Partial sequence characterization and ontogenetic expression of genes involved in lipid metabolism in the tropical gar (Atractosteus tropicus)

The tropical gar (Atractosteus tropicus) is an economically and socially important freshwater fish species from southeastern Mexico, with high aquaculture potential. This study determined the expression of acetyl‐CoA carboxylase (ACC1), fatty acid synthase (FAS), and carnitine palmitoyl transferase 1 (CPT1C) in several tissues of adult specimens, as well as in early ontogeny stages of tropical gar. Partial sequences of all genes were obtained for brain, liver, muscle, mesenteric adipose, kidney, testis, gill, and heart in males. Specific qPCR primers were used to compare the expression between tissues and also during larviculture, starting with embryos (0 days after hatching, [DAH]), and at 5, 10, 15, 20, 25, and 30 DAH. Mesenteric adipose tissue, liver, and muscle showed maximum FAS, ACC1, and CPT1C expression. Additionally, the highest expression of lipogenic genes was detected in embryos (0 DAH), decreasing between 5 and 10 DAH, then increasing at 15 DAH, and ultimately decreasing again from 20 DAH onwards. Based on these results, it can be concluded that genes encoding ACC1, FAS, and CPT1C are expressed differentially across tissues of tropical gar adults, and are regulated during larval development.     

Evaluation of carbohydrate/lipid ratios on growth and metabolic response in tropical gar (Atractosteus tropicus) juvenile

Five experimental diets with different carbohydrates/lipid ratio (CHO:L, 0.75, 1.28, 2.10, 2.52, 4.63) were formulated. Fish were randomly assigned by triplicate (10 fish per tank, 0.50 ± 0.01 g). Growth was registered every 15 days until end of the experiment (45 days). Samples were taken for analysis of chemical carcass composition, blood chemistry, glycogen and lipid liver content, digestive and metabolic enzyme activities. Results showed that survival, growth performance parameters and plasma glucose were not affected by treatments (p > 0.05). Lipids of carcass and liver, as well as triglycerides and plasma cholesterol increase significantly as CHO:L ratio decreased. While an inversely proportional tendency was observed for carcass protein and liver glycogen (p < 0.05). Digestive enzymes did not show significant differences among treatments (p > 0.05). Finally, Hexokinase (HK), glucokinase (GK), phosphoenol pyruvate carboxykinase (PEPCK) and fructose‐1,6‐biphosphase (FBPase) showed high regulation by carbohydrates up to the CHO:L ratio of 2.10 (p < 0.05), while pyruvate kinase (PK) activity was not significantly affected by the CHO:L ratio. By the other side, glucose‐6‐phosphate dehydrogenase (G6PD) tends to significantly increase as the CHO:L ratio increases. We conclude that A. tropicus fry have a high capacity to utilize carbohydrates substituting lipids as energy source in balanced diets.     

Assessment of dietary lipid sources in tropical gar,Atractosteus tropicus larvae: Growth parameters and intermediary lipogenic gene expression

Five experimental diets containing different lipid sources, fish oil (D1), soybean lecithin (D2), corn oil (D3), canola oil (D4) and olive oil (D5), were evaluated in Atractosteus tropicus larvae for the relative gene expression of the enzymes fatty acid synthase (fas), acetyl‐CoA carboxylase 1 (acc1) and carnitine palmitoyltransferase 1C (cpt1c), in addition to their effects on larval growth, survival and cannibalism during a 30‐day feeding trial. Higher growth and survival were obtained in treatments D1 and D2, and lower performance in diets D3, D4 and D5. The highest levels of expression of fas and acc1 occurred in larvae fed with D1, which contained high amounts of n‐3 long‐chain polyunsaturated fatty acids (LC‐PUFA), mainly DHA and EPA FA are regulators of lipogenesis. The higher cpt1c expression in plant‐based diets is attributed to the fact that these diets are rich in α‐linolenic acid (ALA) and low DHA, EPA and ARA levels that favour ß‐oxidation. In conclusion, the diets with fish oil (D1) and soybean lecithin (D2) were the best treatments for larval growth, survival and cannibalism and thus appear to meet both lipid and energy requirements of A. tropicus larvae, meanwhile the use of vegetable oils influences the expression of intermediary lipogenic genes.     

