Expression and activity of trypsin and pepsin during larval development of the spotted rose snapper Lutjanus guttatus

The present study aimed to describe and understand the development of the digestive system in spotted rose snapper (Lutjanus guttatus) larvae from hatching to 40 days post-hatch (dph). The mouth opened between 2 and 3 dph, at that moment the digestive tract was barely differentiated into the anterior and posterior intestine, although the liver and pancreas were already present. Gastric glands were observed until 20 dph, followed by the differentiation of the stomach between 20 and 25 dph. Trypsinogen expression and trypsin activity were detected at hatching, increasing concomitantly to larval development and the change in the type of food. Maximum levels of trypsinogen expression were observed at 25 dph, when animals were fed with Artemia nauplii, and maximum trypsin activity was detected at 35 dph, when larvae were fed with an artificial diet. On the other hand, pepsinogen gene expression was detected at 18 dph, two days before pepsin enzymatic activity and appearance of gastric glands. Maximum pepsin activity was also observed at 35 dph. These results suggest that in this species weaning could be initiated at an earlier age than is currently practiced (between 28 and 30 dph), since larvae of spotted rose snapper develop a functional stomach between days 20 and 25 post-hatch.     

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (E_h) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 810±100 μmol/kg by 63 dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing E_h from −239±1 to −262±7 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and E_h were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.     

Thyroid hormone receptor α and regulation of type 3 deiodinase

Mice deficient in thyroid hormone receptor α (TRα) display hypersensitivity to thyroid hormone (TH), with normal serum TSH but diminished serum T(4). Our aim was to determine whether altered TH metabolism played a role in this hypersensitivity. TRα knockout (KO) mice have lower levels of rT(3), and lower rT(3)/T(4) ratios compared with wild-type (WT) mice. These alterations could be due to increased type 1 deiodinase (D1) or decreased type 3 deiodinase (D3). No differences in D1 mRNA expression and enzymatic activity were found between WT and TRαKO mice. We observed that T(3) treatment increased D3 mRNA in mouse embryonic fibroblasts obtained from WT or TRβKO mice, but not in those from TRαKO mice. T(3) stimulated the promoter activity of 1.5 kb 5'-flanking region of the human (h) DIO3 promoter in GH3 cells after cotransfection with hTRα but not with hTRβ. Moreover, treatment of GH3 cells with T(3) increased D3 mRNA after overexpression of TRα. The region necessary for the T(3)-TRα stimulation of the hD3 promoter (region -1200 to -1369) was identified by transfection studies in Neuro2A cells that stably overexpress either TRα or TRβ. These results indicate that TRα mediates the up-regulation of D3 by TH in vitro. TRαKO mice display impairment in the regulation of D3 by TH in both brain and pituitary and have reduced clearance rate of TH as a consequence of D3 deregulation. We conclude that the absence of TRα results in decreased clearance of TH by D3 and contributes to the TH hypersensitivity.     

Nucleotide Enrichment of Live Feed: A Promising Protocol for Rearing of Atlantic Cod Gadus morhua Larvae

We investigated the effect of two commercial nucleotide products (NT1 and NT2), administered through live feed, on growth and stress tolerance of Atlantic cod larvae. Expression of genes related to muscle growth (igf-1, igf1r, igf-2, fst, fgf6, myod, and myhc) and nucleotide metabolism (uox, hprt, ndk, and uck) was evaluated during larval development. In addition, the expression of genes related to stress (hif-1α, hif-2α, hif-3α, and mb) was studied after an air exposure stress test. The enrichment of rotifers with nucleotides did not reveal any difference in nucleotide profiles, the exception being the RNA level of the NT1-enriched group that was significantly higher than the unenriched rotifer. Unenriched Artemia showed poor nucleotide profiles compared to enriched Artemia since 5' UMP, 5' GMP, and 5' AMP were observed only in the nucleotide groups. At 38?days post-hatch (dph), NT1 group had significantly higher dry weight (3.1?±?0.1?mg) than the control (CON; 2.3?±?0.1?mg). The treatments did not produce any significant differences in the expression of the key myogenic genes. Among the genes associated with nucleotide metabolism, ndk was down-regulated in NT1 at 38 dph. In the air exposure test, survival was significantly higher in the CON (77?±?6?%) than in NT1 (48?±?3?%) and NT2 (50?±?3?%). After air exposure, mb was expressed at lower levels in NT2 group, hif-2α was induced in NT1 group, and hif-3α was upregulated in all groups. Our findings indicate that the improvement in the nucleotide profile of Artemia upon nucleotide enrichment could eventuate in the rapid growth of larvae.     

Ontogeny of the gastrointestinal tract and selected digestive enzymes in cobia Rachycentron canadum (L.)

