DNA Metabarcoding of Amazonian Ichthyoplankton Swarms

Abstract

Tropical rainforests harbor extraordinary biodiversity. The Amazon basin is thought to hold 30% of all river fish species in the world. Information about the ecology, reproduction, and recruitment of most species is still lacking, thus hampering fisheries management and successful conservation strategies. One of the key understudied issues in the study of population dynamics is recruitment. Fish larval ecology in tropical biomes is still in its infancy owing to identification difficulties. Molecular techniques are very promising tools for the identification of larvae at the species level. However, one of their limits is obtaining individual sequences with large samples of larvae. To facilitate this task, we developed a new method based on the massive parallel sequencing capability of next generation sequencing (NGS) coupled with hybridization capture. We focused on the mitochondrial marker cytochrome oxidase I (COI). The results obtained using the new method were compared with individual larval sequencing. We validated the ability of the method to identify Amazonian catfish larvae at the species level and to estimate the relative abundance of species in batches of larvae. Finally, we applied the method and provided evidence for strong temporal variation in reproductive activity of catfish species in the Ucayalí River in the Peruvian Amazon. This new time and cost effective method enables the acquisition of large datasets, paving the way for a finer understanding of reproductive dynamics and recruitment patterns of tropical fish species, with major implications for fisheries management and conservation.

Metabarcoding by capture using a single COI probe (MCSP) to identify and quantify fish species in ichthyoplankton swarms

“…In this study, we will compare the result from this new approach to a previous approach using four probes designed for siluriforms [ 21 ]. In the following text, we will use the term single-probe for the COI from Danio and the term siluriform probes for the four probes previously developed [ 21 ].…”

“…These two batches were randomly divided into two subsets. The first subset was used to perform individual DNA extractions and were then Sanger sequenced [ 21 ]. These individual DNAs (270 individuals from the Marañon River and 102 from the Napo River) were pooled in equimolar quantities to constitute mock samples (Mar-Mock and Nap-Mock).…”

“…We previously successfully used enrichment by capture to develop a method allowing quantification of siluriform species in Amazonian ichthyoplankton samples, using four different probes distributed in the main clades of the phylogeny of Amazonian siluriform species [ 21 ]. Yet, siluriform species represent only ~20% of the Amazonian fish diversity.…”

DNA metabarcoding of Neotropical ichthyoplankton: Enabling high accuracy with lower cost

“…This observation reinforces the idea that more sequences of fish species must be deposited in databases, especially in river basins with rich fish fauna as the Upper Paraná River. Another positive aspect is that, compared with DNA barcoding (Arroyave and Stiassny 2014, Becker et al 2015, Frantine-Silva et al 2015, our method can trigger significant advances in the quality of ecological studies in fish, as observed in some recent studies with metabarcoding (Guillerault et al 2017, Maggia et al 2017, Mariac et al 2018, Barbato et al 2019 and, mainly, to ensure significant reduction in labor time and cost per specimen reaching 80% of reduction (Shokralla et al 2015). Despite some differences in obtaining DNA samples, both environmental DNA (eDNA) and DNA metabarcoding from bulk samples are subject to false negatives and false positives (Ficetola Gentile et al 2015Gentile et al , 2016Divoll et al 2018).…”

“…To solve this problem, DNA metabarcoding, based on next generation sequencing (NGS) could offer an alternative approach, allowing a reduction of labor time as well as costs. Although Neotropical ichthyofauna metabarcoding studies are scarce (Maggia et al 2017, Mariac et al 2018, Sales et al 2019, this technique has been utilized in several ecological studies on ichthyoplankton and also to identify gut contents (Cruaud et al 2017, Guillerault et al 2017, Maggia et al 2017, Divoll et al 2018, Barbato et al 2019).…”