Fructooligosaccharide supplementation in diets for tropical gar (Atractosteus tropicus) juvenile: Effects on morphophysiology and intestinal barrier function

Supplementary fructooligosaccharides (FOS) were evaluated on growth, survival, gut morphology, digestive enzyme activity and expression of intestinal barrier genes in tropical gar (Atractosteus tropicus). Four treatments were designed to include FOS (5, 10, 15 and 20 g/Kg) and were compared with a control (0 g/Kg) and the current commercial trout diet. Ten fish were stocked (0.25 ± 0.01 g and 4.2 ± 0.4 cm total length) per experimental unit by triplicate in a 45‐day experiment. Fish fed with 5 and 10 g/Kg of FOS had the highest average weight and total average length. Fish fed 5 g/Kg FOS showed the best growth indexes (absolute weight gain: 2.16; specific growth rate: 4.96; and feed conversion rate: 1.23) and survival (96.66%) and also had the highest acid protease, chymotrypsin and leucine aminopeptidase activities. Fish fed 5 g/Kg FOS had a high absorption area and accumulation of lipids in the liver. Finally, relative overexpression of occluding (occ), nucleotide‐binding oligomerization domain‐2 (nod2) and mucin 2 (muc2) was detected in fish fed 10 and 15 g/Kg FOS. The supplementation between 5 and 10 g/Kg FOS in diets for A. tropicus juveniles could be beneficial for growth, digestive enzyme activities, gut morphology and intestinal barrier function.     

Digestive enzymatic activity on Tropical gar (Atractosteus tropicus) larvae fed different diets

Digestive enzymatic activity and growth performance on tropical gar (Atractosteus tropicus) larvae fed Artemia nauplii (LF), frozen adult Artemia (AB), an artificial diet (AF) with 46% protein and 16% lipids and a starvation group (SG) from first feeding (5?days after hatching-5 DAH) to 34 DAH were studied. All larvae under starvation (SG) died at 15 DAH. By the end of the experimental period, morphological variables (total length, wet weight and specific growth rate) were significant in larvae fed AF compared to LF and AB. All enzymes studied in the experiment were present since the start of exogenous feeding (including pepsin) and the enzymatic activity varied with the diets. Low levels of enzymatic activity were observed until the 29 DAH; however, after this moment, there was a significant increase (eightfold), particularly for the AF treatment. In vitro protein digestibility tests performed with enzymatic extracts showed that artificial diets with 52% protein and 14% lipids were better digested by larvae before 30 DAH, while diets with 45% protein and 11% lipids were better digested after this age. Taking into account the better growth performance, higher enzymatic activity and better protein digestibility obtained, artificial diets can be used since the start of exogenous feeding on tropical gar larvae, as in other lepisosteids.     

Weaning of alligator gar (Atractosteus spatula) larvae to artificial diets

Growth performance and survival of alligator gar (Atractosteus spatula) larvae fed Artemia nauplii, two artificial diets (with different protein content and buoyancy) and a control under starvation at first feeding [5 days after hatching (5 DAH)] were studied. All larvae under starvation (C) died at 10 DAH, while survival was near 60% for the rest of the treatments at the end of the experimental period. By the end of the experimental period morphological variables (total length, snout length, wet weight and dry weight) were better in larvae fed the artificial diets compared to live food. These results were confirmed by means of nucleic acid indexes and digestive enzymatic activity. The importance of the relationship between diet size and snout gape was evidenced from these results. Differences between artificial diets were attributed to different digestibility values. Alligator gar larvae are able to grow and survive satisfactorily when fed artificial diets from the start of exogenous feeding, provided that these have an adequate size, buoyancy and quality.     

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4 % with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80 % with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8 % with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.     

Digestive enzyme activity during early larval development of the Cuban gar Atractosteus tristoechus

