Ontogenetic development and digestive functions in the long snouted seahorse hippocampus guttulatus

Resumo

Introduction The high demand of seahorses for aquarium trade, traditional Chinese medicine (TCM) and as souvenirs, along with the destruction and degradation of coastal habitats (seagrass beds, coral reefs and mangroves), and accidental captures, has raised many concerns about the medium-term viability of seahorse’s natural populations (Hippocampus spp.) (Lourie et al. 1999; Vincent 1996; Vincent et al. 2011; Kumaravel et al. 2012). Currently, all seahorse species are included in Appendix II of CITES (Convention on International Trade in Endangered Species) (CITES 2002), which regulates the legal import and export of seahorses, both alive and dry. The ex-situ production of seahorses has the potential to represent a valid partial alternative to individuals captured in the wild, although, for most species such activity is still a relatively new field of knowledge, which needs to implement the appropriate methodological techniques. The knowledge of morphological development and growth patterns of cultivated fishes is of fundamental importance for the optimization of the production in captivity and for the determination of the offspring quality in controlled restocking or population reinforcement programs. The initial ontogenetic development includes complex processes of cell growth and differentiation that develop on different time-scales for each species. The high mortality of newborn at early developmental stages (from 6 to 15 days after birth), mainly due to the low digestion efficiency and absorption of nutrients from the diet (Celino et al. 2012; Willardino et al. 2012; Blanco et al. 2015), represents a major bottleneck in the rearing of seahorses. The European long snouted seahorse Hippocampus guttulatus Cuvier 1829 is a recent candidate in the ornamental trade and its rearing will help in both the experimental assessment of ecological hypotheses and further development of conservational plans. Many aspects such as the effect of temperature, diet and enzymatic activities have been recently investigated; allowing to successfully breeding H. guttulatus in captivity. In spite of these studies, there is still a general lack of information on many important morphological and physiological aspects of the early development of these animals. Seahorses are species of agastric teleosts, which lack of a functional stomach (Wilson and Castro 2010). In such species, digestion takes place mainly in the intestine (Rønnestad et al. 2013), which develops during the ontogeny from a short and straight tube into a long and segmented duct. The exocrine pancreas synthesizes and secretes digestive enzymes such as proteases, glucosidases and lipases into the intestine, facilitating the process of decomposition of nutrients from the diet into easily absorbed molecules (Zambonino and Cahu 2001). The production of digestive mucosubstances in many marine teleost species is mainly related to glycoconjugates (GCs), molecular components secreted by goblet cells throughout the digestive tract. As reported in other agastric species, the numerous goblet cells present in the oesophagus, secreting neutral, sulphated and sialylated GCs, could be considered as a morphological adaptation that replaces a functional stomach (Jaroszewska et al. 2008). Consequently, neutral GCs are probably involved in the enzymatic digestion and absorptive processes (Domeneghini 1998; Radaelli et al. 2000). Otherwise, sulphated GCs and sialic acid residues might be involved in the regulation of protein and peptide transfer, as well as in the protection from bacteria and other pathogens (Díaz et al. 2003). The specialization and number of these cells vary throughout the ontogenetic development and intestinal region (Domeneghini 1998). Recent studies carried out in juveniles of H. guttulatus show that the levels of some digestive enzymes (trypsin, chitinase and α-amylase) increase from 15 to 30 days after birth, while the levels of lipase were high since birth (Blanco et al. 2015). According to Planas (2012), the survival of newly released seahorses during the first week of life is independent of the type of diet supplied. Even though, the energetic requirements must be satisfied as soon as possible with an appropriated diet in order to prevent high mortalities after the first week. In support to this hypothesis, results from several authors demonstrate that the supply of copepods from 0 to 5 days after birth improves the subsequent survival of seahorses juveniles (Payne and Rippingale 2000; Sheng et al. 2006; Olivotto et al. 2008b; Blanco 2014; Blanco et al. 2015; Planas et al. 2017a). The scientific literature considers newly released seahorses as juveniles completely developed and physiologically functional at the onset of exogenous feeding (from the release to the male’s brood pouch). Up to date, the high mortality of seahorses at early developmental stages (from 0 to 15 days after birth –DAB) has only been related with nutritional factors and the digestibility of the diet. Nonetheless, the hypothesis of an incomplete morphogenesis at those