Development of digestive enzymes in larvae of Mayan cichlid Cichlasoma urophthalmus

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6?days after hatching: dah), larvae were fed Artemia nauplii until 15?dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60?dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60?dah. Most digestive enzymes were present from yolk absorption (5?C6?dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8?dah up to 20?dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5?kDa) were detected at 8?dah using SDS?CPAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf?=?0.54) for acid proteases (pepsin-like) from 3?dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13?dah.     

Digestive enzyme activities during early ontogeny in Common snook (Centropomus undecimalis)

Common snook (Centropomus undecimalis) is one of the most important marine species under commercial exploitation in the Gulf of Mexico; for this reason, interest in developing its culture is a priority. However, larviculture remains as the main bottleneck for massive production. In this sense, our objective was to determine the changes of digestive enzymes activities using biochemical and electrophoretic techniques during 36?days of Common snook larviculture fed with live preys (microalgae, rotifers, and Artemia). During larviculture, all digestive enzymatic activities were detected with low values since yolk absorption, 2?days after hatching (dah) onwards. However, the maximum values for alkaline protease (6,500?U?mg?protein^?1), trypsin (0.053?mU?×?10^?3?mg?protein^?1), and Leucine aminopeptidase (1.4?×?10^?3?mU?mg?protein^?1) were detected at 12 dah; for chymotrypsin at 25 dah (3.8?×?10^?3?mU?mg?protein^?1), for carboxypeptidase A (280?mU?mg?protein^?1) and lipase at 36 dah (480?U?mg?protein^?1), for ??-amylase at 7 dah (1.5?U?mg?protein^?1), for acid phosphatases at 34 dah (5.5?U?mg?protein^?1), and finally for alkaline phosphatase at 25 dah (70?U?mg?protein^?1). The alkaline protease zymogram showed two active bands, the first (26.3?kDa) at 25 dah onwards, and the second (51.6?kDa) at 36 dah. The acid protease zymogram showed two bands (RF?=?0.32 and 0.51, respectively) at 34 dah. The digestive enzymatic ontogeny of C. undecimalis is very similar to other strictly marine carnivorous fish, and we suggest that weaning process should be started at 34 dah.     

Development of digestive enzyme activity in larvae of spotted sand bass <Emphasis Type="Italic">Paralabrax maculatofasciatus</Emphasis>. 1. Biochemical analysis

Spotted sand bass Paralabrax maculatofasciatus is a potential aquaculture species in Northwest Mexico. In the last few years it has been possible to close its life cycle and to develop larviculture technology at on pilot scale using live food, however survival values are low (11%) and improvements in growth and survival requires the study of the morpho-physiological development during the initial ontogeny. In this research digestive activity of several enzymes were evaluated in larvae, from hatching to 30 days after hatching (dah), and in live prey (rotifers and Artemia), by use of biochemical and electrophoretic techniques. This paper, is the first of two parts, and covers only the biochemical analysis. All digestive enzyme activities were detected from mouth opening; however the, maximum activities varied among different digestive enzymes. For alkaline protease and trypsin the maximum activities were detected from 12 to 18 dah. Acid protease activity was observed from day 12 onwards. The other digestive enzymes appear between days 4 and 18 after hatching, with marked fluctuations. These activities indicate the beginning of the juvenile stage and the maturation of the digestive system, in agreement with changes that occur during morpho-physiological development and food changes from rotifers to Artemia. All enzymatic activities were detected in rotifers and Artemia, and their contribution to enhancement the digestion capacity of the larvae appears to be low, but cannot be minimised. We concluded that the enzymatic equipment of P. maculatofasciatus larvae is similar to that of other marine fish species, that it becomes complete between days 12 and 18 after hatching, and that it is totally efficient up to 25 dah.     

