From zero to ossified: Larval skeletal ontogeny of the Neotropical Cichlid fish Cichlasoma dimerus.

The identification of skeletal elements, the analysis of their developmental sequence, and the time of their appearance during larval development are essential to broaden the knowledge of each fish species and to recognize skeletal abnormalities that may affect further fish performance. Therefore, this study aimed to provide a general description of the development of the entire skeleton highlighting its variability in Cichlasoma dimerus. Larvae of C. dimersus were stained with alcian blue and alizarin red from hatching to 25 days posthatching. Skeletogenesis began with the endoskeletal disk and some cartilage structures from the caudal fin and the splachnocranium, while the first bony structure observed was the cleithrum. When larvae reached the free-swimming and exogenous feeding stage, mostly bones from the jaws, the branchial arches, and the opercle series evidenced some degree of ossification, suggesting that the ossification sequence of C. dimerus adjusts to physiological demands such as feeding and ventilation. The caudal region was the most variable regarding meristic counts and evidenced higher incidence of bone deformities. In conclusion, this work provides an overview of C. dimerus skeletogenesis and lays the groundwork for further studies on diverse topics, like developmental plasticity, rearing conditions, or phylogenetic relationships.

Potential applications of recombinant bifidobacterial proteins in the food industry, biomedicine, process innovation and glycobiology.

Bifidobacterial proteins have been widely studied to elucidate the metabolic mechanisms of diet adaptation and survival of Bifidobacteria, among others. The use of heterologous expression systems to obtain proteins in sufficient quantities to be characterized has been essential in these studies. L. lactis and the same Bifidobacterium as expression systems highlight ways to corroborate some of the functions attributed to these proteins. The most studied proteins are enzymes related to carbohydrate metabolism, particularly glycosidases, due to their potential application in the synthesis of neoglycoconjugates, prebiotic neooligosaccharides, and active metabolites as well as their high specificity and efficiency in processing glycoconjugates. In this review, we classified the recombinant bifidobacterial proteins reported to date whose characterization has demonstrated their usefulness or their ability to produce a product of commercial interest for the food industry, biomedicine, process innovation and glycobiology. Future directions for their study are also discussed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00957-1.

Expression of ion transport proteins and routine metabolism in juveniles of tropical gar (Atractosteus tropicus) exposed to ammonia.

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.

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.

The larval attachment organ of the tropical gar Atractosteus tropicus Gill, 1863 (Lepisosteiformes: Lepisosteidae).

Larval attachment organs (LAOs) are unicellular or multicellular organs that allow larvae to adhere to a substrate before yolk-sac absorption and the free-swimming stage. This study documents the LAO of tropical gar, Atractosteus tropicus, using a combination of scanning electron microscopy and light microscopy. It is shown that the LAO of A. tropicus is a super-organ surrounded by a wall and containing at its centre many smaller multicellular organ units, each comprised of attachment and support cells. Attachment cells are secretory and house large vacuoles filled with a glycoprotein. At hatching, the super-organ is well developed and occupies almost the entire anteroventral surface of the head. During subsequent development, the smaller individual units begin to regress, until at 6 days post-hatching the super-organ and its individual units are no longer visible.

Gonadotropin-inhibitory hormone (GnIH) distribution in the brain of the ancient fish Atractosteus tropicus (Holostei, Lepisosteiformes).

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.

Partial characterization of digestive proteases in juveniles of Microphis brachyurus (short-tailed pipefish) (Syngnathiformes: Syngnathidae)

Short-tailed pipe fish (Microphis brachyurus) is a freshwater organism with high economic potential for the aquarium hobby, so it is necessary to implement methods to promote its culture through studies of digestive physiology. General activities of acid and alkaline proteases were evaluated, as well as the effect of pH, temperature and inhibitors. The optimal pH of stomach proteases was 2, while the optimal pH of intestinal proteases was 10. Optimal temperature for the acidic proteases was 35 ºC, while for alkaline proteases it was 45 ºC. Thermal stability showed high resistance at 35 ºC for both acid and alkaline proteases (above 100% residual activity). Acid proteases are resistant at pH 2 (50% of residual activity), meanwhile alkaline proteases were highly resistant at pH 10 (90% of residual activity). Acid proteases were inhibited by 80% with pepstatin A and alkaline proteases were inhibited with TLCK and TPCK for trypsin (75%) and chymotrypsin (80%), respectively. Finally, metallo-proteases were 75% partially inhibited some serine proteases by 75% with EDTA. In conclusion, M. brachyurus has a good digestive capacity, since they can degrade a wide variety of proteins due to their greater proteolytic activity.(AU)