The ontogenesis of digestive enzymes (proteases, amylases, lipases, and phosphatases) in Cuban gar Atractosteus tristoechus was determined in larvae between 5 and 18 days after hatching (DAH). Variations in specific activities of most enzymes were related to the transition from endogenous to exogenous feeding and to the transition from the larval to the juvenile stage. Alkaline protease activity was not detected until 8 DAH in contrast to acid protease activity, which was quantifiable at 5 DAH. Acid protease activity was consistently higher than alkaline protease activity, indicating the presence of a functional stomach in the early stages of larval development. The acid protease activities of larvae and adults were compared by means of zymogram analysis. Four acid protease bands were found in adults (two more than in larvae). This result is the first time that more than one band of acid proteolytic activity has been found in Lepisosteidae. High lipase activity indicated the importance of lipid utilization, particularly during yolk-sac absorption. In contrast to the other enzymes studied, amylase activity was consistently low, probably due to the strictly carnivorous diet of gar larvae and their low capacity for carbohydrate digestion. High activities of aminopeptidase and acid and alkaline phosphatases suggest intestinal absorption. This result, together with the existence of a short gut and a lower proteolytic activity in the intestine than in the stomach, suggest that most of the proteolytic activity takes place in the stomach, while the primary function of the intestine is nutrient uptake.     

Using cornstarch in microparticulate diets for larvicultured tropical gar (<Emphasis Type="Italic">Atractosteus tropicus</Emphasis>)

Aquaculture in Mexico has been developed by the cultivation of commercial species. In Tabasco, the cultivation of native species is mainly limited by the lack of nutrition studies to support its crop profitability. Among these species is the tropical gar (Atractosteus tropicus), which has great potential for cultivation. However, the nutritional value of carbohydrates in diets for this species which contribute to improved growth and survival, have not been evalulated,. Thus, in the present investigation, isoprotein and isolipid diets have been designed based on the substitution of cellulose by corn starch (D1: 0 % starch–15 % cellulose, D2: 7.5 % starch–7.5 % cellulose and D3: 15 % starch–0 % cellulose) and compared with a commercial trout diet (45 % protein and 16 % lipids). A total of 1800 larvae (0.008 ± 0.002 g and 10.5 ± LT 0.126 mm) were used, distributed in a recirculation system in order to evaluate growth and survival for 30 days. The results show higher growth and survival of 97 % of larvae fed the D3 diet, while cannibalism in the species was mitigated. Major digestive enzyme activities occurred (acid protease, alkaline protease, trypsin, chymotrypsin, leucine aminopeptidase, carboxypeptidase A, lipase, α-glucosidase and amylase) for larvae fed D3. It is concluded that the contribution of corn starch (15 %) replacing cellulose in the diet improves growth and survival of this species.     

Partial characterization of digestive proteases of the three‐spot cichlid Cichlasoma trimaculatum (Günter 1867)

Partial characterization of digestive proteases in the three‐spot cichlid Cichlasoma trimaculatum juveniles was conducted. It was determined that there is higher alkaline proteases activity (3.95 ± 0.32 IU mg^?1 protein) compared to acidic proteases (2.01 ± 0.57 IU mg^?1 protein). Optimal temperature for alkaline proteases is 60 °C which resulted in more thermostability to temperature changes. On the other hand, optimal temperature for acidic proteases is 50 °C. Optimal pH for acidic proteases was pH 2, while for alkaline proteases, it was pH 10, which resulted in more stability in relation to pH changes than acidic protease. The use of specific inhibitors and the SDS‐PAGE electrophoresis analysis revealed seven types of bands for alkaline proteases, which make evident the main presence of serine proteases. In acidic proteases, more than 98 g kg^?1 of the activity was inhibited with pepstatin A inhibitor. Therefore, it is evident that C. trimaculatum digestion is composed by acidic and alkaline proteases; thus, it should be considered an omnivorous fish.     

Stimulatory effect of thyroid hormones improves larval development and reproductive performance in alligator gar (Atractosteus spatula) and spotted gar (Lepisosteus oculatus)

Development of alligator gar (Atractosteus spatula) and spotted gar (Lepisosteus oculatus) larvae was evaluated after exposure of embryos to thyroid hormones (TH) by two different methods of administration. For the first method, alligator gar embryos were placed in a water bath with triiodothyronine (T₃; 1 ppm for 2 h), and control treatment embryos were placed in a water bath without T₃. For the second method, spotted gar adult males and females were injected with different TH during induced spawning: a group was injected with T₃ [20 mg kg^?1 body weight (BW)], a second group with thyroxine (T₄; 20 mg kg^?1 BW), a third group with thyroid‐stimulating hormone (TSH; 4 IU kg^?1BW) and a control group with dimethyl sulfoxide. Both methods resulted in increases in the concentrations of T₃ and T₄ in embryos of both species. An increase in the alkaline enzymatic activity of TH‐treated larvae was observed as well as an increase in the RNA:DNA, RNA:Dry weight and DNA:Dry weight ratios at hatching. Some positive effects of TH application were: a significant increase in the hatching rate of spotted gar larvae from TH‐injected parents and a higher percentage of successful spawns. Evenness of larval sizes and a higher survival rate were observed in alligator gar larvae. Snout development was accelerated by TH treatment in both species.     