Immunocytochemical localization of gonadotropins during the development of XX and XY Nile tilapia

Gonadal development and steroidogenesis in teleosts is regulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Earlier studies in tilapia have shown that FSH-β and LH-β appear by 14 days after hatching (dah), results from the current study corroborate with these previous reports in tilapia. Here we demonstrate the appearance of LH in pituitary between 14 and 20 dah. In addition to this the present study primarily focuses on any possible differences in appearance of LH-β and FSH-β immunoreactivity between XX and XY population of Nile tilapia. LH immunoreactivity was found to be lower in pituitary of XX fish when compared to XY fish. The development of FSH-β immunoreactivity in pituitary of the Nile tilapia is also presented. Overall, it remains to be established what significance these findings on the appearance of gonadotropins hold for sex differentiation in tilapia. F. Sakai and I. Swapna contributed equally.     

Histological evaluation of the elimination of Artemia nauplii from larval rearing protocols on the digestive system ontogeny of shi drum (Umbrina cirrosa L.)

The influence of the absence of Artemia nauplii from larval diet protocols on growth and digestive system ontogeny was studied using histological techniques in the shi drum (Umbrina cirrosa). One group of larvae was reared using the standard intensive rearing protocol, which offers a combination of enriched rotifers (Brachionus plicatilis), Artemia spp. nauplii and artificial diet (Std-group). Another group was reared using the same protocol, but without the offering of Artemia nauplii (group No-Artemia). The ontogenesis of the digestive system from hatching to metamorphosis was a very rapid process, and there were no differences between the two feeding regimes in the temporal appearance of the various components of the digestive system. The first organised presence of the hepatic and pancreatic tissue appeared at 2–3 d after hatching (dah), suggesting that these organs function from a very early developmental stage. In the No-Artemia larvae between 13 and 29 dah there was a reduction in the height of enterocytes in the intestinal mucosa, a progressive flattening of the primary intestinal folds in the anterior and posterior intestine and a decrease in lipid stores in the liver, suggesting a period of relative starvation. However, by the end of the study at 41 dah, there were no significant differences in body length, intestinal morphology or liver lipid stores between larvae reared under the two feeding regimes. The study suggests that the diet may influence the maturation and/or function, but not the ontogeny of the digestive system. Furthermore, the rapid differentiation of the digestive system in shi drum and the prompt recovery of the No-Artemia larvae from the symptoms of starvation by 29 dah, indicate a plasticity during ontogenesis and the ability of larvae to adapt to artificial diets at very early developmental stages.     

Digestive enzyme activity at different developmental stages of blackspot seabream, Pagellus bogaraveo (Brunnich 1768)

Blackspot seabream, Pagellus bogaraveo (Brunnich), has been identified as a potential species to diversify European aquaculture production. Although rearing aspects have been widely investigated, little information exists on the nutritional requirements for this species. The aim of this study was to build up information on the activity of digestive enzymes at certain developmental stages of blackspot seabream in order to understand the nutritional needs of larvae and post larvae. Fish larvae were reared from hatching to 55 days after hatching (dah), and the feeding plan consisted in rotifers (5–35 dah), Artemia naupli (30–35 dah) metanaupli (35–45) and Gemma microdiet (45–55 dah). At 7, 11, 21, 45 and 55 days after hatching (dah), pooled samples of fish larvae were collected for analysis of trypsin, amylase, lipase, alkaline phosphatase and leucine–alanine peptidase activity. Up to 21 dah, the whole larvae body was used for enzymatic analysis, whereas in older larvae only the dissected abdominal cavity was used. Blackspot seabream body dry weight growth was exponential, increasing from 60 μg at 5 dah to 30±9.7 mg at 55 dah. Amylase specific activity decreased significantly during development, exhibiting at 11 dah (0.6 U mg^?1 protein) an average value 2.7 times lower than at 7 dah, and remaining stable between 45 and 55 dah (0.7 U mg protein^?1). Trypsin specific activity remained constant until 21 dah (between 38 and 44 mU mg protein^?1), which could be related to the larvae feeding regime. At later stages of development, lipase‐specific activity exhibited a significant increase (P<0.05), being three times higher at 55 dah (8 U mg protein^?1) than at 45 dah. The total activity of the studied digestive enzymes increased significantly during larval development (until 21 dah), whereas afterwards only lipase and leucine–alanine peptidase increased significantly between 45 and 55 dah. The pattern of digestive enzymes activity was related to organogenesis and the type of food used at different developmental stages.     

