Isolation, identification, and gene expression analysis of the main digestive enzymes during ontogeny of the Neotropical catfish Pseudoplatystoma punctifer (Castelnau, 1855)
Introduction
The early life stages of fish constitute a very sensitive phase during which morphogenesis occurs in a very short time period and optimal feeding and nutrition at mouth opening are key for survival and growth (Yúfera and Darias, 2007); however, very little is known about the nutritional requirements of fish larvae (Holt, 2011). Studying the natural diets of developing larvae can be difficult, and indirect approaches under culture conditions, such as the characterization of the ontogeny of the digestive system, have been widely used to better understand their developing digestive capacities, and nutritional requirements and conditions (Gisbert et al., 2008; Lazo et al., 2011; Rønnestad et al., 2013; Zambonino Infante et al., 2008; Zambonino Infante and Cahu, 2001). The development of the digestive system is species-specific and is a genetically programmed process that is affected by the general life history and reproductive strategy of each species, and by a variety of abiotic and biotic factors, such as water temperature and food availability and composition (Lazo et al., 2011; Rønnestad et al., 2013; Zambonino Infante et al., 2008). In the context of aquaculture, knowledge of the interspecific differences in the relative timing of the differentiation, development, and functionality of the digestive tract and accessory glands during early life stages is essential to develop feeding protocols adapted to the physiological stages of development of each species. The most common approach to estimate the digestive capacities of fish has been the determination of the activity of digestive enzymes by biochemical analysis. Studies evaluating larval digestive performance are generally focused on the appearance of the activity of pancreatic enzymes before the onset of exogenous feeding, the enzymatic maturation of the brush border of enterocytes, and the appearance of pepsin activity in gastric fish, which mark the transition from the larval to the juvenile mode of digestion (Lazo et al., 2011; Rønnestad et al., 2013; Yúfera et al., 2018; Zambonino Infante et al., 2008). Among the different enzymes involved in digestion, α-amylase (EC 3.2.1.1) is synthesized in the exocrine pancreas and is key for the digestion of complex carbohydrates in fish (Cahu and Zambonino Infante, 1994; Darias et al., 2006; Ma et al., 2005; Moyano et al., 1996). The phospholipases A2 (PLA2, EC 3.1.1.4) are essential lipolytic enzymes that hydrolyze phospholipids to generate free fatty acids and lysophospholipids (Dennis, 1994). The pancreatic phospholipase A2-IB (PLA2-IB) is one of the different types of secretory PLA2 (sPLA2) and it is considered the most important digestive enzyme in marine fish (Cahu et al., 2003; Rønnestad et al., 2013). The pancreatic enzyme trypsin (EC 3.4.21.4) is considered the most important alkaline proteolytic enzyme in early life stages of fish and it also plays a key role in activating other pancreatic enzymes in the gut lumen (Rønnestad et al., 2013). Chymotrypsin (EC 3.4.21.1) is another important pancreatic proteolytic enzyme, whose activity is complementary to that of trypsin. During the digestive system ontogeny of gastric fish, trypsin, and chymotrypsin are responsible of protein digestion in the alkaline environment of the intestine until the stomach is formed. At that time, a third proteolytic enzyme, pepsin, appears. Among the two main classes identified, pepsin A and C, the first one is the predominant form, and several isoforms exist in gastric fish (Kapoor et al., 1976). They are responsible for the initial and partial hydrolysis of proteins in the stomach in the presence of an acidic environment. Its precursor, pepsinogen, is produced and secreted by the gastric glands of the stomach, where it is activated by hydrochloric acid (Darias et al., 2005; Darias et al., 2007a; Douglas et al., 1999; Gawlicka et al., 2001). Lipoprotein lipase (LPL, EC 3.1.1.34) is a key regulator of lipid metabolism that hydrolyzes triglyceride-rich lipoproteins transported in the bloodstream as chylomicrons and very-low-density lipoproteins, and the released fatty acids are taken up by the tissues for oxidation or storage (Mead et al., 2002). Contrary to the activity of the main digestive enzymes, the ontogenetic expression pattern of the genes encoding for these enzymes has been studied in relatively few fish species, even though basic knowledge on the molecular mechanisms underlying the function and modulation of the enzymatic hydrolysis of the various dietary macronutrients is necessary to better understand the process of digestion in fish (Yúfera et al., 2018).
