Identification and expression analysis of transcripts involved in taurine biosynthesis during early ontogeny of tropical gar Atractosteus tropicus.

In fishes, the availability of taurine is regulated during ontogenetic development, where its endogenous synthesis capacity is species dependent. Thus, different pathways and involved enzymes have been described: pathway I (cysteine sulfinate-dependent pathway), cysteine dioxygenase type 1 (cdo1) and cysteine sulfinic acid decarboxylase (csad); pathway II (cysteic acid pathway), cdo1 and glutamic acid decarboxylase (gad); and pathway III (cysteamine pathway), 2-aminoethanethiol dioxygenase (ado); whereas taurine transporter (taut) is responsible for taurine entry into cells on the cell membrane and the mitochondria. This study determined if the tropical gar (Atractosteus tropicus), an ancient holostean fish model, has the molecular mechanism to synthesize taurine through the identification and analysis expression of transcripts coding for proteins involved in its biosynthesis and transportation, at different embryo-larvae stages and in different organs of juveniles (31 dah). We observed a fluctuating expression of all transcripts involved in the three pathways at all analyzed stages. All transcripts are expressed during the beginning of larval development; however, ado and taut show a peak expression at 9 dah, and all transcripts but csad decreased at 23 dah, when the organism ended the larval period. Furthermore, at 31 dah, we observed taut expression in all examined organs. The transcripts involved in pathways I and III are expressed differently across all organs, whereas pathway II was only observed in the brain, eye, and skin. The results suggested that taurine biosynthesis in tropical gar is regulated during its early development before first feeding, and the pathway might also be organ-type dependent.

Addition of ß-glucans in diets for tropical gar (Atractosteus tropicus) larvae: effects on growth, digestive enzymes and gene expression of intestinal epithelial integrity and immune system.

The effect of ß-glucans 1,3/1,6 from Saccharomyces cerevisiae yeast at different inclusion percentages (0.0, 0.2, 0.4, 0.6, and 0.8%) in the diet for tropical gar (Atractosteus tropicus) larvae was evaluated on growth, digestive enzyme activity and, relative expression of the immune system genes. The bioassay started on the third day after hatching (DAH) and lasted 21 days, using a total of 1500 larvae of 0.055 ± 0.008 g and, a total length of 2.46 ± 0.26 cm. Larviculture was carried out in a recirculation system with 15 tanks of 70 L using a density of 100 organisms per experimental unit. No significant differences in larval growth were observed by the inclusion of ß-glucans (p > 0.05). Digestive enzymes showed changes in lipase and trypsin activities, presenting higher values in fish fed 0.6% and 0.8% ß-glucans diets compared to the other treatments (p < 0.05). Leucine-aminopeptidase, chymotrypsin, acid phosphatase, and alkaline phosphatase activity showed higher activities in larvae fed with a 0.4% ß-glucan diet compared to the control group. The relative expression of intestinal membrane integrity (mucin 2) muc-2, (occludins) occ, (nucleotide-binding oligomerization domain) nod-2, and immune system lys (lysosome) genes showed over-expression in larvae fed the 0.4% ß-glucan diet to the rest of the treatments (p < 0.05). The inclusion of ß-glucans at 0.4-0.6% in diets for A. tropicus larvae could improve larviculture, as effects on the increase in the activity of several digestive enzymes and the expression of genes of the immune system.

Hypoxia- and hyperoxia-related gene expression dynamics during developmental critical windows of the tropical gar Atractosteus tropicus.

Aquatic hypoxia is both a naturally-occurring and anthropogenically-generated event. Fish species have evolved different adaptations to cope with hypoxic environments, including gill modifications and air breathing. However, little is known about the molecular mechanisms involved in the respiration of embryonic and larval fishes during critical windows of development. We assessed expression of the genes hif-1α, fih-1, nhe1, epo, gr and il8 using the developing tropical gar as a piscine model during three developmental periods (fertilization to hatch, 1 to 6 days post hatch (dph) and 7 to 12 dph) when exposed to normoxia (~7.43 mg/L DO), hypoxia (~2.5 mg/L DO) or hyperoxia (~9.15 mg/L DO). All genes had higher expression when fish were exposed to either hypoxia or hyperoxia during the first two developmental periods. However, fish continuously exposed to hypoxia had increased expression of the six genes by hatching and 6 dph, and by 12 dph only hif-1α still had increased expression. The middle developmental period was the most hypoxia-sensitive, coinciding with several changes in physiology and morphology. The oldest larvae were the most resilient to gene expression change, with little variation in expression of the six genes compared. This study is the first to relate the molecular response of an air-breathing fish to oxygen availability to developmental critical windows and contributes to our understanding of some molecular responses of developing fish to changes in oxygen availability.

