Tropical Gar Atractosteus Tropicus Research Articles

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.

Inclusion of Mannan-Oligosaccharides in Diets for Tropical Gar Atractosteus tropicus Larvae: Effects on Growth, Digestive Enzymes, and Expression of Intestinal Barrier Genes

Mannan-oligosaccharides (MOS) are non-digestible carbohydrates, and their use in aquaculture as prebiotics is well documented. The objective of this work was to test whether MOS supplemented in the diet of A. tropicus larvae (2, 4, and 6 g kg−1) influence growth parameters, the activity of digestive enzymes, and the expression of genes related to the intestinal barrier. The highest total length was observed in larvae fed 6 g kg−1 MOS compared to control larvae. Trypsin activity increased with the addition of MOS to the diets, but leucine aminopeptidase activity only increased with 6 g kg−1 MOS. Lipase and α-amylase activities increased in larvae fed with 2 and 4 g kg−1 MOS. The expression of zo-2 was higher with the 6 g kg−1 MOS treatment. The cl-3 transcripts were lower with 2 g kg−1 MOS but higher with 6 g kg−1 MOS. All tested concentrations of MOS increased the expression of muc-2. In this study, incorporating mannan-oligosaccharides into the diet of A. tropicus larvae had a positive effect, and the concentration of 6 g kg−1 produced the best results. Therefore, including this prebiotic in the diets for the culture of A. tropicus larvae is suitable.

Morphological and cardiac alterations after crude oil exposure in the early-life stages of the tropical gar (Atractosteus tropicus).

Fish development can be affected by environmental pollutants such as crude oil (anthropogenic or natural sources), causing alterations especially in cardiac function and morphology. Most such studies have focused on saltwater species, whereas studies in freshwater fishes are scant. The objective of the current study was to evaluate the effects of crude oil exposure (as 0, 5, 10, 15, or 20% high-energy water accommodated fractions, HEWAF) on cardiac function and edema formation during two early periods of development (embryo and eleuteroembryo, 48 h each) individually using the tropical gar Atractosteus tropicus as a model. Embryos did not exhibit alterations in body mass, total length, condition factor, and cardiac function as a function of oil. In contrast, eleuteroembryos proved to be more sensitive and exhibited increased body mass, total length, and condition factor, decreased heart rate and phenotypic alterations such as cardiac dysmorphia (tubular hearts) and spine curvature at high concentrations of HEWAF. Moreover, edema formation was observed in both stages This study shows different functional responses of A. tropicus after crude oil exposure and provides useful information of the developmental impacts of these compounds on the early life stages of freshwater tropical fishes.

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.

Modelling the potential distribution of two tropical freshwater fish species under climate change scenarios

Abstract Climate change is causing shifts in the distribution patterns of freshwater fish at various spatio‐temporal scales. Tropical freshwater fish are vulnerable, especially in areas where a high impact of climate change is predicted; thus, there is an increasing need to predict these shifts to determine conservation and adaptation strategies. Ecological niche models offer a reliable way to predict the effects of climate change on species distribution. Potential shifts in the distribution of tropical fish were tested under two scenarios (4.5 – moderate and 8.5 – extreme) with three general circulation models for years 2050 and 2070 using maximum entropy software using as models two predatory species – the tropical gar Atractosteus tropicus and the giant cichlid Petenia splendida. The potential distribution of both species was associated with warm and humid–sub‐humid conditions. Future projections showed a higher availability of suitable areas for both species resulting from the expansion of warmer conditions in the middle and upper basins of the Central American mountain range and centre of the Yucatan Peninsula. Ecological niche models of keystone or umbrella species such as A. tropicus and P. splendida could be useful to support conservation plans of protected areas. The potential distribution of both species covers areas of high suitability including six important biosphere reserves in Mexico, three protected areas in Guatemala and part of the Mesoamerican biological corridor. Despite the potential expansion of the present distribution range suggested by the models, it is important to consider the biological and ecological requirements of the species and the ecological implications of these potential shifts in distribution. Both scenarios could have several implications at genetic, population, and ecosystem levels.

Expression of long-chain polyunsaturated fatty acids biosynthesis genes during the early life-cycle stages of the tropical gar Atractosteus tropicus.

Long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), including eicosapentaenoic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), are essential in multiple physiological processes, especially during early development of vertebrates. LC-PUFA biosynthesis is achieved by two key families of enzymes, fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl). The present study determined the expression patterns of genes encoding desaturases (fads1 and fads2) and elongases (elovl2 and elovl5) involved in the LC-PUFA biosynthesis during early life-stages of the tropical gar Atractosteus tropicus. We further analyzed the fatty acid profiles during early development of A. tropicus to evaluate the impact of Fads and Elovl enzymatic activities. Specific oligonucleotides were designed from A. tropicus transcriptome to perform qPCR (quantitative polymerase chain reaction) on embryonic and larval stages, along with several organs (intestine, white muscle, brain, liver, heart, mesenteric adipose, kidney, gill, swim bladder, stomach, and spleen) collected from juvenile specimens. Fatty acid content of feeds and embryonic and larval stages were analyzed. Results show that fads1, fads2, elovl2 and elovl5 expression was detected from embryonic stages with expression peaks from day 15 post hatching, which could be related to transcriptional and dietary factors. Moreover, fads1, fads2 and elovl2 showed a higher expression in intestine, while elovl5 showed a higher expression in liver, suggesting that the tropical gar activates its LC-PUFA biosynthetic machinery to produce ARA, EPA and DHA to satisfy physiological demands at crucial developmental milestones during early development.

Survival, Growth, and Development in the Early Stages of the Tropical Gar Atractosteus tropicus: Developmental Critical Windows and the Influence of Temperature, Salinity, and Oxygen Availability

Alterations in fish developmental trajectories occur in response to genetic and environmental changes, especially during sensitive periods of development (critical windows). Embryos and larvae of Atractosteus tropicus were used as a model to study fish survival, growth, and development as a function of temperature (28 °C control, 33 °C, and 36 °C), salinity (0.0 ppt control, 4.0 ppt, and 6.0 ppt), and air saturation (control ~95% air saturation, hypoxia ~30% air saturation, and hyperoxia ~117% air saturation) during three developmental periods: (1) fertilization to hatch, (2) day 1 to day 6 post hatch (dph), and (3) 7 to 12 dph. Elevated temperature, hypoxia, and hyperoxia decreased survival during incubation, and salinity at 2 and 3 dph. Growth increased in embryos incubated at elevated temperature, at higher salinity, and in hyperoxia but decreased in hypoxia. Changes in development occurred as alterations in the timing of hatching, yolk depletion, acceptance of exogenous feeding, free swimming, and snout shape change, especially at high temperature and hypoxia. Our results suggest identifiable critical windows of development in the early ontogeny of A. tropicus and contribute to the knowledge of fish larval ecology and the interactions of individuals × stressors × time of exposure.

Quality Evaluation of Reference Gene Expression on Different Tissues in Adults of Tropical Gar Atractosteus tropicus

Tropical gar (Atractosteus tropicus) is an ancestral subtropical fish species in southeastern Mexico, which has great potential as a model species for physiological, biomedical and genomic studies. The quantification of gene expression through RT-qPCR is one of the most commonly used techniques, due to its precision, sensitivity and high performance, particularly in gene expression to compare between cells, tissues and organs; as well as different populations, stages of development, metabolism, among other conditions. This study analyzed the stability and normalization of six commonly used reference genes such as alpha elongation factor (ef1-α), beta-actin (actb), 18S ribosomal RNA (18s rrna), beta-2-microglobulin (b2m), tubulin alpha (α-tub) and glyceraldehyde 3-phosphate dehydrogenase (gapdh) in the different tissues of the intestine, muscle, gill, stomach, brain and liver in adult males of A. tropicus from reared in captivity through three BestKeeper, geNorm and NormFinder algorithms. Based on our results we can conclude that in the three BestKeeper, NormFinder and geNorm algorithms, the most stable genes are ef1, followed by 18s rrna and actb where the gene stability will depend on specific tissue to analyze in tropical gar A. tropicus adults.

Trypsin gene expression in adults and larvae of tropical gar Atractosteus tropicus.

Trypsin gene (try) expression levels were quantified in different organs of wild and captive tropical gar (Atractosteus tropicus) adults, and changes in expression during initial ontogeny of the species were determined. RNA was extracted from the pancreas, and cDNA was synthesized and later amplified by endpoint PCR using oligonucleotides designed from different try sequences of fish registered in GenBank. Subsequently, specific oligonucleotides were designed from the partial sequences. Gene expression was measured after RNA extraction and synthesis of the cDNA of 11 organs (liver, pancreas, stomach, esophagus, intestine, pyloric caeca, brain, muscle, gills, gonad, and kidney) of captive and wild adults. Likewise, samples of A. tropicus larvae were taken on days 0 (embryo), 5, 10, 15, 20, 25, and 30days after hatching (DAH), the RNA was extracted, and the synthesis of cDNA was carried out to measure real-time gene expression (qPCR). The results showed that the highest relative try expression occurred mainly in the esophagus, liver, stomach, and pancreas of both wild and captive adult fish; however, captive organisms had a higher try expression level than wild fish. Although try expression during initial ontogeny was high in embryos (0 DAH), it did not reach the maximum value until 15 DAH. It was concluded that try expression levels in captive adults are due to the high protein content in the balanced feed (trout diet). The highest try expression level during larviculture was detected at 15 DAH, which indicates that A. tropicus larvae have a mature digestive system and can efficiently hydrolyze proteins from feed at this developmental stage.