The ontogeny of the digestive system of cobia Rachycentron canadum from hatching to 22 days post-hatch (dph) (20·1 mm standard length) was examined with light microscopy. The activities of selected pancreatic enzymes were also determined during this period in order to optimize current rearing methods for this species. At hatching (3·6 mm), the digestive tract consisted of a relatively undifferentiated, straight tube positioned dorsally to the yolk sac. The major morphological changes in the digestive tract primarily occurred over the first 1–4 dph (3·6–4·4 mm). During this time, larvae began exogenous feeding (3 dph) and the digestive tract differentiated into five histologically distinct regions: buccopharynx, oesophagus, stomach anlage, anterior intestine and posterior intestine. Yolk reserves were exhausted by 5 dph (4·5 mm) and the oil globule began rapidly decreasing in size disappearing entirely by 9–10 dph (6·3–6·8 mm). Gastric glands differentiated at this time, and by 12 dph (8·1 mm) surface mucous cells of the stomach anlage stained positive for neutral mucosubstances. By 16 dph (11·6 mm), the blind sac (fundic region) of the stomach formed as did the pyloric caecae which initially appeared as a single protrusion of the anterior intestine just ventral to the pyloric sphincter. Generally, enzyme activities (U larva⁻¹) for amylase (0·0–1·8), chymotrypsin (0·0–7902·4), trypsin (0·2–16·6) and lipase (9·3–1319·0) were measurable at or soon after hatching and increased steadily from c. 8–22 dph (5·7–20·1 mm). The results of this study are discussed in terms of current and future weaning practices of this species.     

Development of a qRT-PCR assay to determine the relative mRNA expression of two different trypsins in Atlantic cod (Gadus morhua)

A fluorescence resonance energy transfer (FRET) based quantitative RT-PCR method (qRT PCR) was developed in this study for measuring the mRNA expression of trypsins Y and I in the Atlantic cod. Atlantic cod beta-actin was used as the reference gene and standard curves were created for quantification of the mRNA expression levels. For yet unknown reasons, the Atlantic cod (Gadus morhua) produces several trypsins with different characteristics. Trypsin I is the most common and best characterized of these but trypsin Y is a recently discovered enzyme. The recombinant form of trypsin Y was found to have unique characteristics relative to trypsin I. The native form of trypsin Y has proven difficult to isolate from the cod and activity assays do not distinguish between the activities of trypsin I and trypsin Y. The results show that trypsin Y mRNA is expressed in a very low copy number relative to that of trypsin I (ratio of 1:1340), which may explain the difficulty of isolating the native form of trypsin Y.     

Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus)

Histological, biochemical and molecular techniques were used to describe the functional development of the pancreas in winter flounder (Pleuronectes americanus) with specific reference to the expression of three trypsinogen genes. The pancreas was identified shortly following hatch, appearing as a compact structure situated dorsal and slightly posterior to the liver. As the larval fish approached metamorphosis, the pancreas became diffuse, spreading throughout the mesentery surrounding the stomach, the upper intestine and the pyloric caecae. Trypsin 2 expression was detected from 5 days post-hatch (dph). Two other related trypsinogen genes isolated from the pyloric caecae (Trypsin 1) and the intestine (Trypsin 3) showed contrasting results. Trypsin 1 showed very low levels of expression and only in late larval stages and metamorphosis. Trypsin 3 showed expression only after 20 dph. In order to determine tissue-specific expression of the three trypsinogen genes, the RNA from seven gastrointestinal-associated tissues was examined. Trypsin 1 and Trypsin 2 expression was most notably associated with the pyloric caecae, cardiac stomach, pyloric stomach and the rectum, although some variation in expression level between tissues was observed. Trypsin 3 expression had a narrower tissue distribution and was only associated with the pyloric caecae and the rectum. The tissue expression patterns observed here are likely due in part to the diffuse nature of the pancreas. Trypsin-like activity was evident from hatch and continued at significant levels through to at least 25 dph.     

Early development of New Zealand hapuku Polyprion oxygeneios eggs and larvae

This study describes for the first time the normal development of New Zealand hapuku Polyprion oxygeneios embryos and larvae reared from fertilization to 11 days post-hatch (dph) at a constant temperature. Fertilized eggs were obtained from natural spawnings from communally reared captive wild broodstock. Eggs averaged 2 mm in diameter and had single or multiple oil globules. Embryos developed following the main fish embryological stages and required an average of 1859·50 degree hours post-fertilization (dhpf) to hatch. The newly hatched larvae (4·86 mm mean total length, L(T) ) were undifferentiated, with unpigmented eyes, a single and simple alimentary tube and a finfold that covered the entire body. Larvae relied on the energy from the yolk-sac reserves until 11 dph (7·33 mm mean L(T) ), when yolk-sac reabsorption was almost completed. Some of the major developmental stages from hatching to yolk-sac reabsorption were eye pigmentation (5 dph), upper jaw formation (7 dph), lower jaw formation (8 dph) and mouth opening (8-9 dph). By 9 dph, the digestive system consisted of pancreas, liver, primordial stomach, anterior and posterior gut; therefore, P. oxygeneios larvae would be capable of feeding on live prey. The developmental, morphological and histological data described constitutes essential baseline information on P. oxygeneios biology and normal development.     

