Choice of markers and biosampler species
Our results emphasize the importance of combining several markers to
enhance species detection and confidence in the results (Alberdi et al.
2019; Polanco et al. 2021; Tournayre et al. 2024). As expected,MiFish and Teleo showed substantial overlap, detecting
more than 70% of species in common and yielding highly similar
frequencies of occurrences in biosamplers. Both markers identified
nearly the same number of fish species, though 10 species were detected
exclusively by one or the other. These unique species were found in a
small number of digestive contents, suggesting that these differences
likely reflect the rarity of these species in the environment, or at
least their lower consumption by crustaceans, rather than variations in
primer robustness or discriminatory power. The third marker tested
(MG2 ) preferentially amplified invertebrates and detected
significantly fewer fish species than the 12S rRNA markers. While it
appears less effective for fish monitoring, it may still prove helpful
for validating the identity of the biosampler species or to inventory
other taxonomic groups (Tournayre et al., 2024). Although using multiple
markers can increase the time and cost of molecular analyses,
laboratory-processing time can be reduced by pooling stomach contents
while ensuring reliable detection through large sequencing depth.
Another important practical consideration is the choice of natural
samplers based on their abundance and distribution range. For
neotropical rivers, Macrobrachium brasiliense and Macrobrachium
olfersii which are widely distributed across South America and can be
easily collected in large numbers, seem to be ideal candidates,
collectively detecting 94% of the total dDNA fish diversity.
Understanding the spatial ecology and feeding habits of potential
biosamplers can help guide the selection of species (or species
combinations) that will yield the most comprehensive inventory
(Calvignac-Spencer et al., 2013). This knowledge can help assess the
spatial and temporal resolution of the dietary data, which depends on
the home range size and digestion rate of organisms (Feller 2006; Prog,
Pihl, and Rosenberg 1984). In this context, an interesting perspective
is to compare scavenger crustacean dDNA and the increasingly used water
eDNA approaches to determine which one provides the best insight into
local assemblages in large rivers (Cantera, Decotte, et al., 2022;
Cantera et al., 2023b, 2023a; Coutant et al., 2023; Zinger et al.,
2020). Indeed, while water eDNA provides a snapshot of biodiversity,
dietary DNA may offer a more temporally integrated signal due to gut
retention time and circumvents several issues commonly encountered with
water eDNA in turpid river systems, such as high sediment loads that
limit filtration capacity and high concentrations of organic matter that
can cause PCR inhibition.