El pez pipa (Microphis brachyurus) es un organismo dulceacuícola con alto potencial económico para la acuarofilia; sin embargo, es necesario implementar su cultivo a través de estudios de fisiología digestiva. Se evaluó el efecto del pH, temperatura e inhibidores sobre las actividades enzimáticas de proteasas ácidas y alcalinas. El pH óptimo de proteasas estomacales es de 2, mientras que el de proteases intestinales es de 10. La temperatura óptima de proteasas ácidas es de 35 ºC y las alcalinas de 45 ºC. La estabilidad térmica para proteasas ácidas y alcalinas es a los 35 ºC (más de 100% de actividad residual). La estabilidad a los diferentes pH de las proteasas ácidas es en 2 (50 % de la actividad residual), mientras que para las proteasas alcalinas es en 10 (90 % de la actividad residual). Las proteasas ácidas fueron inhibidas en 80% con pepstatina A y las proteasas alcalinas fueron altamente inhibidas con TLCK para tripsina (75%) y TPCK quimitripsina (80%). Finalmente, las metaloproteasas fueron inactivadas con EDTA en 70%. En conclusión, M. brachyurus tiene una buena capacidad digestiva al degradar una amplia variedad de proteinas debido a su alta actividad proteolítica.(AU)

Effect of organophosphorus pesticides in juveniles of Litopenaeus vannamei: alteration of glycogen, triglycerides, and proteins.

The presence of pesticides has recently been reported in shrimp farms adjacent to agricultural areas on the east coast of the Gulf of California. This study assessed the possible effect of organophosphorus pesticides in the coastal environment of Sinaloa, México, using the white shrimp Litopenaeus vannamei as a bioindicator since their presence, abundance or behavior indicate some process or state of the system in which they live. Sublethal bioassays were performed on shrimps in intermolt state using commercial brands of organophosphorus pesticides, chlorpyrifos (0.0015 mg l-1), diazinon (0.0120 mg l-1), methamidophos (1.207 mg l-1), azinphos-methyl (0.0101 mg l-1), and methyl parathion (0.0075 mg l-1) were tested. Results showed reductions in glycogen, triglycerides, and total protein concentrations in shrimp muscle, except for the diazinon treatments, in which an increase in triglyceride level was detected. The observed alterations in the three cellular components were probably due to the metabolic compensation mechanism of the shrimp in reaction to the stress produced by organophosphorus pesticides, which act as endocrine disruptors. The establishment of continuous environmental monitoring programs of the coastal zone of Northwestern Mexico is strongly recommended.

Developmental changes in oxygen consumption and hypoxia tolerance in the heat and hypoxia-adapted tabasco line of the Nile tilapia Oreochromis niloticus, with a survey of the metabolic literature for the genus Oreochromis.