Distribution of cholecystokinin-immunoreactive cells in the gut of developing Atlantic cod Gadus morhua L. larvae fed zooplankton or rotifers

One of the main gastrointestinal hormones, cholecystokinin (CCK), was studied in order to advance understanding of the control of the digestive process in Atlantic cod Gadus morhua larvae after onset of first feeding. Larvae were fed either natural zooplankton or enriched rotifers in similar rearing systems and sampled from hatching to 22 days post-hatch (dph). CCK was visualized by immunohistochemistry and the first CCK-immunoreactive (IR) cells were detected at 8 dph corresponding to 6 days after first feeding. The CCK-IR cells were mostly found in the anterior midgut, and the number of CCK-IR cells was lower in the posterior midgut. They were also present in the hindgut of some of the larvae, but not in the foregut. No clear differences were found in the ontogenetic appearance and the distribution pattern of CCK-IR cells between the two dietary treatments. This indicates that the onset of CCK production in the gut as well as the spatial distribution of the CCK-IR cells is not differentially affected by these diets. To what extent the hormone production itself is influenced by dietary factors needs to be studied by more sensitive methods.     

Organogenesis of the digestive tract in the white seabream,Diplodus sargus. Histological and histochemical approaches

The ontogeny of the digestive tract of the white seabream, Diplodus sargus during the larval development up to day 45 post-hatching (dph) has been studied using histological and histochemical techniques. The oesophageal goblet cells appeared around 6 dph and contained neutral and acid mucosubstances (PAS/diastase-PAS and Alcian Blue pH 2.5 positive reactions). An incipient stomach can be distinguished from 2 dph but the first sign of gastric gland development was detected around 13-15 dph, increasing in number and size by 22-23 dph. Gastric glands were concentrated in the cardiac stomach region and they had a high content of protein rich in tyrosine, arginine and tryptophan. Acidophilic supranuclear inclusions related to pynocitosis of proteins, were already observed in the intestinal cells of the posterior intestine around 4-6 dph (exogenous feeding) and they were present until 25 dph. The intestinal mucous cells appeared between 15-18 dph and contained a mixture of neutral and acid mucosubstances/glycoconjugates, carboxylated ones being more abundant than the sulphated ones. The stomach and gastric glands were fully developed by the first month of life marking the beginning of digestive features characteristic of the juvenile stage. Around 4-6 dph, glycogen, proteins and neutral lipids were observed in the granular cytoplasm of hepatocytes. Strongly acidophilic zymogen granules were also present, at this time, in the basophilic cytoplasm of the exocrine pancreatic acinar cells and contained abundant proteins, especially rich in arginine, tyrosine and tryptophan.     

Expression of the oligopeptide transporter, PepT1, in larval Atlantic cod (Gadus morhua)

The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter, PepT1. This study evaluates the expression of PepT1 in larval Atlantic cod (Gadus morhua) during the three weeks following the onset of exogenous feeding. Larval Atlantic cod were fed either wild captured zooplankton or enriched rotifers. cDNA was prepared from whole cod larvae preceding first feeding and at 1000 each Tuesday and Thursday for the following three weeks. Spatial and temporal expression patterns of PepT1 mRNA were compared between fish consuming the two prey types using in situ hybridization and quantitative real-time PCR. Results indicated that PepT1 mRNA was expressed prior to the onset of exogenous feeding. In addition, PepT1 was expressed throughout the digestive system except the esophagus and sphincter regions. Expression slightly increased following first-feeding and continued to increase throughout the study for larvae feeding on both prey types. When comparing PepT1 expression in larvae larger than 0.15-mg dry mass with expression levels in larvae prior to feeding, no differences were detected for larvae fed rotifers, but the larvae fed zooplankton had significantly greater PepT1 expression at the larger size. In addition, PepT1 expression in the zooplankton fed larvae larger than 0.15-mg dry mass had significantly greater expression than rotifer fed larvae of a similar weight. Switching prey types did not affect PepT1 expression. These results indicate that Atlantic cod PepT1 expression was slightly different relative to dietary treatment during the three weeks following first-feeding. In addition, PepT1 may play an important role in the larval nutrition since it is widely expressed in the digestive tract.     