Development of testis and digestive tract in longnose gar (Lepisosteus osseus) at the onset of exogenous feeding of larvae and in juveniles

The aim of this study was to describe the ontogenetic development of the testis and the alimentary tract in longnose gar (Lepisosteus osseus) related to fish size and age at the onset of exogenous feeding and late ontogenesis. Using light microscopy, testes were first detected histologically by the appearance of primordial germ cells 9 days after the first exogenous feeding [31–31.5 mm total body length(TL)] and presumptive seminiferous tubules (maleness characteristic) in fish of 107 mm TL. The present histological studies indicated that the alimentary tract of lepisosteids is completely functional at the beginning of exogenous feeding, several days before the completion of yolk absorption. Based on these results, we have concluded that garfish larvae/juveniles can be effectively trained to consume formulated diets at early stages, after an initial feeding of live food for 2–3 days (23.5 mm TL). Our findings provide evidence of the first controlled rearing of longnose gar using live and formulated diets, providing the possibility of experimental work with this non‐teleost fish.     

Ontogenetic shifts in prey selection and foraging behaviour of larval and early juvenile alligator gar (Atractosteus spatula)

The early foraging ecology of alligator gar (Atractosteus spatula) is poorly documented, with little information available on young‐of‐year food habits or ontogenetic diet shifts. We conducted laboratory experiments to quantify prey selection and foraging behaviours of larval and early juvenile alligator gar (16–80 mm TL) simultaneously offered zooplankton, chironomid larvae and one of three densities of fish prey. The smallest size groups of alligator gar consumed zooplankton almost exclusively, but with increasing size, selection for zooplankton declined and selection for fish prey increased. At higher densities of fish prey, alligator gar exhibited lower selection for zooplankton and positively selected for fish at smaller sizes. Ingestion efficiencies for chironomids were considerably lower than for zooplankton or fish prey, resulting in low rates of consumption and negative selection for chironomids by all size groups of alligator gar. Fish prey elicited a different foraging response from alligator gar than zooplankton or chironomids, as alligator gar pursued and struck at fish over longer distances than for other prey types. With increasing size, alligator gar used a wider vertical range of the water column for foraging, changed their strike tactics and exhibited decreased handling times for zooplankton and fish. These observations indicate that alligator gar undergo several functional and behavioural changes during early ontogeny that facilitate a rapid transition to piscivory, but fish prey density strongly affects prey consumption patterns and the size at which alligator gar transition to piscivory.     

Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita, during early ontogeny

Digestive enzymes of freshwater fish Labeo rohita (family Cyprinidae; class Actinopterygii; infraclass Teleostei; order Cypriniformes) were studied during ontogenic development. Amylase, protease and lipase activities showed a polynomial relationship with age of rohu, whereas trypsin, chymotrypsin and lipase activities exhibited exponential trends with the age of rohu larvae. SDS‐PAGE of crude enzyme extract revealed that the proteases of higher molecular weight (MW) appeared during early ontogeny, whereas low MW proteases were observed at a later stage. Substrate SDS‐PAGE supported the quantitative study of protease activities as evidenced with the increasing number and intensity of activity bands with the age of fish. The number of protease activity bands observed in 4, 10, 12 and 24 DAH (days after hatching) larvae were 5, 7, 8 and 9, respectively. Inhibition of protease activities with soybean trypsin inhibitor (SBTI) (58.6–81.2%), phenyl methyl sulphonyl fluoride (PMSF) (55.6–70%), N‐α‐p‐tosyl‐l ‐lysine chloromethyl ketone (TLCK) (41.1–52.3%) and N‐tosyl‐l ‐phenylalanine chloromethyl ketone (TPCK) (27.9–44.5%) indicated the presence of serine proteases, trypsin‐ and chymotrypsin‐like enzymes in rohu larvae. Inhibition with SBTI and TPCK showed power, TLCK and ethylenediaminetetraacetic acid showed exponential, whereas PMSF showed polynomial relationships during the study period.     