Development of visual cells in the Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis>

The development of rod and cone photoreceptor cells was investigated in the retinas of Pacific bluefin tuna larvae and juveniles, using RET-P1 monoclonal antibody labeling to identify photoreceptors. At 60 h after hatching, which was about when feeding began, opsin (presumably green opsin (Rh2)) was expressed in the outer segments of cone cells. At 15 days after hatching (dah), although many labeled cone cells were observed in the dorsal retina, the same type of cone cells had partially appeared in the ventral retina. The presence of rod cell bodies was confirmed by the expression of Rh1 opsin at 15 dah. At 21 dah, the presence of outer segments of rod cells was confirmed by the expression of Rh1 opsin and by morphology. The observations suggest that the cone cells were substantially operable upon the development of their outer segment at around the beginning of the post-larval stage, and the rod cells began to function at around 15 to 21 dah, before and during metamorphosis.     

Morphological and histological changes in digestive tract development during starvation in the miiuy croaker

A histological method was used to describe the ontogenetic development of the digestive tract of laboratory-reared miiuy croaker (Miichthys miiuy) and to evaluate the effects of short-term food deprivation on the morphology and histology of the digestive tract. Larvae and juveniles were maintained at 24 °C in a thermostatically controlled system. Three starvation experiments were conducted during different developmental stages: 1–7 days after hatching (dah; prior to benthic swimming); 26–35 dah (during settling); and 42–53 dah (after benthic swimming). According to the structural changes in the ontogenetic development of the digestive tract, three stages were observed. The first stage was from hatching to 3 dah; the digestive tract was undifferentiated in newly hatched larvae and then showed remarkable morphological changes and differentiation. During this period, larvae depended on endogenous nutrition. The second stage (4–20 dah) was a critical period in which larvae transitioned from endogenous feeding to exogenous feeding and the digestive tract fully differentiated into the buccopharynx, oesophagus, stomach, anterior intestine and posterior intestine. Goblet cells and vacuoles appeared in the digestive tract, and pharyngeal teeth and taste buds developed. During the third stage (20–36 dah), the gastric glands developed and the stomach differentiated into the fundic, cardiac and pyloric regions. At 25 dah, pyloric caeca developed and mucosal folds and spiral valves were clearly distinguishable. After 30 dah, the digestive tract did not undergo any noticeable differentiation, indicating the complete development of the digestive system. The wet weight and SGR (specific growth rate) of miiuy croaker larvae and juveniles greatly decreased when they were deprived of food, and compensatory growth was observed in re-feeding juveniles. The livers of starved larvae and juveniles were atrophied and dark coloured, the intestines were transparent and thin, and the stomach cubages were reduced. The histological effects of starvation were mainly evident in the degeneration of cells in digestive organs, as seen in the shrinkage and separation of cells and the loss of intercellular substances in the liver, pancreas, intestine and stomach. These changes became more severe with increased duration of starvation. In addition, the histological structure of the digestive tracts of starved larvae and juveniles partly recovered after re-feeding, and the effects of starvation on miiuy croaker were age dependent.     