The genus Oreochromis is among the most popular of the tilapiine cichlid tribe for aquaculture. However, their temperature and hypoxia tolerance, if tested at all, is usually tested at temperatures of 20-25°C, rather than at the considerably higher temperatures of 30-35°C typical of tropical aquaculture. We hypothesized that both larvae and adults of the heat and hypoxia-adapted Tabasco-line of the Nile tilapia Oreochromis niloticus would be relatively hypoxia-tolerant. Oxygen consumption rate ( M ˙ O 2 ), Q10 and aquatic surface respiration (ASR) was measured using closed respirometry at 2 (c. 0.2 g), 30 (c. 2-5 g), 105 c. (10-15 g) and 240 (c. 250 g) days of development, at 25°C, 30°C and 35°C. M ˙ O 2 at 30°C was inversely related to body mass: c. 90 µM O2 g-1 /h in larvae down to c. 1 µM O2 g-1 /h in young adults. Q10 for M ˙ O 2 was typical for fish over the range 25-35°C of 1.5-2.0. ASR was exhibited by 50% of the fish at pO2 of 15-50 mmHg in a temperature-dependent fashion. However, the largest adults showed notable ASR only when pO2 fell to below 10 mmHg. Remarkably, pcrit for M ˙ O 2 was 12-17 mmHg at 25-30°C and still only 20-25 mmHg across development at 35°C. These values are among the lowest measured for teleost fishes. Noteworthy is that all fish maintain equilibrium, ventilated their gills and showed routine locomotor action for 10-20 min after M ˙ O 2 ceased at near anoxia and when then returned to oxygenated waters, all fish survived, further indicating a remarkable hypoxic tolerance. Remarkably, data assembled for M ˙ O 2 from >30 studies showed a > x2000 difference, which we attribute to calculation or conversion errors. Nonetheless, pcrit was very low for all Oreochromis sp. and lowest in the heat and hypoxia-adapted Tabasco line.

Partial characterization of digestive proteases in sheepshead, Archosargus probatocephalus (Spariformes: Sparidae)

Digestive proteases were partially characterized in sheepshead juveniles, using biochemical and electrophoretic techniques. Results showed higher activity level of the stomach proteases (2.39 ± 0.02 U mg protein-1) compared to the intestinal proteases (1.6 ± 0.1 U mg protein-1). The activity of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A was also recorded. The optimum temperature of the stomach proteases was recorded at 45 °C, while for intestinal proteases was recorded at 55 °C. Stomach proteases showed less stability to temperature changes than intestinal proteases. An optimum pH of 2 was recorded for stomach proteases with high stability under acidic conditions, while an optimum pH of 9 was recorded for intestinal proteases showing high stability under alkaline conditions. Stomach proteases were inhibited around 78% with Pepstatin A, indicating the presence of pepsin as the main protease. The stomach proteases zymogam revealed one active band with Rf of 0.49, this enzyme was completely inhibited by Pepstatin A. The intestinal proteases zymogram revealed four active proteases (51.3, 34.9, 27.8 and 21.2 kDa) that were inhibited by TLCK, which mainly represent a trypsin-like serine proteases. It can be conclude that digestion in sheepshead can be considered as a carnivorous species with an omnivorous tendency.(AU)

Se caracterizaron parcialmente las proteasas digestivas de juveniles del sargo, utilizando técnicas bioquímicas y electroforéticas. Los resultados muestran mayores niveles de actividad en las proteasas estomacales (2.39 ± 0.02 U mg proteina-1) comparados con los de las proteasas intestinales (1.6 ± 0.1 U mg protein-1), también se registró la actividad de tripsina, quimotripsina, leucina aminopeptidasa y carboxipeptidasa A. La temperatura óptima de las proteasas estomacales fue de 45 °C, mientras que la de las proteasas intestinales fue de 55 °C. El pH óptimo fue de 2 para las proteasas estomacales con alta estabilidad a condiciones ácidas, mientras que el pH óptimo para las proteasas intestinales fue de 9, mostrando una alta estabilidad en condiciones alcalinas. Las actividades de las proteasas estomacales fue inhibida en un 78% con Pepstatina A, lo que indica la presencia de pepsina, como principal proteasa. El zimograma de proteasas estomacales reveló una sola banda con actividad proteasa, con Rf de 0.49, completamente inhibida por Pepstatina A. El zimograma de proteasas intestinales reveló cuatro bandas (51.3, 34.9, 27.8 y 21.2 kDa). Todas las bandas se inhibieron con TLCK, lo que muestra la presencia principalmente de serina proteasas tipo tripsina. Se concluye que la digestión del sargo puede ser considerada como la de una especie carnívora con tendencia al omnivorismo.(AU)

In vitro digestibility and proteases inhibitory effect of several feedstuffs for Parachromis dovii juveniles and P. dovii hybrid larvae.

