Dataset Information

Application of real-time PCR for the identification of the endangered species Galemys pyrenaicus through faecal samples.

ABSTRACT:

Background

Currently, many micromammals are important targets for study. The endangered Galemys pyrenaicus is an outstanding example. Globally, their populations have suffered a substantial decline in last 20 years. In the surveyed area, the capture of desman is legally forbidden due to the high conservation concerns. Reason by non-invasive sampling through faeces is proposed for its monitoring. Furthermore, the confusion between faeces from desman and Mediterranean water shrews must be considered. Thus, the aim of this study was focused on developing RT-PCR assays to determine the presence of Galemys pyrenaicus and N. a. anomalus from non-invasive samples.

Methods and results

The study was conducted in the mountains of the System Central of Extremadura (Spain). A total of 186 samples were collected from 2018 to 2021 by experts where historically reported and/or our previous studies confirmed their presence. RT-PCR assays using hydrolysis probes were designed to detect genetic material from both desman and Mediterranean water shrews and its specificity was confirmed. The reliability of the method was further assessed by PCR sequencing of mitochondrial Cyb and d-loop, resulting fully compatible with the RT-PCR approach. Intraspecific phylogenetic relationship was reported to improve knowledge about mtDNA variability in the desman from the Central System.

Conclusions

We demonstrated that RT-PCR gives a gold opportunity to further map the species using faeces which minimizes disturbance and reports both population status and individual presence. Cost-effective RT-PCR combined with field-collected faeces allows us to better investigate the full range of occurrence of the species.

SUBMITTER: Ripa A

PROVIDER: S-EPMC10770224 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Publications

Application of real-time PCR for the identification of the endangered species Galemys pyrenaicus through faecal samples.

Ripa Adriana A Díaz-Caballero José A JA Palacios-González María Jesús MJ Espinosa Antonio A García-Zapata Juan Luis JL Fernández-Garcia José Luis JL

Molecular biology reports 20240105 1

<h4>Background</h4>Currently, many micromammals are important targets for study. The endangered Galemys pyrenaicus is an outstanding example. Globally, their populations have suffered a substantial decline in last 20 years. In the surveyed area, the capture of desman is legally forbidden due to the high conservation concerns. Reason by non-invasive sampling through faeces is proposed for its monitoring. Furthermore, the confusion between faeces from desman and Mediterranean water shrews must be ...[more]

PMID: 38180618

Similar Datasets

Project description:BackgroundSpecies with strict ecological requirements may provide new insights into the forces that shaped the geographic variation of genetic diversity. The Pyrenean desman, Galemys pyrenaicus, is a small semi-aquatic mammal that inhabits clean streams of the northern half of the Iberian Peninsula and is endangered in most of its geographic range, but its genetic structure is currently unknown. While the stringent ecological demands derived from its aquatic habitat might have caused a partition of the genetic diversity among river basins, Pleistocene glaciations would have generated a genetic pattern related to glacial refugia.ResultsTo study the relative importance of historical and ecological factors in the genetic structure of G. pyrenaicus, we used mitochondrial and intronic sequences of specimens covering most of the species range. We show, first, that the Pyrenean desman has very low levels of genetic diversity compared to other mammals. In addition, phylogenetic and dating analyses of the mitochondrial sequences reveal a strong phylogeographic structure of a Middle Pleistocene origin, suggesting that the main lineages arose during periods of glacial isolation. Furthermore, both the spatial distribution of nuclear and mitochondrial diversity and the results of species distribution modeling suggest the existence of a major glacial refugium in the northwestern part of the Iberian Peninsula. Finally, the main mitochondrial lineages show a striking parapatric distribution without any apparent exchange of mitochondrial haplotypes between the lineages that came into secondary contact (although with certain permeability to nuclear genes), indicating incomplete mixing after the post-glacial recolonization. On the other hand, when we analyzed the partition of the genetic diversity among river basins, the Pyrenean desman showed a lower than expected genetic differentiation among main rivers.ConclusionsThe analysis of mitochondrial and intronic markers in G. pyrenaicus showed the predominant effects of Pleistocene glaciations on the genetic structure of this species, while the distribution of the genetic diversity was not greatly influenced by the main river systems. These results and, particularly, the discovery of a marked phylogeographic structure, may have important implications for the conservation of the Pyrenean desman.

Project description:BackgroundThe diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.MethodsTwo multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC.ResultsMultiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.ConclusionsOur multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.

Project description:Molecular techniques allow non-invasive dietary studies from faeces, providing an invaluable tool to unveil ecological requirements of endangered or elusive species. They contribute to progress on important issues such as genomics, population genetics, dietary studies or reproductive analyses, essential knowledge for conservation biology. Nevertheless, these techniques require general methods to be tailored to the specific research objectives, as well as to substrate- and species-specific constraints. In this pilot study we test a range of available primers to optimise diet analysis from metabarcoding of faeces of a generalist aquatic insectivore, the endangered Pyrenean desman (Galemys pyrenaicus, É. Geoffroy Saint-Hilaire, 1811, Talpidae), as a step to improve the knowledge of the conservation biology of this species. Twenty-four faeces were collected in the field, DNA was extracted from them, and fragments of the standard barcode region (COI) were PCR amplified by using five primer sets (Brandon-Mong, Gillet, Leray, Meusnier and Zeale). PCR outputs were sequenced on the Illumina MiSeq platform, sequences were processed, clustered into OTUs (Operational Taxonomic Units) using UPARSE algorithm and BLASTed against the NCBI database. Although all primer sets successfully amplified their target fragments, they differed considerably in the amounts of sequence reads, rough OTUs, and taxonomically assigned OTUs. Primer sets consistently identified a few abundant prey taxa, probably representing the staple food of the Pyrenean desman. However, they differed in the less common prey groups. Overall, the combination of Gillet and Zeale primer sets were most cost-effective to identify the widest taxonomic range of prey as well as the desman itself, which could be further improved stepwise by adding sequentially the outputs of Leray, Brandon-Mong and Meusnier primers. These results are relevant for the conservation biology of this endangered species as they allow a better characterization of its food and habitat requirements.

Dataset Information

Application of real-time PCR for the identification of the endangered species Galemys pyrenaicus through faecal samples.

Background

Methods and results

Conclusions

Publications

Application of real-time PCR for the identification of the endangered species Galemys pyrenaicus through faecal samples.

Similar Datasets

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