stages has never been considered. If this was the case, the role of GCs in the early development of the digestive system could be critical. Other aspects that have not been yet investigated in seahorses concern the understanding of how copepods improve the digestive efficiency and the processes of nutrient absorption in the absence of a functional stomach. The understanding of all these aspects is essential to carry out an efficient breeding, by contributing to the implementation of optimal rearing conditions. Considering this, the following hypothesis have been established for the present Doctoral Thesis: Hypotheses H.i) Seahorses at birth are morphologically similar to the definitive phenotype but not fully developed; H.ii) The digestive efficiency at early stages of development (from 0 to 15 days after birth, DAB) is not proportional to the morphological development; H.iii) The supply of an inadequate diet at early stages alters the proper development of the digestive system; H.iv) The biochemical composition of goblet cells in the digestive system changes during the ontogeny; H.v) Unforeseen morphological changes in the anatomy of the digestive tract can increase the digestive efficiency. Objectives In order to evaluate these hypotheses the following objectives have been proposed: O.i) To describe the general development of most vital organs during the ontogeny (from 0 to 60 DAB) of H. guttulatus through the application of histological techniques (Chapter 3); O.ii) To determine the effect of the diet (copepods and Artemia spp.) on the biochemical composition of gut and liver of H. guttulatus through conventional histology and infra-red spectroscopy (FPA-FTIR) technique (Chapter 4); O.iii) To describe the digestive functions in H. guttulatus juveniles through conventional histochemistry (Chapter 5); O.iv) To describe the morphological development of the digestive system through high resolution tomography (µ-CT) and 3D image reconstruction (Chapter 5). Results and Discussion In order to accomplish objective O.i, 295 juveniles of H. guttulatus at different developmental stages (from 0 to 60 DAB) were subjected to histological procedures during a research stay at the University of Padova (Italy), obtaining 600 histological sections, stained with Haematoxylin-Eosin. These sections were later analysed during a research stay at the Laboratory of Histology and Histochemistry at the National University of Mar de Plata (Argentina). This analysis showed that, although most vital organs were already present in juveniles after male’s pouch release, they underwent important transformations during the first two months of live (0-60 DAB). Newborn of H. guttulatus were morphologically similar to adults, with some yolk reminiscences surrounding the intestine. At that stage, the nuclei of enterocytes were still undergoing mitosis in the midgut (anterior intestine), with apical acidophilic inclusions and large supranuclear vacuoles in the hindgut (posterior intestine). Digestive glands (liver, pancreas and gallbladder) were already formed at birth, with the exocrine pancreas characterized by numerous granules of zymogen. The first oogonia was observed at 5 DAB. The cranial kidney contributed to the hematopoietic activity throughout the development of this organ since birth, although the lymphoid organs developed in a later stage of the ontogeny (DAB 15). From 5 DAB, lipid deposits accumulated in liver parenchyma and persisted until DAB 15. At that stage (15 DAB), oesophageal longitudinal folds and the intestinal wrinkles in the midgut increased noticeably in length and the specialization of the digestive tract occurred with the development of the first intestinal loop and mucosal folding. From 20 DAB onward, lipids were already mobilised from the liver and oocytes attained the perinuclear stage. The fovea emerged in the eyes at 30 DAB, in coincidence with the change from pelagic to benthic behaviour. At that stage, the most interesting feature was the formation of the second intestinal loop, indicating a progressive enlargement of the intestinal absorption surface. In females, oocytes were observed in a cortical alveoli stage at 60 DAB, indicating a mature condition, while male gonads were never observed during the whole study (0-60 DAB). These results showed that the intestine suffered important morphological changes during the ontogeny, with a progressive increase of the absorption surface. Wilson and Castro (2010) reported that intestinal folds, which increase the absorption surface of nutrients, also favour the mixture of the food with pancreatic enzymes in the intestine. Therefore, the observed morphological changes are likely reflecting an increase of the digestive capacity of seahorses. The presence of acidophilic granules in newborn results from the uptake of proteins through the process of pinocytosis (Falk-Petersen 2005). Otherwise, the absorption of lipids in the intestine is reduced at the beginning of the exogenous feeding, and only a small proportion of lipids absorbed from the food is incorporated into lipoprotein particles, appearing as supranuclear vacuoles (Izquierdo et al. 2000). According to