Viral nerve necrosis in hatchery-produced fry of Asian seabass Lates calcarifer: sequential microscopic analysis of histopathology

We studied the natural progression of viral nerve necrosis (VNN) in larvae of Asian seabass Lates calcarifer Bloch from 0 to 40 days post-hatch (dph). The hatchlings were reared in the vicinity of a confirmed nodavirus-affected older batch. Using light and electron microscopy (EM), we made a sequential analysis of histopathological manifestations in nerve tissue and other organs. There were no changes from the day of hatching until 4 dph. Larvae at 4 dph had viral particles in the intramuscular spaces underlying the skin, but the nerve cells of the brain were normal. The first signs of necrosis of the brain cells were observed at 6 dph. EM observations revealed characteristic membrane-bound viral particles measuring 30 nm in the cytoplasm of nerve cells of the brain, spinal cord and retina. Histological samples of fry examined when group mortalities reached 20 to 35% revealed highly vacuolated brains, empty nerve cell cytoplasm and viral particles in the intercellular spaces. Viral particles occurred extensively in the intramuscular spaces and the epidermal layers. These observations were corroborated by positive immunostaining of the virus-rich intramuscular spaces. EM studies also revealed progressive necrotic changes in the cells harboring the virus. Results emphasize the need to maintain hygiene in the hatchery environment and to develop strategies for prevention of disease spread among cohabiting seabass and other susceptible fish larvae. Intramuscular localization of the nodavirus in both preclinical healthy-looking and post-clinical moribund larvae suggests that virus neutralization strategies during larval development could be effective in controlling VNN-associated mortalities.     

Ontogeny of American paddlefish lymphoid tissues

The temporal and spatial distribution of American paddlefish Polyodon spathula immune cell populations was determined using enzyme cytochemistry and immunohistochemistry. Monocytes and macrophages were present in the renal haematopoietic tissue, spleen, meningeal myeloid tissue, cardiac myeloid tissue and lamina propria of the spiral valve at 7 days post-hatch (dph). Immature lymphocytes were present in the renal haematopoietic tissue, spleen, meningeal myeloid tissue, cardiac myeloid tissue, thymus and lamina propria of the spiral valve at 7 dph. Type A lymphocytes (T-cell like) were demonstrated in the thymus by 21 dph. Type B immunoglobulin positive lymphocytes (B-cell like) were present in the renal haematopoietic tissue, cardiac myeloid tissue and lamina propria of the spiral valve by 7 dph, the thymus at 21 dph, the spleen by 56 dph, and were not observed in the meningeal myeloid tissue of paddlefish aged 7–28 dph. Granulocytes were present in the renal haematopoietic tissue, thymus, spleen, meningeal myeloid tissue, cardiac myeloid tissue and lamina propria of the spiral valve by 7 dph. The spleens in 7–28 dph fish were predominately red pulp. Differentiation of leukocytic and erythrocytic compartments (white and red pulp, respectively) was not apparent in the spleen until 56 dph. Remarkable thymic cortical and medullary differentiation was not yet present at 28 dph, and the thymus was not sampled at 56, 84 or 112 dph. The cardiac myeloid tissue was not developed until 56 dph. Peyer's patches were present in the lamina propria by 56 dph. Paddlefish lympho-myeloid structures are therefore slow to develop, and vaccination procedures should be performed at 2–4 months post-hatch.     

Histological development of the digestive system of Mayan cichlid Cichlasoma urophthalmus (Günther 1862)

The ontogeny of the digestive tract in Cichlasoma urophthalmus was studied by means of optical microscopy from hatching to 30 days post‐hatching (dph; 855 degree days, dd). The development of the digestive system in this precocial species was a very intense and asynchronous process, which proceeded from both distal ends interiorly. At hatching, the digestive tract consisted of a straight tube with a smooth lumen dorsally attached to the yolk‐sac. The digestive accessory glands were already differentiated and eosinophilic zymogen granules were visible in the exocrine pancreas. At the onset of exogenous feeding between 5 and 6 dph (142.5–171.0 cumulative thermal units, CTU), the buccopharynx, oesophagus, intestine, liver and pancreas were almost completely differentiated, with the exception of the gastric stomach that completed its differentiation between 11 and 14 dph (313.5–399.0 CTU). The development of gastric glands at 14 dph and the differentiation of the stomach in the fundic, cardiac and pyloric regions at 19 dph (541.5 CTU) were the last major events in digestive tract development and designated the onset of the juvenile period. Remnants of yolk were still detected until 16 dph (456.0 CTU), indicating a long period of mixed nutrition that lasted between 10 and 11 days (285.0–313.5 CTU). The results of the organogenesis of larvae complement previous data on the functionality of the digestive system and represent a useful tool for establishing the functional systemic capabilities and physiological requirements of larvae to ensure optimal welfare and growth under aquaculture conditions, which might be useful for improving current larval rearing practices for this cichlid species.