Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers

Abstract

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring.However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.

Multiplexing PCR allows the identification of within‐species genetic diversity in ancient eDNA

“…First, a set of multiplex PCR primers can be used to improve the taxonomic resolution of a group of interest (Côté et al., 2016) or resolve cryptic species identifications (Brosseau et al., 2019), compared to single‐barcode methods. Second, combining multiple metabarcoding primer sets into a single multiplex PCR can greatly increase the taxonomic information obtained from a single amplification run (De Barba et al., 2014; Schuette et al., 2022; Weber et al., 2023). A traditional metabarcoding approach can achieve similar results by running multiple distinct metabarcoding runs using different primer sets (Epp et al., 2012; Garcés‐Pastor et al., 2022; Willerslev et al., 2014); however, this comes at the cost of additional laboratory expenses and, especially in the case of sed aDNA, usage of available DNA extracts.…”

DNA metabarcoding reveals the dietary profiles of a benthic marine crustacean, Nephrops norvegicus

“…Thus, there is considerable scope for improvement to identify species that are concealed by amplification bias. One solution to overcome host amplification is simply to target specific taxonomic groups such as vertebrates using a well-established 12S rRNA assay [ 18 , 52 ] and the 18S rRNA assay [ 53 ]. This would greatly enhance the resolution of fish species detected to distinguish the proportion of discards that might contribute to the Nephrops diet.…”

“…However, a multimarker assay would be needed to obtain a holistic view of eukaryotic diversity in the diet. Therefore, an alternative solution is the design of a blocking primer which can block amplification of host DNA in a complex sample [ 53 , 54 ]. This can help preferentially bind non-host DNA through incorporating nucleotide mismatches for Nephrops in the primer with a C3 spacer on the 3′ end which blocks extension of the host PCR amplification [ 55 , 56 ].…”

Towards exhaustive community ecology via DNA metabarcoding

“…We excluded strictly methodological studies (e.g., testing the performance of primers), reviews and meta‐analyses, and studies focusing on intraspecific evolutionary patterns. After a detailed screening, we also excluded diet studies, and studies on symbionts or parasites (overall, 72 studies evaluated; see Table S1a) because none of them attempted exhaustive reconstruction of communities, and they used primers focusing on the taxa assumed to be the diet or the symbionts of target organisms (see Weber et al, 2023).…”

“…Nonetheless, just obtaining the list of taxa living in a specific environment provides little insight on how they interact, and analyses of biotic interactions involving a large number of taxa remain extremely challenging. In addition to species occurrences, metabarcoding studies can provide direct information on species interactions, for instance through the analysis of diet, parasites and the host‐associated microbiota (Alberdi et al, 2019; Bass et al, 2015; Ravindran, 2019; Roslin & Majaneva, 2016; Taberlet et al, 2018; Weber et al, 2023), but direct observations of interaction can only focus on a few taxa, and are not enough to reconstruct what happens across all the trophic levels. In the last years, novel frameworks have been proposed for the multitrophic and multitaxa analysis of communities in absence of direct observation of interactions, on the basis of species traits, phylogenetic information and machine learning algorithms (Fricke et al, 2022; Gravel et al, 2019), even though a lot of work remains to be done to assess their power, strengths and limitations (Burian et al, 2021; D'Amen et al, 2018; Fricke et al, 2022; Gravel et al, 2019).…”