Determination of optimal prey for rearing the Tropical Gar Atractosteus tropicus (Lepisosteiformes: Lepisosteidae) / Determinación de presas óptimas para la crianza del pejelagarto Atractosteus tropicus (Lepisosteiformes: Lepisosteidae)

Abstract Larval feeding studies of both ornamental and consumable fish species are important for formulating successful management, and culture strategies for conservation purposes. In the present study, we evaluated prey selectivity for the tropical gar Atractosteus tropicus in the larval stage (first 8 weeks) using the zooplankton Artemia fransiscana, Daphnia pulex and Moina macrocopa as prey following the hypothesis that prey selection of the fish species is related not only to prey species preferences but to the difference in prey densities present in the environment. Functional responses were tested at prey densities of 0.2, 0.5, 1.0, 2.0, 4.0 and 8.0 ind. mL-1 and analyzed using Manly's α. For prey selectivity, we used the three zooplankton species at three different densities. In these two experiments the fish larvae were allowed to feed for 45 min. To quantify feeding behavior (encounters, attacks, captures, ingestions, rejections) we used a density of 1 ind. mL-1 using each prey species based on 10 minutes of direct observation. Our results showed a functional response Type II for A. tropicus preying mostly on A. franciscana and M. macrocopa. The Manly's α index showed that M. macrocopa and A. franciscana are the most preys selected. The values for encounters for the three prey species were relatively constant during the eight weeks. Encounter values for the cladocerans were low in comparison to A. franciscana; however, high success in capture and ingestion was observed for all prey species used. Our results from the functional response experiments supports the hypothesis that A. tropicus is an active predator presenting a functional response of a carnivorous fish and the shift in prey selection suggests that even at low prey availability, A. tropicus is able to manipulate and feed on zooplankton of wide range in size. Also, according to our results, we suggest the use of a mix of A. franciscana and M. macrocopa to feed A. tropicus in culture systems in concentrations ≈ 2 ind. mL-1 during the first 3 weeks of age and then shift to M. macrocopa from the 4 week. Our results, in conjunction with studies on the survivorship of the juveniles would aid in conservation efforts and improve the production of gars in aquaculture. Rev. Biol. Trop. 66(3): 1018-1033. Epub 2018 September 01.

Resumen Estudios sobre alimentación en etapa larvaria en peces ornamentales como de consumo son importantes para formular estrategias exitosas de manejo y cultivo con fines de conservación. En el presente estudio evaluamos la selectividad de presa del pejelagarto Atractosteus tropicus en etapas larvarias (primeras 8 semanas) utilizando el zooplancton Artemia fransiscana, Daphnia pulex y Moina macrocopa como presas, probando la hipótesis de que la selección de presas en peces está relacionada no solamente a la preferencia por la especie de presa sino también a la diferencia en la densidad de presas en el ambiente. También se probó la respuesta funcional con densidades de presa de 0.2, 0.5, 1.0, 2.0, 4.0 y 8.0 ind. mL-1. Para la selección de presa, utilizamos las tres especies de zooplancton en tres densidades diferentes. En ambos experimentos, las larvas de los peces se alimentaron por 45 min. Para cuantificar el comportamiento alimenticio (encuentros, ataques, capturas, ingestiones y rechazos), se usó una densidad de 1 ind. mL-1utilizando cada especie de presa mediante observación directa por 10 minutos. Nuestros resultados muestran que A. tropicus presenta una respuesta funcional Tipo II, depredando principalmente A. franciscana y M. macrocopa. El índice α de Manly mostró que las especies M. macrocopa and A. franciscana fueron las mayormente seleccionadas. Los valores de encuentros para las tres especies de presa fueron relativamente constantes a lo largo de las ocho semanas. Los valores de ataque, captura e ingestión para los cladóceros fueron bajos; sin embargo, se observó gran éxito en la captura e ingestión para todas las especias de presa utilizadas. Nuestros resultados sobre los experimentos de respuesta funcional soportan la hipótesis de que A. tropicus es un depredador activo y presenta una respuesta funcional relacionada con especies de peces carnívoras y que el cambio en la selección de presas sugiere que aún a bajas densidades y disponibilidad de presas, A. tropicus es capaz de manipular y alimentarse de presas de diferentes tamaños. También, sugerimos el uso de una mezcla de A. franciscana y M. macrocopa para alimentar a A. tropicus en sistemas de cultivo en concentraciones ≈ 2 ind. mL-1 durante las primeras tres semanas de edad y después cambiar a M. macrocopa a partir de la cuarta semana. Nuestros resultados, junto con estudios de sobrevivencia de los juveniles pueden ayudar a generar esfuerzos de conservación y mejorar la producción de pejelagarto en acuicultura.

Sex-specific differences in transcriptome profiles of brain and muscle tissue of the tropical gar.

