Dataset Information

Molecular assays to reliably detect and quantify predation on a forest pest in bats faeces.

ABSTRACT: Targeted molecular methods such as conventional PCR (cPCR) and quantitative PCR (qPCR), combined with species-specific primers and probes, are widely applied for pest species detection. Besides, the potential of qPCR to quantify DNA in samples makes it an invaluable molecular tool to infer the predation levels on specific prey by analysing predators' stools. Nevertheless, studies on the diet of bats failed to find any empirical relationship, and it remains to be evaluated. Thus, we developed and evaluated two species-specific PCR assays to detect and quantify DNA of a major forest pest, the pine processionary, Thaumetopoea pityocampa, in bats' faeces. Further, we empirically compared a range of different known DNA concentrations (input) of the target species mixed with mocks and bat faecal samples against DNA abundances yielded by qPCR (output) for a quantitative assessment. Overall, cPCR showed a lower detection rate than qPCR, but augmenting the replicate effort from one to three replicates led to a greater increase in the detection rate of the cPCR (from 57 to 80%) than the qPCR (from 90 to 99%). The quantitative experiment results showed a highly significant correlation between the input and output DNA concentrations (t = 10.84, p < 0.001) with a mean slope value of 1.05, indicating the accuracy of our qPCR assay to estimate DNA abundance of T. pityocampa in bat faeces. The framework of this study can be taken as a model to design similar assays applicable to other species of interest, such as agricultural pests or insects of public health concern.

SUBMITTER: Baroja U

PROVIDER: S-EPMC8831491 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular assays to reliably detect and quantify predation on a forest pest in bats faeces.

Baroja Unai U Garin Inazio I Vallejo Nerea N Caro Amaia A Ibáñez Carlos C Basso Andrea A Goiti Urtzi U

Scientific reports 20220210 1

Targeted molecular methods such as conventional PCR (cPCR) and quantitative PCR (qPCR), combined with species-specific primers and probes, are widely applied for pest species detection. Besides, the potential of qPCR to quantify DNA in samples makes it an invaluable molecular tool to infer the predation levels on specific prey by analysing predators' stools. Nevertheless, studies on the diet of bats failed to find any empirical relationship, and it remains to be evaluated. Thus, we developed and ...[more]

PMID: 35145165

Similar Datasets

Project description:IntroductionThe latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype-tolerance using digital PCR.MethodsA subtype-B-specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype-tolerance in digital PCR (Bio-Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide.ResultsHIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R2 = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R2 = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty-nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG.ConclusionsThe results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non-B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.

Project description:Sampling methods that are both scientifically rigorous and ethical are cornerstones of any experimental biological research. Since its introduction 30 years ago, the method of using plasticine prey to quantify predation pressure has become increasingly popular in biology. However, recent studies have questioned the accuracy of the method, suggesting that misinterpretation of predator bite marks and the artificiality of the models may bias the results. Yet, bias per se might not be a methodological issue as soon as its statistical distribution in the samples is even, quantifiable, and thus correctable in quantitative analyses. In this study, we focus on avian predation of lepidopteran larvae models, which is one of the most extensively studied predator-prey interactions across diverse ecosystems worldwide. We compared bird predation on plasticine caterpillar models to that on dead caterpillars of similar size and color, using camera traps to assess actual predation events and to evaluate observer accuracy in identifying predation marks a posteriori. The question of whether plasticine models reliably measure insectivorous bird predation remained unanswered, for two reasons: (1) even the evaluation of experienced observers in the posterior assessment of predation marks on plasticine models was subjective to some extent, and (2) camera traps failed to reflect predation rates as assessed by observers, partly because they could only record evidence of bird presence rather than actual predation events. Camera traps detected more evidence of bird presence than predation clues on plasticine models, suggesting that fake prey may underestimate the foraging activity of avian insectivores. The evaluation of avian predation on real caterpillar corpses was probably also compromised by losses to other predators, likely ants. Given the uncertainties and limitations revealed by this study, and in the current absence of more effective monitoring methods, it remains simpler, more cost-effective, ethical, and reliable to keep using plasticine models to assess avian predation. However, it is important to continue developing improved monitoring technologies to better evaluate and refine these methods in order to advance research in this field.

Dataset Information

Molecular assays to reliably detect and quantify predation on a forest pest in bats faeces.

Publications

Molecular assays to reliably detect and quantify predation on a forest pest in bats faeces.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets