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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.

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Tropical gar (Atractosteus tropicus) thrives in aquatic habitats with high levels of total nitrogen (TAN) and unionized ammonia (NH3). However, the tolerance of TAN and NH3, the excretion mechanisms involved, and the effects of these chemicals on routine metabolism are still unknown. Therefore, our objectives were to assess the acute toxicity of TAN and NH3 in A. tropicus juveniles after a 96-h exposure (LC50-96 h) to NH4Cl and after chronic exposure to two concentrations (15% and 30% of LC50-96 h TAN) for 12 days, as well as to evaluate the transcriptional effects associated with Rhesus proteins (rhag, rhbg, rhcg) and ion transporters (NHE, NKA, NKCC, and CFTR) in gills and skin; and to determine the effects of TAN and NH3 on routine metabolism through oxygen consumption (µM g-1 h-1) and gill ventilation frequency (beats min-1). LC50-96 h values were 100.20 ± 11.21 mg/L for TAN and 3.756 ± 0.259 mg/L for NH3. The genes encoding Rhesus proteins and ion transporters in gills and skin showed a differential expression according to TAN concentrations and exposure time. Oxygen consumption on day 12 showed significant differences between treatments with 15% and 30% TAN. Gill ventilation frequency on day 12 was higher in fish exposed to 30% TAN. In conclusion, A. tropicus juveniles are highly tolerant to TAN, showing upregulation of the genes involved in TAN excretion through gills and skin, which affects routine oxygen consumption and energetic cost. These findings are relevant for understanding adaptations in the physiological response of a tropical ancestral air-breathing fish.

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The Holostei group occupies a critical phylogenetic position as the sister group of the Teleostei. However, little is known about holostean pituitary anatomy or brain distribution of important reproductive neuropeptides, such as the gonadotropin-inhibitory hormone (GnIH). Thus, the present study set out to characterize the structure of the pituitary and to localize GnIH-immunoreactive cells in the brain of Atractosteus tropicus from the viewpoint of comparative neuroanatomy. Juveniles of both sexes were processed for general histology and immunohistochemistry. Based on the differences in cell organization, morphology, and staining properties, the neurohypophysis and three regions in the adenohypophysis were identified: the rostral and proximal pars distalis (PPD) and the pars intermedia. This last region was found to be innervated by the neurohypophysis. This organization, together with the presence of a saccus vasculosus, resembles the general teleost pituitary organization. A vast number of blood vessels were also recognized between the infundibulum floor of the hypothalamus and the PPD, evidencing the characteristic presence of a median eminence and a portal system. However, this well-developed pituitary portal system resembles that of tetrapods. As regards the immunohistochemical localization of GnIH, we found four GnIH-immunoreactive (GnIH-ir) populations in three hypothalamic nuclei (suprachiasmatic, retrotuberal, and tuberal nuclei) and one in the diencephalon (prethalamic nucleus), as well as a few scattered neurons throughout the olfactory bulbs, the telencephalon, and the intersection between them. GnIH-ir fibers showed a widespread distribution over almost all brain regions, suggesting that GnIH function is not restricted to reproduction only. In conclusion, the present study describes, for the first time, the pituitary of A. tropicus and the neuroanatomical localization of GnIH in a holostean fish that exhibits a similar distribution pattern to that of teleosts and other vertebrates, suggesting a high degree of phylogenetic conservation of this system.

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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 30 days 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.

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Abstract:The tropical gar A. tropicus plays an important ecological role as it regulates other fish stocks in different water bodies in Southeastern México. Nevertheless, wild populations are declining, and one conservation alternative is the aquaculture production and basic knowledge of reproductive biology; for males, this requires the study of germ and somatic structures of testes, to characterize the reproductive cycle, and to provide basic knowledge for exploitation and conservation models and strategies. With this aim, a total of 24 males with an average sL = 47.2 cm were collected from wild populations from the Laguna Pomposú, municipality of Jalpa de Mendez (18°19' - 93°01'12" W), Tabasco, Mexico. Fish were collected with a trawl net and were transported live to the Tropical Aquaculture Laboratory, División Académica de Ciencias Biológicas (DACBiol), Universidad Juárez Autónoma de Tabasco (UJAT). Males were killed by prolonged immersion in MS222. Testes samples were collected from each specimen and were processed using the standard histological procedures, that consisted of dehydration in an ascending ethanol series, xylol, embedding in paraffin, sectioning at 7 µm, and staining with hematoxylin-eosin (HE). The diameter of 20 seminiferous tubules (Dst), height of germinal epithelium (Hge), gonadosomatic index (GSI) and gonad volume (gV) were determined monthly. Based on morphometric and morpho-physiological characteristics, the testes consisted of a network of anastomosed tubules with non-restricted cystic spermatogenesis, and a permanent germinal epithelium. This is the first report of a permanent germinal epithelium in A. tropicus. Five reproductive classes were histologically identified: Class I Regressed; Class II Early Maturation; Class III Mid Maturation; Class IV Late Maturation; Class V Regression. Monthly GSI, gV and Dst values were lower in January and February, the testis showed spermatozoa remains and a regenerating discontinuous germinal epithelium. In March spermiogenesis increased and proliferation of spermatogonia decreased. Male tropical gar followed a seasonal reproductive cycle, indicated by the monthly variation of the reproductive classes and the reproductive season processes observed, and for which temperature and rainfall seem to stimulate reproductive activity and spermiation. Rev. Biol. Trop. 64 (4): 1597-1609. Epub 2016 December 01.

Resumen:A. tropicus tiene un papel ecológico importante, como regulador de otras poblaciones de peces, en los cuerpos de agua de México, pero sus poblaciones silvestres se reducen. Una alternativa de conservación es el cultivo, el cual requiere caracterizar el ciclo reproductivo por medio del estudio de estructuras germinales y somáticas de los testículos, conocimientos que son básicos para formar modelos de aprovechamiento y conservación. Se capturaron mensualmente tres machos sexualmente maduros (N = 24), con un promedio de sL = 47.2 cm en Laguna de Pomposú, Jalpa de Méndez (18°19´59" N - 93°01´12" W), Tabasco, México, de octubre 2009 a septiembre 2010. La técnica de captura fue red de arrastre, se transportaron vivos al laboratorio de acuicultura tropical, DACBiol, UJAT. Los machos recolectados se sacrificaron con baños de inmersión en sobredosis de MS222, los testículos se procesaron para análisis histológico. Se determinó mensualmente el diámetro de 20 túbulos seminíferos (Dst), altura de epitelio germinal (Hge), índice gonadosomático (GSI) y volumen de gónada (gV). Características morfo-fisiológicas del testículo muestran que está constituido de una red de túbulos anastomosados con espermatogénesis quística no restringida, y un epitelio germinal permanente, de nuestro conocimiento es la primera vez que se reporta este tipo de epitelio en Holostei (Lepisosteiformes: Lepisosteidae). Se identificaron cinco clases reproductivas: Clase I Recrudescencia, Clase II Madurez temprana, Clase III Madurez intermedia, Clase IV Maduración tardía, Clase V Regresión, que al contrastarlo con el valor mensual de los indicadores sexuales "GSI, gV, Dst" muestra un patrón de variación; durante enero-febrero se presentan valores bajos, se observa un epitelio germinal discontinuo en regeneración; durante marzo se incrementa la proliferación de espermatogonias disminuyendo la espermatogénesis. Los machos de A. tropicus muestran una actividad reproductora estacional anual, explicado por las variaciones mensuales de los indicadores reproductores, donde la temperatura y la precipitación parecen tener un papel importante como factores que estimulan la actividad reproductora y por tanto la espermiación.

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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 5 days 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 24.9 kDa) 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.

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En el sureste de México A. tropicus es una especie de gran importancia ecológica, económica, cultural y para acuicultura, sin embargo, sus poblaciones silvestres han disminuido a causa de diversas actividades antropogénicas, por lo que se caracterizó el ciclo reproductor de A. tropicus, a partir de los cambios en los elementos germinales y somáticos durante la ovogénesis y la estructura de toda la gónada, información valiosa para el mejor manejo y aprovechamiento de la especie. Mensualmente se capturaron con redes de arrastres, seis hembras sexualmente maduras (N=72), durante un ciclo anual (octubre 2003-septiembre 2004), en poblaciones silvestres de la laguna de Pomposu del municipio de Jalpa de Méndez; Tabasco, México. Las hembras fueron sacrificadas por decapitación y los ovarios se procesaron por métodos histológicos convencionales, se determinó el diámetro entre 10-20 ovocitos en los diferentes estadios de desarrollo al azar. Se determinó mensualmente el índice gonadosomático (IGS), volumen gonadal (gV) y el diámetro del folículo (fD). Además, se identificaron ocho estadios de desarrollo de la ovogénesis: I: cromatina nucléolo, II: perinucléolo temprano, III: perinucléolo avanzado, IV: alvéolos corticales, V: vitelogénesis temprana, VI: vitelogénesis media, VII: vitelogénesis avanzada, VIII: maduración final. El ciclo reproductor la constituye cuatro clases: I.-Proliferativa, II.-Maduración inicial, III.-Maduración tardía, IV.-Regresión-proliferación. El análisis de la información obtenida de los valores encontrados del IGS, gV, fD, estadios de la ovogénesis y clases reproductivas, indican que en diciembre se reinicia el crecimiento de los folículos (previtelogenesis), de mar-zo-julio se presenta la captación del vitelo (vitelogenesis) y en agosto-noviembre se presenta la máxima actividad reproductora, al contrastar este patrón con los factores ambientales (temperatura, precipitación) se sugiere que estos pueden funcionar como un gatillo ambiental, en el desarrollo de la ovogénesis y el desove.

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: I: 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. Rev. Biol. Trop. 60 (4): 1857-1871. Epub 2012 December 01.

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Biomarkers are a widely applied approach in environmental studies. Analyses of cholinesterase (ChE), glutathione S-transferase (GST) and lipid peroxidation (LPO) are biomarkers that can provide information regarding early effects of pollutants at different biochemical levels on an organism. The aim of this study was to evaluate the biomarker approach on a Costa Rican native and relevant species. For this, larvae of gar (Atractosteus tropicus) were exposed to the organophosphorus nematicide, ethoprophos. Acute (96hr) exposure was conducted with pesticide concentrations ranging from 0.1μg/L to 1 500μg/L. The 96hr LC50 calculated was 859.7μg/L. After exposure, three biomarkers (ChE, GST and LPO) were analyzed in fish that survived the acute test. The lowest observed effect concentration (LOEC) regarding ChE activity inhibition was 50μg/L. This concentration produced a significant inhibition (p<0.05) of the enzyme by 20%. The highest concentration tested without showing any effect on ChE activity and therefore considered as no observed effect concentration (NOEC) was 10μg/L. Ethoprophos concentration of 400μg/L caused a ChE inhibition by 79%. In this study, no significant variations (p>0.05) in GST activity and LPO were observed in A. tropicus larvae after exposure to ethoprophos.

El proceso de reproducción inducida de Atractosteus tropicus es útil para la acuicultura y la reintroducción en zonas donde las poblaciones silvestres se han reducido considerablemente. En larvas de esta especie se evaluó la toxicidad aguda, así como la respuesta de tres biomarcadores: actividad colinesterasa (ChE), actividad de Glutation S-transferasa (GST) y peroxidación de lípidos (LPO). Asimismo, se realizaron exposiciones agudas (96hr) a etoprofos (nematicida organofosforado), en donde se utilizaron concentraciones entre 0.1μg/L y 1 500μg/L del nematicida. La concentración letal 50 (LC50) calculada fue de 859.7μg/L; la máxima concentración sin efecto en los organismos (NOEC) 10μg/L y la concentración más baja en la cual se observó algún efecto (LOEC) 50μg/L. A esa concentración, el efecto observado fue una reducción significativa (p<0.05) en la actividad de la ChE. Una concetración de etoprofos de 400μg/L causó una inhibición del 79% en la actividad ChE. La actividad GST y la LPO no mostraron una respuesta significativa (p>0.05) luego de la exposición de los organismos a etoprofos.

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Karyotype of the tropical gar Atractosteus tropicus Lepisosteiformes: Lepisosteidae) and chromosomal variation in their larval and adults. 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 micro-chromosomes (*t). The fundamental number was FN = 80 chromosome arms. We saw no sexual differences on chromosome structure. Rev. Biol. Trop. 57 (3): 529-539. Epub 2009 September 30.

El cariotipo del pejelagarto Atractosteus tropicus se describe por medio de tinción Giemsa de 295 preparaciones cromosómicas en mitosis a partir de 120 larvas y 15 adultos (5 hembras y diez machos) de la población que habita en Tabasco, sureste de México. El número diploide 2n=56 cromosomas se dio en 281 dispersiones cromosómicas de un total de 445 muestras, de ellas 73 dispersiones provinieron de 206 metafases larvales, y 208 dispersiones de 239 metafases de adultos. Se observó variación en el número de cromosomas desde 46 hasta 64 elementos cromosómicos (larvas). Las metafases con 58 cromosomas fueron el segundo valor más abundante, después del número diploide 2n=56 (35%). En los adultos esa variación no fue relevante. Esta variación cromosómica se relaciona con microcromosomas móviles. El cariotipo se determinó de seis dispersiones cromosómicas bien definidas de tres hembras y tres machos; y el cariotipo promedio se integró de ocho pares de cromosomas metacéntricos (m), cuatro pares de cromosomas submetacéntricos (sm), ocho pares de telocéntricos (t), y ocho pares de microcromosomas telocéntricos (*t) El número fundamental observado en el cariotipo típico promedio fue NF = 80 brazos cromosómicos. No se detectaron diferencias sexuales.

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