Covering more than 6,000,000 km2, the Amazon basin is home to the richest fish fauna in the world with 2406 valid species, 1402 of which are endemic (Jézéquel et al., 2020); however, to our knowledge, no data has been reported on the molecular basis of the early digestive physiology of any Amazonian fish species. Fish is the main source of proteins, essential fatty acids, and micronutrients for the local population, especially for low-income families, and per capita fish consumption is one of the highest in the world (Isaac and de Almeida, 2011). Fish populations are increasingly faced with numerous threats such as pollution, deforestation, hydropower dams, invasive species, and overfishing (Carolsfeld et al., 2003; Winemiller et al., 2016). To counter-balance these effects, aquaculture has been developing steadily for the last decades to contribute to the food needs of a fast growing population (FAO, 2020). Among the cultured species, the highly prized species of the genus Pseudoplatystoma Bleeker, 1862 (maximum total lengths of up to 140 cm (Buitrago-Suárez and Burr, 2007)) are the most produced catfish species in South America, and Brazil is the largest producer (IBGE, 2020; Valladão et al., 2018). Production mostly relies on interspecific hybrids (e.g., P. reticulatum x P. corruscans) for their better growth performance, and more recently, on intergeneric hybrids between Pseudoplatystoma spp. and omnivorous catfish species such as Leiarius marmoratus or Phractocephalus hemioliopterus, since they are less cannibalistic during early life stages, readily accept compound diets, and exhibit faster growth rates than the Pseudoplatystoma spp. parent species (Hashimoto et al., 2012). However, the production of hybrids entails risks for the environment and the aquaculture industry. Hybrids have been frequently detected in natural environments and, in the case of the interspecific hybrids, are contaminating natural stocks due to their fertility (Hashimoto et al., 2013). Additionally, some genetic monitoring studies have revealed that the production, trade, and management of these hybrids are currently uncontrolled in Brazil, as broodstocks are often mistakenly composed of interspecific hybrids and even post-F1 hybrids, causing economic losses (Hashimoto et al., 2015). In this context, in order to achieve sustainability in Pseudoplatystoma spp. aquaculture, genetic improvement programs and culture techniques should be developed for pure species seeking to obtain similar performances as those of hybrids (Alves et al., 2014).
In order to increase basic knowledge on the molecular basis of the ontogeny of the digestive system of commercially important Amazonian fish species, the aim of this study was to understand the molecular phylogeny of the main digestive enzyme precursors and to analyze their ontogenetic expression pattern in Pseudoplatystoma punctifer (Castelnau, 1855). This is a carnivorous migratory catfish species widely distributed in the Amazon basin in Bolivia, Brazil, Colombia, Ecuador, Peru, and Venezuela (Buitrago-Suárez and Burr, 2007) with high potential for aquaculture diversification in the region. We previously analyzed the histological development of the digestive system (Gisbert et al., 2014) and the ontogeny of the digestive enzyme activity (Castro-Ruiz et al., 2019) of this species and in this study we focused on the molecular ontogeny. For that purpose, the digestive enzyme precursors of α-amylase (amy), phospholipase A2 (sPLA2-IB), lipoprotein lipase (lpl), trypsinogen (try), chymotrypsinogen (ctr), and pepsinogen (pga) of this species were isolated, partially sequenced, and identified, with gene expression patterns characterized from 3 to 24 days post-fertilization (dpf).
Section snippets
Fish rearing and feeding protocol
Pseudoplatystoma punctifer larvae were obtained by hormonal-induced spawning of a sexually mature couple of genitors (♀: 4.73 kg; ♂: 1.15 kg) maintained in captivity at the Instituto de Investigaciones de la Amazonia Peruana (IIAP, Iquitos, Peru). The female and male were injected intramuscularly with the synthetic hormone Conceptal® (Intervet, Huixquilucan, México) at 2.6 ml kg−1 and 1 ml kg−1 BW, respectively. Hormone injections were administered in two doses: a first one at 10% and 50% of
Growth and survival
Growth during the ontogeny of P. punctifer followed an exponential curve TL (mm) = 4.181 e0.23×T (r2 = 0.97, P < 0.05) (Fig. 1). Survival rate was 95% and 49% at the end of the Artemia feeding period (17 dpf) and at the end of the experiment (24 dpf), respectively.
Sequences and phylogenetic analyses
The size of the partial nucleotide sequences isolated for P. punctifer is indicated in Table 1. Since the aim of this work was to analyze the expression patterns of these genes during ontogeny, obtaining the full-length cDNAs was not
Discussion
This study provides the first comprehensive analysis of the transcriptional ontogeny of some of the most important digestive enzymes of an Amazonian fish species of the genus Pseudoplatystoma and gives insights into the molecular phylogeny of the digestive enzymes and the development of the digestive capacities and feeding preferences during the early life stage of P. punctifer.
Overall, the phylogenetic relationships of the protein sequences of the studied digestive genes of P. punctifer
Conclusions
The gene expression of the digestive enzymes analyzed during the development of P. punctifer followed the typical profile of a carnivorous species with the exception of amy, which increased during development. Based on this, it is suggested that P. punctifer displays an omnivorous feeding behavior with a preference towards carnivory during the early life stage. The gene expression results, together with those previously obtained at the protein activity level (Castro-Ruiz et al., 2019), showed
Funding
This work was funded by the International Joint Laboratory ‘Evolution and Domestication of the Amazonian Ichthyofauna’ (LMI EDIA, IRD-IIAP-UAGRM, France, Peru and Bolivia) and the IRTA. D.C.-R. benefited from a travel grant from the National Fund for Scientific, Technological Development and Technological Innovation (FONDECYT, Peru) and from a Sud-Nord mobilization grant from the IRD (France).
Declaration of Competing Interest
None.
Acknowledgments
This work has been done within the framework of the network LARVAplus ‘Strategies for the development and improvement of fish larvae production in Ibero-America’ (117RT0521) funded by the Ibero-American Program of Science and Technology for Development (CYTED, Spain).
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