Parachromis dovii, a native cichlid from Costa Rica, is highly appreciated for its size and flesh quality. Further, P. dovii easily accept inert feed from the beginning of exogenous feeding; however, its growth is low compared to live food. For this reason, evaluation of several feedstuffs using two in vitro techniques was done. The quantification of the in vitro inhibitory effect of seven plant ingredients on the alkaline protease activity was done using enzymatic extracts from larvae samples of 6, 15, 22 and 30 days after hatching (DAH). The in vitro alkaline digestibility assays were run for six protein sources (from animal and plant origin) using the enzymatic extract from larvae 30 DAH. Independent of fish age, all plant feedstuffs reduced alkaline digestive proteases activity; however, the wheat flour (14.1 % at 6 DAH, 33.4 % at 15 DAH) and broken rice meal (51.6 % at 22 DAH) showed the lowest inhibition percentage of alkaline digestive activity, whereas the highest inhibition percentage was found with soybean and palm kernel meals (92.5 % at 30 DAH and 87.4 %, respectively) (P < 0.05). The alkaline proteases inhibition percentage of feedstuffs varied during larvae ontogeny. From six protein dietary sources tested, tankage and fish meal presented the highest in vitro digestibility values, 113.9 and 74.9 %, respectively. Contrary, the lowest digestibility was found for blood and soybean meals (38.07 and 19.82 %, respectively).

Mitotic karyotype of the tropical freshwater crayfish Procambarus (Austrocambarus) llamasi (Decapoda: Cambaridae).

In Mexico, the biology of Procambarus has been more studied than the biology of other Cambarids because of its diversity and potential use in aquaculture. We determined the karyotype of the Mexican tropical freshwater crayfish Procambarus (Austrocambarus) llamasi from 189 metaphase spreads from gill tissues of 17 adults. They had 98-120 chromosomes (mode 2n=120 chromosomes). There are 60 pairs of monoarm, telocentric chromosomes. Sex chromosomes were not detected and we propose that the P. llamasi karyotype can be used to distinguish this species from other Mexican crayfish. Additionally, we suggest using karyological data in aquaculture and conservation biology.

Mitotic karyotype of the tropical freshwater crayfish Procambarus (Austrocambarus) llamasi (Decapoda: Cambaridae)

In Mexico, the biology of Procambarus has been more studied than the biology of other Cambarids because of its diversity and potential use in aquaculture. We determined the karyotype of the Mexican tropical freshwater crayfish Procambarus (Austrocambarus) llamasi from 189 metaphase spreads from gill tissues of 17 adults. They had 98-120 chromosomes (mode 2n=120 chromosomes). There are 60 pairs of monoarm, telocentric chromosomes. Sex chromosomes were not detected and we propose that the P. llamasi karyotype can be used to distinguish this species from other Mexican crayfish. Additionally, we suggest using karyological data in aquaculture and conservation biology. Rev. Biol. Trop. 58 (2): 655-662. Epub 2010 June 02.

El género Procambarus ha recibido mayor atención en los estudios de los principios fundamentales de su biología debido a su diversidad en el territorio mexicano y potencial uso en acuicultura. El cariotipo típico del acocil tropical mexicano Procambarus (Austrocambarus) llamasi, se estudió mediante 189 dispersiones cromosómicas en metafase del tejido branquial de 17 adultos tratados con la técnica citológica de inmersión. Encontramos un amplio número de cromosomas, que variaron entre 98-120 elementos cromosómicos, con número modal diploide de 2n=120 elementos cromosómicos. El cariotipo del acocil tropical esta constituido por 60 pares de cromosomas monorrámeos, todos los centrómero están en la región telocéntrica de los cromosomas. En las metafases mitóticas de hembras y machos no fueron identificados cromosomas sexuales. Sugerimos considerar la estructura cromosómica del cariotipo como una herramienta citotaxonómica así como el empleo de datos cariológicos para propósitos de acuicultura y conservación del acocil tropical.

[Karyotype of the tropical gar Atractosteus tropicus (Lepisosteiformes: Lepisosteidae) and chromosomal variation in their larval and adults]. / 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.

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

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.