Combining spawn egg counts,individual photo-ID and genetic fingerprinting to estimate the population size and sex ratio of an endangered amphibian

According to the International Union for Conservation of Nature Red List, 41% of the world's amphibian species are threatened with extinction, making them more threatened than any other vertebrate group nowadays. Given the global amphibian crisis, comprehensive understanding of demographics and population trends of declining and threatened species is essential for effective management and conservation strategies. Counting egg spawns is widely used to assess population abundance in pond breeding anurans. However, it is unknown how such counts translate into robust population size estimations. We monitored the breeding activity of the Natterjack toad (Epidalea calamita), combining egg string counts and individual photo-identification with Capture-Mark-Recapture population size and operational sex ratio estimation. Male Natterjack toads were identified by the pattern of natural markings with repeated ID of the same individual confirmed for 10% of the samples using genetic fingerprinting. We identified 647 unique individuals within a closed study population at Caherdaniel, Co Kerry. Population estimates derived from egg string counts estimated a breeding population of 368 females (95% CI 353–384) and Capture-Mark-Recapture estimated a breeding population of 1698 males (95% CI 1000–2397). The female:male sex ratio was conservatively estimated at 1:5 (95% CI 1:3–1:6) where 62% ± 6% of females were assumed to spawn. These substantially departed from any priori assumption of 1:1 which could have underestimated the breeding population by up to 72%. Where amphibian absolute population size estimation is necessary, methods should include empirical survey data on operational sex ratios and not rely on assumptions or those derived from the literature which may be highly population and/or context-dependent.     

Multiplex qPCR assay for simultaneous quantification of CYP51-S524T and SdhC-H152R substitutions in European populations of Zymoseptoria tritici

Demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides are currently relied upon for the control of septoria tritici blotch (STB) in European wheat fields. However, multiple mutations have occurred over time in the genes encoding the targeted proteins that have led to a practical loss of fungicide efficacies. Among the different amino acid substitutions in Zymoseptoria tritici associated with resistance to these fungicides, S524T in CYP51 (DMI target) and H152R in SdhC (SDHI target) are regarded as conferring the highest resistance factors to DMI and SDHI, respectively. To facilitate further studies on the monitoring and selection of these substitutions in Z. tritici populations, a multiplex allele-specific quantitative PCR (qPCR) assay allowing for estimation of both allele frequencies in bulk DNA matrices was developed. The assay was then used on complex DNA samples originating from a spore trap network set up in Belgium, Denmark, Sweden, and Ireland in 2017 and 2018, as well as on leaf samples with symptoms. The S524T allele was present in all field samples and its proportion was significantly higher in Ireland than in Belgium, whereas the proportion of H152R was only sporadically present in both countries. The frequency of S524T varied greatly in the airborne inoculum of all four countries; however, the H152R allele was never detected in the airborne inoculum. The method developed in this study can be readily adopted by other laboratories and used for multiple applications including resistance monitoring in field populations of Z. tritici.