totally resistant toSchistosoma mansoni, and presents a molecular marker of 350 bp by polymerase chain reaction and restriction fragment length polymorphism of the entire rDNA internal transcriber spacer. The scope of this work was to determine the heritage pattern of this marker. A series of cross-breedings between B. tenagophila from Taim (resistant) and B. tenagophila from Joinville, state of Santa Catarina (susceptible) was carried out, and their descendants F1 and F2 were submitted to this technique. It was possible to demonstrate that the specific fragment from Taim is endowed with dominant character, since the obtained segregation was typically mendelian.

Angiostrongylus costaricensis, A. cantonensis e A. vasorum: Diferenciação, detecção no hospedeiro intermediário e filogenia utilizando a PCR-RFLP, Multiplex PCR e sequenciamento da região espaçadora transcrita interna do DNA ribossomal

Angiostrongylus costaricensis, A. cantonensis e A. vasorum possuem importância humana e veterinária. O exame dos moluscos infectados com esses parasitos é feito após digestão do molusco e sedimentação das larvas (L3). Essas larvas são inoculadas em roedores para obtenção dos vermes adultos que são identificados morfologicamente. Em decorrência dessas dificuldades, desenvolvemos um método molecular para a detecção de larvas desses parasitos utilizando a técnica de PCR-RFLP com iniciadores direcionados para as regiões conservadas da região espaçadora transcrita interna dois do DNA ribossomal (ITS2 do rDNA nuclear) e citocromo oxidase subunidade I do DNA mitocondrial (COI do DNAmt). A enzima de restrição ClaI foi a que forneceu os resultados mais precisos para a primeira região e a RsaI para a segunda. Para a reconstrução filogenética desses helmintos sequenciamos a região ITS2 do rDNA. As topologias das árvores foram ligeiramente diferentes. Na árvore de máxima parcimônia (MP) foi observada a formação de dois grupos: I) A. costaricensis, II) A. cantonensis e A. vasorum. Nas árvores de agrupamento de vizinhos (NJ) e máxima verossimilhança (ML) também houve a formação de dois grupos: I) A. costaricensis e A. vasorum, II) A. cantonensis

Nas três árvores, o grupo de A. costaricensis foi dividido em dois ramos, separando as populações do Brasil e da Costa Rica, sustentados por alto valor de “bootstrap”. As topologias das árvores e os valores de distância dessas populações sugerem que elas estão se diferenciando, provavelmente, devido à distância geográfica. Os resultados obtidos sugerem a existência de um único gênero: Angiostrongylus. De fato, as espécies A. cantonensis e A. costaricensis consideradas por alguns autores como pertencentes ao gênero Parastrongylus mostraram-se distantes entre si e mais próximas de A. vasorum. A partir das sequências desses nematóides, foram desenhados dois iniciadores. Esses iniciadores associados a um par de iniciador direcionado para a região ITS do hospedeiro intermediário, foram utilizados em uma única reação, denominada multiplex-PCR. Foram obtidos perfis específicos para cada nematóide. O amplicon de ITS2 de A. costaricensis do Brasil foi de 400 pb, de A. costaricensis da Costa Rica 480 pb, de A. cantonensis um fragmento duplo de 480-500 pb e de A. vasorum 450 pb. Com essa mesma metodologia detectou-se A. costaricensis em Sarasinula marginata experimentalmente infectada, sendo obtido um amplicon do parasito de 400 pb, associado ao amplicon do molusco de 1000 pb

Angiostrongylus costaricensis, A. cantonensis e A. vasorum: diferenciação, detecção no hospedeiro intermediário e filogenia utilizando a PCR-RFLP, Multiplex PCR e sequenciamento da região espaçadora transcrita interna do DNA ribossomal / Angiostrongylus costaricensis, A. cantonensis and A. vasorum: differentiation, tracing the intermediate host and phylogeny using PCR-RFLP, multiplex PCR and sequencing of internal transcribed spacer of ribosomal DNA

Angiostrongylus costaricensis, A. cantonensis e A. vasorum possuem importância humana e veterinária. O exame dos moluscos infectados com esses parasitos é feito após digestão do molusco e sedimentação das larvas (L3). Essas larvas são inoculadas em roedores para obtenção dos vermes adultos que são identificados morfologicamente. Em decorrência dessas dificuldades, desenvolvemos um método molecular para a detecção de larvas desses parasitos utilizando a técnica de PCR-RFLP com iniciadores direcionados para as regiões conservadas da região espaçadora transcrita interna dois do DNA ribossomal (ITS2 do rDNA nuclear) e citocromo oxidase subunidade I do DNA mitocondrial (COI do DNAmt). A enzima de restrição ClaI foi a que forneceu os resultados mais precisos para a primeira região e a RsaI para a segunda. Para a reconstrução filogenética desses helmintos sequenciamos a região ITS2 do rDNA. As topologias das árvores foram ligeiramente diferentes. Na árvore de máxima parcimônia (MP) foi observada a formação de dois grupos: I) A. costaricensis, II) A. cantonensis e A. vasorum. Nas árvores de agrupamento de vizinhos (NJ) e máxima verossimilhança (ML) também houve a formação de dois grupos: I) A. costaricensis e A. vasorum, II) A. cantonensis. Nas três árvores, o grupo de A. costaricensis foi dividido em dois ramos, separando as populações do Brasil e da Costa Rica, sustentados por alto valor de “bootstrap”. As topologias das árvores e os valores de distância dessas populações sugerem que elas estão se diferenciando, provavelmente, devido à distância geográfica. Os resultados obtidos sugerem a existência de um único gênero: Angiostrongylus. De fato, as espécies A. cantonensis e A. costaricensis consideradas por alguns autores como pertencentes ao gênero Parastrongylus mostraram-se distantes entre si e mais próximas de A. vasorum. A partir das sequências desses nematóides, foram desenhados dois iniciadores. Esses iniciadores associados a um par de iniciador direcionado para a região ITS do hospedeiro intermediário, foram utilizados em uma única reação, denominada multiplex-PCR. Foram obtidos perfis específicos para cada nematóide. O amplicon de ITS2 de A. costaricensis do Brasil foi de 400 pb, de A. costaricensis da Costa Rica 480 pb, de A. cantonensis um fragmento duplo de 480-500 pb e de A. vasorum 450 pb. Com essa mesma metodologia detectou-se A. costaricensis em Sarasinula marginata experimentalmente infectada, sendo obtido um amplicon do parasito de 400 pb, associado ao amplicon do molusco de 1000 pb.