Deplano (1991), the presence of lipid vacuoles observed in the hindgut of H. guttulatus at early stages of development may be due to the immaturity of the enterocytes, which accumulate lipoproteins that will be mobilized in more advanced stages. An important function of the liver is the production of bile, which is stored in the gallbladder for the future breakdown of lipids by the effect of biliary and pancreatic enzymes (Diaz et al. 2002; Rønnestad et al. 2013). In the present study, lipoproteins began to accumulate in the liver of H. guttulatus from 5 DAB, reaching maximum levels at DAB 15. The reduction in hepatic lipid deposition observed from 20 DAB suggested that the ability to metabolize lipids becomes efficient from that age. In support to the hypothesis H.i and H.ii, the present study demonstrated that, although newborn seahorses are released from the male’s brood pouch in an advanced developmental stage, major organs become functional only from the first month of age, in coincidence with the beginning of benthic behaviour (Olivotto et al. 2011). In conclusion, the anatomy of the intestine, an immature digestive system and a reduced lipid transport activity in gut and liver prior to days 20 and 30 are important factors involved in the low digestion efficiency observed in the early stages of development of H. guttulatus. For the determination of the effect of the diet in H. guttulatus (O.ii), histological and biochemical composition of gut and liver was assessed in three groups of newborn, fed with different diets: copepods from 0 to 10 DAB (group C), Artemia nauplii until 10 DAB (group A), copepods from 0 to 5 DAB and Artemia nauplii from 6 to 10 DAB (group CA). From DAB 11, all groups were fed with 24h Artemia metanauplii until 30 DAB. In addition, fatty acid composition was analysed in Artemia nauplii, 24h Artemia metanauplii and copepods. Seahorses fed with copepods showed larger survival and growth rates than the other two groups. At histological level, large lipid vacuoles were found on DAB 5 in the intestine of seahorses belonging to group C, while these vacuoles were smaller in group CA, and absent in group A. In more advanced stages of development (30 DAB), differences in intestinal morphology disappeared. On the other hand, from DAB 10, groups fed with diets including Artemia nauplii (A and CA) showed significant accumulations of hepatic lipids, absent in the individuals of group C. In more advanced stages (20 DAB), no differences in the hepatic parenchyma were observed between treatments. The analysis of the distribution of the lipids, phospholipids, proteins and glycogen in the liver, carried out with FPA-FTIR infrared spectroscopy techniques, showed that during the first 10 DAB, the absorbance related to lipids was higher in groups A and CA, while phospholipids were more abundant in groups C and CA. In more advanced stages (30 DAB), the highest values of glycogen absorbance were recorded for group A, the absorbance values of phospholipids were considerably higher in group C and, finally, proteins showed similar values of absorbance in all the experimental groups. The quality of the lipid source in the diet can affect the development of the gastrointestinal tract and the activity of the digestive enzymes (Zambonino and Cahu 2001; Morais et al. 2007). Although the proportion of carbohydrates and total lipids analysed in the present study was higher in Artemia nauplii compared to copepods, it is know that Artemia is richer in n-3 HUFA triacylglycerols, whereas copepods provide phosphoacylglycerols rich in n-3 HUFA (Sargent et al. 1989; Morais et al. 2007; Rasdi and Quin 2016). Moreover, the high level of digestive enzymes contained in copepods contribute to the larval fish digestion (Zaleha et al. 2012). Feeding in early stages of development with preys rich in phosphoacylglycerols and with high levels of HUFA n-3, improves both digestion and lipid transport, promoting the growth and survival of larvae (Izquierdo et al. 2000; Rasdi and Quin 2016). Seahorses ingest the prey by suction, and the pharynx performs mechanical breakage before entering the intestine, facilitating in this way the action of digestive enzymes in the pancreas and intestine. Due to their hardness, Artemia nauplii pass largely indigested through the gut during the early development of seahorses, whereas copepods could be more efficiently digested at the same developmental stage. Another important factor is the longer retention time of copepods in the intestinal chambers, compared to Artemia nauplii (Govoni et al. 1986), therefore, the digestion and absorption of nutrients results more efficient in copepods. Results achieved through the FPA-FTIR analysis showed that the biochemical composition of the liver change with the diet, highlighting the impact that the nutritional quality of the different live preys has in the early development of seahorses, and supporting H.iii. Consequently, the higher growth rates and survivals observed in the present study in juveniles of seahorses fed with copepods are most likely due, among other reasons, to the higher content and larger proportion of phosphoacylglycerols HUFA n-3 