BACKGROUND: The tropical gar (Atractosteus tropicus) is the southernmost species of the seven extant species of gar fishes in the world. In Mexico and Central America, the species is an important food source due to its nutritional quality and low price. Despite its regional importance and increasing concerns about overexploitation and habitat degradation, basic genetic information on the tropical gar is lacking. Determining genetic information on the tropical gar is important for the sustainable management of wild populations, implementation of best practices in aquaculture settings, evolutionary studies of ancient lineages, and an understanding of sex-specific gene expression. In this study, the transcriptome of the tropical gar was sequenced and assembled de novo using tissues from three males and three females using Illumina sequencing technology. Sex-specific and highly differentially expressed transcripts in brain and muscle tissues between adult males and females were subsequently identified. RESULTS: The transcriptome was assembled de novo resulting in 80,611 transcripts with a contig N50 of 3,355 base pairs and over 168 kilobases in total length. Male muscle, brain, and gonad as well as female muscle and brain were included in the assembly. The assembled transcriptome was annotated to identify the putative function of expressed transcripts using Trinotate and SwissProt, a database of well-annotated proteins. The brain and muscle datasets were then aligned to the assembled transcriptome to identify transcripts that were differentially expressed between males and females. The contrast between male and female brain identified 109 transcripts from 106 genes that were significantly differentially expressed. In the muscle comparison, 82 transcripts from 80 genes were identified with evidence for significant differential expression. Almost all genes identified as differentially expressed were sex-specific. The differentially expressed transcripts were enriched for genes involved in cellular functioning, signaling, immune response, and tissue-specific functions. CONCLUSIONS: This study identified differentially expressed transcripts between male and female gar in muscle and brain tissue. The majority of differentially expressed transcripts had sex-specific expression. Expanding on these findings to other developmental stages, populations, and species may lead to the identification of genetic factors contributing to the skewed sex ratio seen in the tropical gar and of sex-specific differences in expression in other species. Finally, the transcriptome assembly will open future research avenues on tropical gar development, cell function, environmental resistance, and evolution in the context of other early vertebrates.

Corticosteroid and progesterone transactivation of mineralocorticoid receptors from Amur sturgeon and tropical gar.

The response to a panel of steroids by the mineralocorticoid receptor (MR) from Amur sturgeon and tropical gar, two basal ray-finned fish, expressed in HEK293 cells was investigated. Half-maximal responses (EC50s) for transcriptional activation of sturgeon MR by 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol and aldosterone, and progesterone (Prog) were between 13 and 150 pM. For gar MR, EC50s were between 8 and 55 pM. Such low EC50s support physiological regulation by these steroids of the MR in sturgeon and gar. Companion studies with human and zebrafish MRs found higher EC50s compared with EC50s for sturgeon and gar MRs, with EC50s for zebrafish MR closer to gar and sturgeon MRs than was human MR. For zebrafish MR, EC50s were between 75 and 740 pM; for human MR, EC50s were between 65 pM and 2 nM. In addition to Prog, spironolactone (spiron) and 19nor-progesterone (19norP) were agonists for all three fish MRs, in contrast with their antagonist activity for human MR, which is hypothesized to involve serine-810 in human MR because all three steroids are agonists for a mutant human Ser810Leu-MR. Paradoxically, sturgeon, gar, and zebrafish MRs contain a serine corresponding to serine-810 in human MR. Our data suggest alternative mechanism(s) for Prog, spiron, and 19norP as MR agonists in these three ray-finned fishes and the need for caution in applying data for Prog signaling in zebrafish to human physiology.

Developmental cardiorespiratory physiology of the air-breathing tropical gar, Atractosteus tropicus.

The physiological transition to aerial breathing in larval air-breathing fishes is poorly understood. We investigated gill ventilation frequency (fG), heart rate (fH), and air breathing frequency (fAB) as a function of development, activity, hypoxia, and temperature in embryos/larvae from day (D) 2.5 to D30 posthatch of the tropical gar, Atractosteus tropicus, an obligate air breather. Gill ventilation at 28°C began at approximately D2, peaking at ∼75 beats/min on D5, before declining to ∼55 beats/min at D30. Heart beat began ∼36-48 h postfertilization and ∼1 day before hatching. fH peaked between D3 and D10 at ∼140 beats/min, remaining at this level through D30. Air breathing started very early at D2.5 to D3.5 at 1-2 breaths/h, increasing to ∼30 breaths/h at D15 and D30. Forced activity at all stages resulted in a rapid but brief increase in both fG and fH, (but not fAB), indicating that even in these early larval stages, reflex control existed over both ventilation and circulation prior to its increasing importance in older fishes. Acute progressive hypoxia increased fG in D2.5-D10 larvae, but decreased fG in older larvae (≥D15), possibly to prevent branchial O2 loss into surrounding water. Temperature sensitivity of fG and fH measured at 20°C, 25°C, 28°C and 38°C was largely independent of development, with a Q10 between 20°C and 38°C of ∼2.4 and ∼1.5 for fG and fH, respectively. The rapid onset of air breathing, coupled with both respiratory and cardiovascular reflexes as early as D2.5, indicates that larval A. tropicus develops "in the fast lane."

The complete mitochondrial DNA of the tropical gar (Atractosteus tropicus).

The mitogenome of the tropical gar, Atractosteus tropicus, (GeneBank accession number KJ531198) has a total length of 16,280 bp, and the arrangement consist of 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 22 transfer RNA similar to other Lepisosteidae family mitogenomes.

Using cornstarch in microparticulate diets for larvicultured tropical gar (Atractosteus tropicus).

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.