Optimum level of dietary lipids for growth, chemical composition and apparent digestibility of lipids for Atractosteus tropicus

Lipids requirement of tropical gar Atractosteus tropicus prejuveniles and juveniles was evaluated on growth, survival, chemical composition and apparent digestibility coefficient (ADC). Four semi-purified experimental diets (5, 10, 15 and 20% of lipids) were obtained with a mix of menhaden oil and soybean lecithin (2:1 ratio). Two trials were carried out, first with 600 prejuveniles of 0.5 g (35 days post-hatching, dph) for 56 days, and the second with 160 juveniles of 15 g (two-months-old) for 42 days. In the first trial, 15 (40.8 g) and 20% (40.2 g) lipid diets showed the best weight. Significant differences were obtained in condition factor (CF), daily lipid intake (DLI), hepatosomatic index (HSI), crude protein, ashes, nitrogen-free extract, moisture and ether extract between treatments. In the second trial, fish fed with the 10% lipid diet obtained the highest weight (89.1 g) compared with the other treatments. Significant differences were recorded for weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR), condition factor (CF), and lipid efficiency rate (LER). Also, significant differences were obtained in crude protein, ether extract, NFE, gross energy, apparent lipid ADC, protein ADC and dry matter ADC. The polynomial models applied to both trials indicated an optimum lipid requirement of 17% for 0.5 g juveniles and of 9% for 15.2 g juveniles.

Determination of optimal prey for rearing tropical gar Atractosteus tropicus (Lepisosteiformes: Lepisosteidae)

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.

Evaluación de alimentos microencapsulados en el cultivo de larvas de pejelagarto (Atractosteus tropicus)

Four microencapsulated diets were evaluated with respect to the growth and survival of tropical gar Atractosteus tropicus larvae. The treatments consisted of four experimental diets and one control diet 1) fish meal, 2) a combination of pork and poultry meals, 3) Nannochloropsis gaditana meal, 4) enzyme preparation and the control treatment 5) Artemia nauplii. The evaluation indicated that the larvae fed the Artemia nauplii obtained the greatest growth and survival (3.93 cm, 0.19 g y 82 % respectively), which justifies a correct culture system operation. While larvae fed microencapsulated diets best values in survival were observed with diets Nannochloropsis gaditana and enzyme preparation (20.0 and 19.2 %). Our results showed that microencapsulated could be used to feed A. tropicus in feasibly form. However, more information concerning to optimize the design and manufacturing are required to improve the growth and survival of organisms.

Primer Designs for Identification and Environmental DNA (eDNA) Detection of Gars

AbstractGars (family Lepisosteidae) play important roles as apex predators in freshwater ecosystems, helping to balance fish populations. Several gar species are exploited as food and game fish, and some species are classified as vulnerable due to habitat loss. New molecular techniques to detect, monitor, and identify environmental DNA (eDNA) from gars might help inform management and conservation efforts for these interesting fish. The goal of this project was to develop and test PCR primers for gars, using specimens of all seven gar species, which are on exhibit at the Belle Isle Aquarium in Detroit, Michigan. Focusing on the mitochondrial gene for cytochrome oxidase I, we first designed primers to amplify DNA from all species of gars (“universal gar primers”) and confirmed their specificity in silico. These primers amplified DNA from all seven species, and species identities were confirmed by sequencing the PCR products. Only one of the three ostensibly Shortnose Gar Lepisosteus platostomus specimens sampled exhibited a Shortnose Gar matriline; two other specimens may be Longnose Gar L. osseus × Shortnose Gar hybrids. Genus‐targeted primers were developed that amplified all Atractosteus species and two of four Lepisosteus species. Species‐specific primers were developed for Longnose Gar, Shortnose Gar, and Tropical Gar Atractosteus tropicus. A primer set that targeted Alligator Gar A. spatula also amplified DNA from Cuban Gar A. tristocheus, but not other species. While the universal gar primers followed by sequencing confirmed the identities of all seven gar species at the Belle Isle Aquarium, PCR with the species‐specific primers enabled direct detection of the presence of DNA from the targeted species in the water in which those species had been maintained. Designing these primers is the first step in developing eDNA markers for gars, which in turn could inform the conservation and management of gar populations.