with respect to Artemia. For this reason and considering physiological criteria, the delivery of copepod in the diet of H. guttulatus juveniles is recommended during the first 15 days of life. The study of the digestive functions (O.iii) was carried out through the biochemical characterization of goblet cells present in the digestive tract of H. guttulatus during the ontogenetic development. The identification of the different types of GCs and the biochemical components of goblet cells has been carried out by means of ten different histochemical techniques during the research stays at the University of Padova (Italy) and Mar del Plata (Argentina). The histochemical composition of goblet cells in the intestine varied with the developmental stage and the considered region. Goblet cells appeared in the digestive tract of H. guttulatus since birth, secreting a large amount of acid and neutral glycoconjugates in buccopharinx and oesophagus, while sulphated and neutral GCs have been detected throughout the digestive tract of the seahorses. The midgut secreted mainly neutral GCs in early stages (< 15 DAB) and acid GCs in more advanced stages, while the hindgut was characterized by the presence of acid GCs throughout the development. Sialic acid residues with O-acyl substituents at C7, C8 or C9 have also been observed in most of the digestive tract with the exception for the hindgut. The histochemical characterization was completed with a 3D reconstruction of the digestive tract, carried out from micro-computed tomography images (μ-CT) during a research stay at the Ghent University (Belgium). The analysis of these images, which complemented the conventional histological analysis, revealed a general increase in the intestinal absorption surface in juveniles of H. guttulatus from 15 DAB and the formation of the first intestinal loop from DAB 20. A second loop was formed around DAB 30 in order to better accommodate the intestinal coil in the visceral cavity. By the end of the experiment (60 DAB), the intestinal tract had increased its size notably and it was packed in a double-eight shape. The different GCs elaborated and secreted by the mucous cells of the digestive tract evidenced a high level of histochemical complexity, related to the different functions that mucus plays. In some teleost species, digestion is described to start in the proximal region of the gastrointestinal tract, and continue in the stomach (Domeneghini 1998). Results obtained in the present study demonstrates that a change in the type of GCs observed in the midgut from 15 DAB, was in correspondence with the increased intestinal absorptive surface observed by 3D image reconstruction technique. This change in the type of secreting cells could be determined by an elder maturity of the enterocytes from that age. This finding might explain the better digestive ability of the intestinal tract observed by enzymatic profiles from 15 DAB (Blanco et al. 2015), which suggest that a morphological change in the anatomy of the digestive tract can increase the digestive efficiency, supporting H.v. Conclusions i) Seahorses at birth are morphologically similar to the definitive phenotype but not fully developed. ii) Stages close to DAB 15 (89 Dºeff) and 30 (177 Dºeff) represent two important transitional points in the development of the digestive system in juveniles of H. guttulatus. At 15 DAB the intestinal absorptive surface increase in length and the GCs changes from neutral to acid. The first intestinal loop appear between 15 and 20 DAB, and the second at 30 DAB. iii) The low digestion efficiency observed in the early stages of development of H. guttulatus is also due to a reduced lipid transport activity and an immature digestive system prior to DAB 20 (118 Dºeff) and 30 (177 Dºeff), respectively. iv) Main organs are fully functional at the age of one month (177 Dºeff) in coincidence with the beginning of benthic behaviour. Females became sexually mature at the age of two months (354 Dºeff). Male gonads were not observed during the study period (<60 days). v) The supply of copepods at early stages significantly improves growth, survival and biochemical composition of liver and gut with respect to Artemia. Copepods are more easily digested and provide the essential fatty acids n-3 HUFA. vi) The different GCs elaborated and secreted by the mucous cells of the digestive tract in H. guttulatus revealed that the glycosylation patterns of GCs vary according to the digestive region and stage of development. vii) Mucosal secretions are mainly composed by carboxylated glycoconjugates (acid GCs); however, sulphated and neutral GCs have also been detected throughout the digestive tract of the seahorses. Sialic acid residues have also been observed in most of the alimentary canal with the exception for the hindgut. viii) The increased intestinal surface, digestion and assimilation of the nutrients from 15 DAB improved the digestive efficiency of H. guttulatus from that age. Inadequate diet delivered to fish when the gastrointestinal motility, digestion and absorption are not completely functional, might influence the proper development of the digestive system.