Changes in digestive enzyme activity during initial ontogeny of bay snook Petenia splendida

Several samples of P. splendida larvae were obtained from eggs until day 60 after hatching (dah) to determine acid and alkaline proteases, trypsin, chymotrypsin, leucine aminopeptidase, α-amylase, lipase, and acid and alkaline phosphatase activities using biochemical techniques. Additionally, SDS–PAGE alkaline protease zymogram and PAGE acid protease zymogram were carried out to identify active isoforms during larviculture. Alkaline protease and chymotrypsin were present at the moment of hatching, increased gradually reaching the maximum values at 35 dah. Trypsin and leucine aminopeptidase activities were low from hatching, increasing gradually as larvae grew. Alkaline protease zymogram showed four zymogens, which appears at different days, remaining present until the end of the larviculture (95.2 kDa at 11 dah, 26.4 kDa at 9 dah, 21.4 kDa at 3 dah, and 23.3 kDa at hatching). Pepsin activity was present at day 7 after hatching and increased progressively until the end of the larviculture. Acid protease zymogram only showed one zymogen (0.65 rf), which appear at 6 dah. Lipase was high at the time of hatching and increased until 15 dah, after which decreased gradually. Amylase was high from the beginning and until 15 dah and then decreased rapidly to almost nothing onward. Alkaline and acid phosphatases presented a high activity at the egg stage, fell slightly during the first feeding and increased again from 20 to 30 dah. Results obtained in this study show that larvae can be fed artificial diets starting on day 10 after hatching.     

Gonadal sex differentiation in protandrous anemone fish, Amphiprion clarkii

To clarify the process of sex differentiation in protandrous anemone fish, histological structure of their gonads was examined from 1 day to 214 days after hatching (dah). Gonadal development was first characterized by differentiation of ovarian tissues at 61 dah, followed by interspersed appearance of testicular tissues in the ovary at 214 dah.     

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker <Emphasis Type="Italic">Miichthys miiuy</Emphasis> larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.     

Effects of salinity on Brazilian flounder Paralichthys orbignyanus from fertilization to juvenile settlement

The Brazilian flounder, Paralichthys orbignyanus, is a promising candidate for aquaculture, especially due to the euryhalinity demonstrated experimentally for large juveniles (3 g) and sub-adults. Flounder are observed in estuaries and were already reared in fresh and salt water, however little is known with respect of salinity tolerance during their early development. The objective of this work was to evaluate the effects of salinity from fertilization to juvenile settlement. Three experiments were conducted to evaluate the effects of salinity. In trial 1 adult flounder were captured in the wild, transferred to the laboratory and induced to spawn. The gametes were hand striped, split in four samples and fertilized with water at 10, 15, 25, and 35‰. Eggs were considered fertilized when the first cell divisions were observed under the microscope. For the trial 2 newly hatched larvae were reared in four salinities (5, 10, 20, and 30‰) and their growth and survival were observed until metamorphosis. In trial 3 larvae and juvenile of different ages (6, 16, 30, 45, and 60 dah — days after hatching) were evaluated for their tolerance to fresh water. Although the fertilization rate was directly proportional to salinity, hatching was successful only in full salt water. Larvae did not survive in low salinity water (5‰) longer than 6 days, whereas growth was improved when larvae were reared at 20 and 30‰. Young larvae cannot survive in salinities below 4‰, but at 30 dah juvenile presented 100% survival in fresh water. The present findings demonstrate the need for high salinity water (30–35‰) for the successful reproduction and incubation of P. orbignyanus eggs. Flounder can be reared successfully at intermediate salinities (20‰) during larviculture, but at lower salinities (5 and 10‰) their survival and growth are impaired. However, immediately after flounder metamorphose into juveniles they survive even in fresh water, demonstrating the strong euryhalinity of this species even at early stages of development.     

Hydrogen peroxide,iodine solution and methylene solution highly enhance the hatching rate of freshwater ornamental fish species