Development of digestive tract and enzyme activities during the early ontogeny of the tropical gar Atractosteus tropicus.

Changes in digestive enzyme activity and histology were studied in Atractosteus tropicus embryos, larvae and juvenile periods. Alkaline protease, chymotrypsin, carboxypeptidase A, lipase and α-amylase were detected in all periods and gradually increased until reaching the maximum peak in juveniles; meanwhile, acid protease was first detected at 5days after hatching (dah) when first feeding started and trypsin and leucine aminopeptidase activities were detected from 19 dah, their values being increased gradually until reaching a maximum value at 31 dah. Acid and alkaline phosphatase activities increased from yolk-sac absorption (3 dah) until day 31 after hatching. Zymogram for acid protease showed two bands in active forms (0.4 and 0.5 Rfs) from day 5 after hatching and a third protease form (0.3 Rf) that appears at 31 dah. Two active forms (26.3 and 24.9kDa) were detected using SDS-PAGE alkaline proteases zymogram at 5 dah, and an additional active form (44.1 kDa) was detected at 7 dah. Regarding the histological development of the digestive system, the exocrine pancreas containing zymogen granules was already visible at 3 dah, whereas at 5 dah first gastric glands were already detected in the stomach. Between 7 and 9 dah, the digestive tract of A. tropicus resembled that of a juvenile specimen with a well-developed and short oesophagus, stomach divided into a glandular and non-glandular (pyloric) stomach, folded intestine with pyloric caeca and a well-developed spiral valve (posterior intestine). Considering this, larvae of A. tropicus are capable of digesting several foods from yolk absorption (3 dah), maximizing its activities at 15 dah, age at which the organisms maximize its capability to absorb nutrients from diets provided.

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles

Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6±12.7g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55°C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.

Histología del ciclo reproductor de hembras del pejelagarto <i>Atractosteus tropicus</i> (Lepisosteiformes: Lepisosteidae) enTabasco, México

In Southeast Mexico, A. tropicus is a species with great ecological, economic, cultural and aquaculture value, however wild populations have diminished due to diverse anthropological causes. The objective of this study was to characterize the reproductive cycle of A. tropicus, with a description of complete gonad structure and the changes in germinal and somatic elements during oogenesis, for better management and use of this species. Six sexually mature females (N=72) were captured monthly with drag nets during one year cycle (October 2003-October 2004) in wild populations of Pomposu lagoon, municipality of Jalpa de Mendez, Tabasco, Mexico. Females were sacrificed by decapitation and the ovaries were processed by standard histological methods; the diameter of 10-20 oocytes taken at random was determined in different stages of development. Gonadosmatic index (GSI), gonadal volume (gV) and follicule diameter (fD) were determined monthly. Among results we could determine eight oogenesis developmental stages: I: chromatin nucleolus stage, II: early perinucleolus stage, III: mid perinucleolus stage, IV: advanced perinucleolus stage, V: early vitellogenesis stage, VI: mid vitellogenesis stage, VII) advanced vitellogenesis stage and VIII: final maturation stage. The reproductive cycle could be divided in four phases: 1: proliferative phase, II: initial maturation phase, III: late maturation phase, IV: regressing phase. Analysis of the data obtained from the IGS, gV and fD values, oogenesis stages and reproductive phases, indicate that in December previtellogenesis phase restarts, from March through July yolk deposition (vitellogenesis) occurs, and from August to November the maximum reproductive activity takes place. Contrasting this pattern with local environmental factors such as temperature and precipitation, we suggested that these may serve as an environmental activator in the development of oogenesis and spawning of this species.

Cariotipo del pejelagarto tropical Atractosteus tropicus (Lepisosteiformes: Lepisosteidae) y variación cromosómica en sus larvas y adultos

The karyotype of the tropical gar Atractosteus tropicus is described from conventional Giemsa-staining of 295 mitotic chromosome slides from 120 larvae and 15 adults (five females and ten males) from Tabasco, southern of Mexico. The diploid number 2n = 56 chromosomes was calculated (73 spreads from 206 larval and 208 adult metaphases). Variation on chromosome number was from 46 (4.4%) to 64 (3.9%) chromosome elements on larval samples, whereas 58 (11.7%) chromosomes were the second most abundant after the diploid number of 2n = 56 (35%). Such variation was related with the presence of mobile microchromosomes. The karyotype was determined from six clear chromosome spreads photographed from three females and three males. The averaged karyotype was integrated by eight pairs of metacentric (m) chromosomes, four submetacentric (sm) pairs, eight telocentric (t) pairs and eight pairs of telocentric microchromosomes (*t). The fundamental number was FN = 80 chromosome arms. We saw no sexual differences on chromosome structure.