Integrative taxonomy: combining molecular and morphological characteristics to identify Lymnaea (Galba) cubensis, intermediate host of Fasciola hepatica / Taxonomia integrativa: combinando características moleculares e morfológicas para identificar Lymnaea (Galba) cubensis, hospedeiro intermediário de Fasciola hepatica

Abstract Despite the epidemiological importance of the Lymnaeidae family regarding transmission of Fasciola hepatica, knowledge about the diversity and distribution of these molluscs and the role of each species in the expansion of fasciolosis remains sparse. Classical morphological (n=10) identification was performed in lymneids from Lagoa Santa, a municipality in the state of Minas Gerais, Brazil, along with molecular and phylogenetic analysis (n=05) based on the partial nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI mtDNA) and ribosomal internal transcribed spacer II (ITS-2 rDNA). The shell morphology made it possible to distinguish the lymneids of Lagoa Santa from Pseudosuccinea columella. Differences found in the penile complex and prostate shape allowed this species to be distinguished from Galba truncatula. However, the homogeneity of reproductive tract characteristics among Lymnaea (Galba) cubensis, L. viator and L. neotropica confirmed that these characteristics show low taxonomic reliability for identifying cryptic species. Genetic divergence analysis for the COI mtDNA gene and ITS-2 region of rDNA revealed greater similarity to Lymnaea (Galba) cubensis. Thus, correct species differentiation is important for monitoring the epidemiological risk of fasciolosis in the state of Minas Gerais, where cases of the disease have increased over recent years.

Resumo Apesar da importância epidemiológica da família Lymnaeidae na transmissão de Fasciola hepatica, o conhecimento sobre a diversidade e a distribuição desses moluscos e o papel de cada espécie, na expansão da fasciolose, ainda é escasso. Realizou-se a identificação morfológica clássica (n=10) em limneídeos de Lagoa Santa, município do estado de Minas Gerais, Brasil, juntamente com a análise molecular e filogenética (n=05), baseada nas sequências parciais de nucleotídeos do gene mitocondrial da subunidade I do citocromo c oxidase (COI mtDNA) e espaçador interno, transcrito do DNA ribossomal II (ITS-2 rDNA). A morfologia da concha possibilitou distinguir os limneídeos de Lagoa Santa de Pseudosuccinea columella. As diferenças encontradas no complexo peniano e na forma da próstata permitiram que essa espécie fosse distinta de Galba truncatula. No entanto, a homogeneidade das características do trato reprodutivo entre Lymnaea (Galba) cubensis, L. viator e L. neotropica confirmou que essas características apresentam baixa confiabilidade taxonômica para a identificação de espécies crípticas. A análise da divergência genética para o gene COI mtDNA e região ITS-2 do rDNA revelou maior similaridade entre os limneídeos de Lagoa Santa com Lymnaea (Galba) cubensis.

Lymnaea cousini , huésped de Fasciola hepatica en el trópico alto andino de Colombia, y sus nuevos haplotipos, confirmados con el marcador mitocondrial del gen de la citocromo oxidasa I / Lymnaea cousini , intermediate host of Fasciola hepatica in the Colombian high tropical Andes, and its new haplotypes confirmed with the mitochondrial marker cytochrome oxidase I

Institución donde se ejecutó el trabajo: Programa de Estudio y Control de Enfermedades Tropicales, PECET, Unidad de Malacología Médica y Trematodos (UMMT), Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia Introducción. La fasciolosis es la enfermedad transmitida por vectores con mayor distribución latitudinal, longitudinal y altitudinal, debido a la capacidad colonizadora del parásito Fasciola hepatica y de sus huéspedes intermediarios, los moluscos limneidos. Estos caracoles se investigan por su importancia epidemiológica, pero su identificación taxonómica es difícil por la similitud fenotípica entre especies. En este sentido, con respecto a Lymnaea cousini , un huésped de F. hepatica en Colombia, existe incertidumbre en razón de su similitud morfológica con L. meridensis , descrita recientemente en Venezuela. Objetivo. Confirmar con el marcador del gen de la citocromo oxidasa I en el ADN mitocondrial COI (ADNmt), el estatus taxonómico de ejemplares morfológicamente caracterizados como L. cousini provenientes de Nariño, Norte de Santander y Santander (Colombia), depositados en la Colección de Moluscos Vectores de la Universidad de Antioquia, VHET N° 37. Materiales y métodos. Para la amplificación del COI mitocondrial, se extrajo ADN total del pie de cada ejemplar con el estuche DNeasy Blood and Tissue (Qiagen ® ). Los productos amplificados se enviaron a secuenciar a Macrogen Inc., Corea. Las 27 secuencias generadas en esta investigación se compararon con secuencias publicadas en el GenBank, incluidas las secuencias de la localidad tipo de L. cousini. Resultados. Se encontraron dos nuevos haplotipos de L. cousini para Colombia. Los especímenes de Nariño correspondían al haplotipo A, referenciado en Ecuador, y los especímenes de Santander y Norte de Santander, a un nuevo haplotipo al que se denominó D. Conclusión. Mediante el marcador mitocondrial del COI , se confirmó que los especímenes pertenecían a la especie L. cousini . Con el hallazgo se duplicó el número de haplotipos conocidos de la especie en Colombia y se amplió su distribución geográfica al suroeste y nordeste de la región altoandina colombiana.

Introduction: Fasciolosis is the disease transmitted by vectors with the highest latitudinal, longitudinal, and altitudinal distribution due to the colonizing capacity of the parasite Fasciola hepatica and its intermediate hosts, Lymnaeidae mollusks. These snails are under research due to their epidemiological importance, but their taxonomic identification is difficult given their interspecific phenotypical similarity. For this reason, there is uncertainty regarding Lymnaea cousini -a host of F. hepatica in Colombia- due to the morphological similarity it has with Lymnaea meridensis , recently described for Venezuela. Objective: To confirm with the COI marker (ADNmt) the taxonomic status of individuals morphologically identified as L. cousini from Nariño, Norte de Santander, and Santander (Colombia), deposited in the Vector Mollusks Collection VHET No. 37 of Universidad de Antioquia. Materials and methods: The amplification of the mitochondrial COI required total DNA extraction of each individual´s foot using the DNeasy Blood and Tissue Kit (Qiagen®). Products amplified were sent for sequencing to Macrogen Inc., Korea. Twenty seven sequences generated in this research were compared to sequences published in the GenBank, including sequences of the type locality of L. cousini . Results: Two new haplotypes of L. cousini were obtained for Colombia. Specimens from Nariño correspond to haplotype A, referenced for Ecuador, and specimens from Santander and Norte de Santander belong to a new haplotype we called haplotype D. Conclusion : By using the mitochondrial COI marker, we confirmed that the species under study did correspond to L. cousini . The number of known haplotypes of the species for Colombia has been duplicated and its geographical distribution has been extended to the southwest and northeast of the Colombian high Andean region.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.