The aim of this study was to evaluate the effect of hydrogen peroxide, iodine solution (PVP) and methylene blue on eggs disinfection of three ornamental fish species, Danio rerio, Pterophyllum scalare and Gymnocorymbus ternetzi. The main idea was to create conditions to enhance the hatching rates. Eggs of each species were exposed to different concentrations of hydrogen peroxide (5, 10, 15 and 25 mg/L), PVP (0.25, 0.5, 0.75 and 1 mg/L) and methylene blue (0.5 1, 2 and 3 mg/L). The optimal doses ranged between species and chemicals: for G. ternetzi, the concentrations that high enhanced the hatching rate were 1 mg/L for the PVP treatment, 25 mg/L for the hydrogen peroxide treatment and 3 mg/L for methylene blue treatment; for P. scalare, the best results were achieved with 25 mg/L for hydrogen peroxide treatment and 3 mg/L for methylene blue treatment. By contrast, for all the different chemical did not increased the D. rerio hatching rate. Results showed that hydrogen peroxide and methylene blue are the most versatile, effective and safe to use in these species. On the other hand, PVP can be used but with many precautions due to very low safety margin. Results clearly show that the optimal concentration of chemicals for eggs disinfection is fish species dependent and it is completely wrong to extrapolate concentrations between different chemicals and fish species. Our study suggests that P. scalare can be used as a model in study of effectiveness of new chemicals with potential to disinfect water and increase hatching rates.     

Immunocytochemical identification of adenohypophyseal cells in the pirarucu (<Emphasis Type="Italic">Arapaima gigas</Emphasis>), an Amazonian basal teleost

The adenohypophysis (AH) of juvenile pirarucu (Arapaima gigas), a representative species of the Osteoglossomorpha (bonytongue fishes, one of the oldest living groups of the teleosts), was studied using histochemical and immunocytochemical methods. The AH is comprised of the pars distalis (PD), without a clear distinction between rostral pars distalis (RPD) and proximal pars distalis (PPD), and the pars intermedia (PI). The neurohypophysis (NH) is positioned on top of the PD and penetrates and branches into the PI. In the most rostral dorsal portion of the PD, adrenocorticotropic cells and fusiform gonadotropic cells were found. In the central PD, scarce prolactin-producing cells and growth-hormone-producing cells were located mainly in the dorsal part, whereas round gonadotropic cells were abundant in the ventral portion of this region. Human thyrotropin immunoreactive cells were not found in the entire AH. In the PI, melanotropic, some adrenocorticotropic, and somatolactin-producing cells were located intermingled surrounding the neurohypophyseal branches. Our results showed that the A. gigas pituitary has some basal characteristics between the ancient Actinopterygii and the more derived teleosts.     

Effects of Photoperiod on Embryos and Larvae of Tawny Puffer,Takifugu flavidus

To determine the optimal condition of photoperiod for embryo development and larval culture of tawny puffer, Takifugu flavidus, the effects of photoperiod on egg hatching and the growth and survival of larvae from 3 to 23 d after hatching (dah) were assessed. The results show that photoperiodic responses varied with the developmental stage during the early development of tawny puffer. Photoperiod did not significantly affect the hatch rate, viability of 24 h post‐hatch larvae, and total mortality rate of tawny puffer embryos; however, it affected the incubation period, which significantly increased with increasing photoperiod from 6 to 24 h. The shortest incubation period was predicted to occur at 3.56 h day length from the quadratic relationship. However, photoperiod strongly affected the growth and survival of the tawny puffer larvae from 3 to 23 dah. Increasing the day length resulted in improvement of the larval growth and survival within the 0–12 h day length range; however, beyond the 12 h day length, a longer day length was not beneficial, and a 24 h continuous light had a negative effect on growth. From the quadratic relationships, the highest growth and survival rates were predicted to occur at 14.16 h and 17.74 h day length, respectively. Therefore, the optimal photoperiods for tawny puffer embryos and larvae were 3–6 and 12–18 h day length, respectively. The results of this study are useful in increasing the production of the species during incubation and larval culture.     

Molecular cloning of the three gonadotropin subunits and early expression of FSHβ during sex differentiation in the nile tilapia, Oreochromis niloticus

Gonadotropin (GTH)α, FSHβ and LHβ cDNAs were cloned from the Nile tilapia. Northern blot analysis detected a single band for each subunit. Preliminary studies indicate that FSHβ is expressed as early as 0 days after hatching (dah) in the fish pituitaries.