Ontogenic changes in the digestive enzyme patterns and characterization of proteases in Indian major carp Cirrhinus mrigala

Digestive enzymes of Cirrhinus mrigala (Ham.) were studied during ontogenic development. Specific amylase activity was detected in first feeding fish. The enzyme activity decreased up to day‐18 and then it increased with the age of fish to reach the highest level on day‐34. Protease activity was 28.61 ± 8.90 mU mg protein^?1 min^?1 on day‐4 and increased with the age throughout the study period. Trypsin activity was 31.86 ± 1.12 mU mg protein^?1 min^?1 on day‐4. The activity decreased up to day‐10 and from day‐12 onwards increased up to day‐26. Chymotrypsin activity was 14.56 ± 2.74 mU mg protein^?1 min^?1on day‐4 and constantly increased up to day‐26. A significant increase in lipase activity was observed between days‐24 and 34. SDS‐PAGE and substrate SDS‐PAGE showed the diversity of protein (17.4–127.8 kDa) and protease activity bands (16.6–88.8 kDa) during ontogenesis. Soybean trypsin inhibitor, phenyl methyl sulphonyl fluoride, N‐α‐p‐tosyl‐l ‐lysine chloromethylketone and N‐tosyl‐l ‐phenylalanine chloromethylketone inhibited the protease activity up to 79.72–97.21, 65.55–94.83, 45.41–75.31 and 40.78–64.72%, respectively. Inhibition study in substrate SDS‐PAGE revealed the abundance of serine proteases and the presence of isoforms of trypsin and chymotrypsin. Ethylenediamine‐tetraacetate showed 5.56–22.78% inhibition of metal ion‐specific enzyme activity.     

Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.     

Alligator gar (Atractosteus spatula,Lacépède 1803) vitellogenin: purification,characterization and establishment of an enzyme‐linked immunosorbent assay

Alligator gar (Atractosteus spatula) is a non‐teleost bony fish distributed in North America. Gar populations have drastically declined as a consequence of habitat deterioration and the lack of regulation for their capture. Control of reproduction is critical for recovering their natural populations. The impossibility to distinguish genders and the determination of sexual maturity have hindered their successful reproduction. This research was aimed at developing an enzyme‐linked immunosorbent assay (ELISA) for the quantitative estimation of vitellogenin (VTG), a female‐specific protein. Plasmatic VTG from 17β‐estradiol (E2)‐induced juveniles and ovary vitellin from adult females were purified and characterized. Polyclonal antibodies against both proteins were produced to develop an ELISA. The immunoassay was validated by quality tests such as sensitivity, parallelism, recovery, reproducibility and specificity. Vitellogenin was determined in different tissues (plasma, mucus, liver and gills) of alligator gar. Vitellogenin and E2 concentrations in female breeders were found to be higher in November, before the spring spawning season. This approach represents a quick, reliable and non‐invasive practical alternative to distinguish genders and evaluate gonad maturation.     

Partial characterization of alkaline proteases from viscera of vermiculated sailfin catfish Pterygoplichthys disjunctivus Weber, 1991

Vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991), a member of the Loricariidae family and an invasive species of several inland waters around the world, possess an enormous digestive tract representing about 10% of fish weight. Thus, the aim of this study was to partially characterize proteases from their digestive tracts. Azocasein digestion of the crude extract of intestine at different pH values and temperatures revealed the presence of alkaline proteases with optimum activities at pH 9.0 and 50°C. Incubation assays of the crude extract with inhibitors such as phenyl methyl sulfonyl fluoride, N-α-p-tosyl-l-lysine chloromethyl ketone, N-tosyl-phenyalanine chloromethyl ketone, benzamidine, pepstatin A and ethylenediamine tetra-acetic acid showed that trypsin and chymotrypsin are the main alkaline proteinases present. Zymography showed that the crude extract of Pterygoplichthys disjunctivus viscera contained proteases with molecular masses ranging from 21.5 to 116 kDa. Trypsin and chymotrypsin were inhibited by the following ions in decreasing order: Hg²⁺, Fe²⁺, Cu²⁺, Li⁺, Mg²⁺, K⁺, while Mn²⁺, and Ca²⁺ had no effect. Activities decreased continuously as the NaCl concentration increased from 0 to 30%. These results constitute important background information for future studies and for the potential biotechnological use of the crude digestive extract from this invasive species.