As technological advancements enhance our ability to study population genetics, we must understand how the intrinsic properties of our datasets influence the decisions we make when designing experiments. Filtering parameter thresholds, such as call rate and minimum minor allele frequency (MAF), are known to affect inferences of population structure in reduced representation sequencing (RRS) studies. However, it is unclear to what extent the impacts of these parameter choices vary across datasets. Here, we reviewed literature on filtering choices and levels of genetic differentiation across RRS studies on wild populations to highlight the diverse approaches that have been used. Next, we hypothesized that choices in filtering thresholds would have the greatest impact when analyzing datasets with low levels of genetic differentiation between populations. To test this hypothesis, we produced seven simulated RRS datasets with varying levels of population structure, and analyzed them using four different combinations of call rate and MAF. We performed the same analysis on two empirical RRS datasets (low or high population structure). Our simulated and empirical results suggest that the effects of filtering choices indeed vary based on inherent levels of differentiation: specifically, choosing stringent filtering choices was important to detect distinct populations that were slightly differentiated, but not those that were highly differentiated. As a result, experimental design and analysis choices need to consider attributes of each specific dataset. Based on our literature review and analyses, we recommend testing a range of filtering parameter choices, and presenting all results with clear justification for ultimate filtering decisions used in downstream analyses.
Parasites may suppress the immune function of an infected host using microRNAs (miRNAs) to prevent protein production. Nonetheless, little is known about the diversity of miRNAs and their mode(s) of action. In this study, we investigated the effects of infection by a parasitic lungworm (Rhabdias pseudosphaerocephala) on miRNA and mRNA expression of its host, the invasive cane toad (Rhinella marina). We compared miRNA and mRNA expression in naive toads that had never been infected by lungworms to toads that were infected with lungworms for the first time in their lives, and to toads that were infected the second time in their lives (i.e., had two consecutive infections). In total, we identified 434 known miRNAs and 106 potential novel miRNAs. Compared to uninfected toads, infected animals upregulated five (single-infection treatment) or four (multiple-infection treatment) miRNAs. Seven of these differentially expressed miRNAs were associated with gene pathways related to the immune response, potentially reflecting immunosuppression of cane toads by their parasites. Infected hosts did not respond with substantial mRNA transcription, with only one differentially expressed gene between control and single-infection hosts. Our study suggests that miRNA-mediated interactions may play a role in mediating the interaction between the parasite and its host. Our findings clarify the role of miRNAs in host-parasite interactions, in a system in which an ongoing range expansion by the host has generated substantial divergence in host-parasite interactions.
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