RESUMEN
The ability to determine the composition and relative frequencies of fish species in large ichthyoplankton swarms could have extremely important ecological applications However, this task is currently hampered by methodological limitations. We proposed a new method for Amazonian species based on hybridization capture of the COI gene DNA from a distant species (Danio rerio), absent from our study area (the Amazon basin). The COI sequence of this species is approximately equidistant from all COI of Amazonian species available. By using this sequence as probe we successfully facilitated the simultaneous identification of fish larvae belonging to the order Siluriformes and to the Characiformes represented in our ichthyoplankton samples. Species relative frequencies, estimated by the number of reads, showed almost perfect correlations with true frequencies estimated by a Sanger approach, allowing the development of a quantitative approach. We also proposed a further improvement to a previous protocol, which enables lowering the sequencing effort by 40 times. This new Metabarcoding by Capture using a Single Probe (MCSP) methodology could have important implications for ecology, fisheries management and conservation in fish biodiversity hotspots worldwide. Our approach could easily be extended to other plant and animal taxa.
Asunto(s)
Código de Barras del ADN Taxonómico/métodos , Peces/clasificación , Larva/genética , Animales , Biodiversidad , Bases de Datos Genéticas , Peces/genética , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
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.
Asunto(s)
Código de Barras del ADN Taxonómico/métodos , Peces/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Larva/genética , Animales , Peces/clasificaciónRESUMEN
The organogenesis of the digestive system was described in the Amazonian pimelodid catfish species Pseudoplatystoma punctifer from hatching (3.5 mm total length, TL) to 41 days post-fertilization (dpf) (58.1 mm TL) reared at 28°C. Newly hatched larvae showed a simple digestive tract, which appeared as a straight undifferentiated and unfolded tube lined by a single layer of columnar epithelial cells (future enterocytes). During the endogenous feeding period, comprised between 20 and 96 h post-fertilization (3.5 to 6.1 mm TL), the larval digestive system experienced a fast transformation with the almost complete development and differentiation of most of digestive organs (buccopahrynx, oesophagus, intestine, liver and exocrine pancreas). Yolk reserves were not completely depleted at the onset of exogenous feeding (4 dpf, 6.1 mm TL), and a period of mixed nutrition was observed up to 6 to 7 dpf (6.8 to 7.3 mm TL) when yolk was definitively exhausted. The stomach was the organ that latest achieved its complete differentiation, characterized by the development of abundant gastric glands in the fundic stomach between 10 and 15 dpf (10.9 to 15.8 mm TL) and the formation of the pyloric sphincter at the junction of the pyloric stomach and the anterior intestine at 15 dpf (15.8 mm TL). The above-mentioned morphological and histological features observed suggested the achievement of a digestive system characteristic of P. punctifer juveniles and adults. The ontogeny of the digestive system in P. punctifer followed the same general pattern as in most Siluriform species so far, although some species-specific differences in the timing of differentiation of several digestive structures were noted, which might be related to different reproductive guilds, egg and larval size or even different larval rearing practices. According to present findings on the histological development of the digestive system in P. punctifer, some recommendations regarding the rearing practices of this species are also provided in order to improve the actual larval rearing techniques of this fast-growing Neotropical catfish species.