Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)

Abstract

Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularian Oikopleura dioica, feeding rates were consistently underestimated in the copepod Calanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach to C. finmarchicus fed Rhodomonas marina significantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.

Use of DNA metabarcoding for stomach content analysis in the invasive lionfish Pterois volitans in Puerto Rico

“…There is currently no precise way to quantify prey in the stomach using metabarcoding. Unfortunately, it cannot be assumed that the number of sequences for each particular species represents the amount of DNA (or number of individuals) contributing to the sample because the quality of that DNA depends on many factors including degradation and digestion rates (Deagle & Tollit 2007, Troedsson et al 2009, Valentini et al 2009b). Thus, quantitative analyses at this time are limited to the frequency of prey occurrence.…”

“…Results are limited or biased to the frequency of occurrence, which still provides useful information when seeking to understand localized effects of an invasive predator. However, the underlying variability in DNA quality, differential breakdown of that DNA during digestion, and differences in digestion stages (Deagle & Tollit 2007, Troedsson et al 2009, Valentini et al 2009b, as well as the objective of identifying several different organisms within the same sample (i.e. the gut) (Valentini et al 2009a), still hinder the quantification aspect in metabarcoding of gut contents.…”

Symbiont type influences trophic plasticity of a model cnidarian–dinoflagellate symbiosis

“…To estimate prey DNA breakdown, all samples were also run in qPCR reactions using primers Af18s-1298F and Af18S-1422R (Leal et al, 2014b). As prey is digested, there is a decreasing quantity of genomic DNA with increasing amplicon size (Troedsson et al, 2009). Thus, the ratio between prey DNA content obtained with the two primer sets amplifying amplicon sizes of 73 and 112 bp will be 0 if all prey DNA is degraded (no prey DNA detected using the primer set amplifying the 112 bp amplicon) and 100 if no prey DNA is broken down (equal amount of prey DNA detected using the two primer sets) (Leal et al, 2014b).…”

Feeding selectivity of bivalve larvae on natural plankton assemblages in the Western English Channel

“…As part of this study, we performed molecular analysis of gut content of bivalve larvae following the bottle incubations, and where there were sufficient larvae, on the gut content of larvae taken directly from the field. Recent studies have used DNA-based techniques for dietary analysis including PCR amplification of 18S genes from the gut content of zooplankton (Nejstgaard et al 2003;Troedsson et al 2007;Nejstgaard et al 2008;Troedsson et al 2009;Durbin et al 2012), including meroplanktonic larvae (Maloy et al 2009;Riemann et al 2010;Fileman et al 2014). If the gut content of the predator can first be removed to reduce coamplification of host DNA, then general or universal primers designed to conserved regions (Holland et al 1991) targeting the 18S gene can be used.…”