von Samson-Himmelstjerna, G.

bioRxiv2022

Evaluation of the nemabiome approach for the study of equine strongylid communities

Basic knowledge on the biology and epidemiology of equine strongylid species remains insufficient although it would contribute to the design of better parasite control strategies. Nemabiome is a convenient tool to quantify and to identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA cistron and its predictive performance and associated biases both remain unaddressed. This study aimed to bridge this knowledge gap using cyathostomin mock communities and comparing performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. The effects of bioinformatic parameters were investigated to determine the best analytical pipelines. Subsequently, barcode predictive abilities were compared across various mock community compositions. The replicability of the approach and the amplification biases of each barcode were estimated. Results were also compared between various types of biological samples, i.e. eggs, infective larvae or adults. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, a reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types, although infective larvae may remain the most tractable in the field. Additional strategies to improve the COI barcode performances are discussed. These results underscore the critical need of mock communities for metabarcoding purposes.

ecology↗

bioRxiv2020

Pharyngeal pumping and tissue-specific transgenic P-glycoprotein expression influence macrocyclic lactone susceptibility in Caenorhabditis elegans

Macrocyclic lactone (ML) resistance has emerged in many parasitic nematodes including the pathogenic horse roundworm Parascaris univalens. The underlying mechanism of ML resistance and the drug penetration routes into the nematodes remain to be elucidated. Drug efflux by P-glycoproteins is considered a potential resistance mechanism but conclusive functional evidence is lacking. To this end, we used a motility assay modified to stimulate pharyngeal pumping (PP) by bacteria or serotonin and tissue-specific expression of Pun- PGP-9 in the free-living model nematode Caenorhabditis elegans. Here, stimulation of PP was identified as an important factor for C. elegans ML susceptibility, increasing the EC50 values of ivermectin by up to 11.1-fold and of moxidectin by 1.2-fold. In this context, intestinal Pun-PGP-9 expression elicited a protective effect against ivermectin and moxidectin only in the presence of PP stimulation, increasing the EC50 values by approximately 3- to 4-fold (ivermectin) or by < 1.3-fold (moxidectin). Conversely, epidermal Pun-PGP-9 expression protected against moxidectin regardless of PP with EC50 fold changes below 1.5 but against ivermectin with a considerable 2.9-fold EC50 increase only when the drug is not actively ingested. Our results highlight the role of active drug ingestion by nematodes for susceptibility and provide conclusive functional evidence for a contribution of P-glycoproteins to ML resistance. Author SummaryParasitic nematode infections pose a serious threat to animal health, in particular in light of the widespread anthelmintic resistance in different nematode species. In equines, the roundworm Parascaris univalens is a major pathogen of foals, exhibiting widespread resistance against macrocyclic lactones (MLs). This represents a particular challenge to animal health, but the underlying mechanisms and drug penetration routes remain mostly unknown. P-glycoprotein ABC-transporters have been linked to ML resistance in several parasitic nematodes. Here we demonstrate by tissue-specific overexpression of Pun-PGP-9 in the free-living model nematode Caenorhabditis elegans their ability to reduce the susceptibility to two commonly used MLs, ivermectin and moxidectin. At the same time, active drug ingestion by pharyngeal pumping (PP) strongly enhanced ivermectin and moderately effects moxidectin susceptibility. In more detail, the effect of intestinal or epidermal Pun-PGP-9 was dependent on active drug ingestion. These observations indicate differences in the drug penetration routes between ML derivatives and allow novel insight into the functional role of P-glycoproteins.

pharmacology and toxicology↗