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The skin of fish is a physicochemical barrier that is characterized by being formed by cells that secrete molecules responsible for the first defense against pathogenic organisms. In this study, the biological activity of peptides from mucus of Seriola lalandi and Seriolella violacea were identified and characterized. To this purpose, peptide extraction was carried out from epidermal mucus samples of juveniles of both species, using chromatographic strategies for purification. Then, the peptide extracts were characterized to obtain the amino acid sequence by mass spectrometry. Using bioinformatics tools for predicting antimicrobial and antioxidant activity, 12 peptides were selected that were chemically produced by simultaneous synthesis using the Fmoc-Tbu strategy. The results revealed that the synthetic peptides presented a random coil or extended secondary structure. The analysis of antimicrobial activity allowed it to be discriminated that four peptides, named by their synthesis code 5065, 5069, 5070, and 5076, had the ability to inhibit the growth of Vibrio anguillarum and affected the copepodite stage of C. rogercresseyi. On the other hand, peptides 5066, 5067, 5070, and 5077 had the highest antioxidant capacity. Finally, peptides 5067, 5069, 5070, and 5076 were the most effective for inducing respiratory burst in fish leukocytes. The analysis of association between composition and biological function revealed that the antimicrobial activity depended on the presence of basic and aromatic amino acids, while the presence of cysteine residues increased the antioxidant activity of the peptides. Additionally, it was observed that those peptides that presented the highest antimicrobial capacity were those that also stimulated respiratory burst in leukocytes. This is the first work that demonstrates the presence of functional peptides in the epidermal mucus of Chilean marine fish, which provide different biological properties when the fish face opportunistic pathogens.
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Aquicultura , Peixes , Muco , Animais , Muco/química , Chile , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Vibrio/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificaçãoRESUMO
Harmful algal blooms (HABs) can produce a variety of noxious effects and, in some cases, the massive mortality of wild and farmed marine organisms. Some HAB species produce toxins that are released into seawater or transferred via food webs (particulate toxin fraction). The objective of the present study was to identify the toxicological effects of subacute exposure to saxitoxin (STX) during embryonic and early larval stages in Seriola rivoliana. Eggs were exposed to dissolved 19 STX (100 µg L-1). The toxic effects of STX were evaluated via the hatching percentage, the activity of three enzymes (protein and alkaline phosphatases and peroxidase), and the expression of four genes (HSF2, Nav1.4b, PPRC1, and DUSP8). A low hatching percentage (less than 5%) was observed in 44 hpf (hours post fertilization) embryos exposed to STX compared to 71% in the unexposed control. At this STX concentration, no oxidative stress in the embryos was evident. However, STX induced the expression of the NaV1.4 channel α-subunit (NaV1.4b), which is the primary target of this toxin. Our results revealed the overexpression of all four candidate genes in STX-intoxicated lecithotrophic larvae, reflecting the activation of diverse cellular processes involved in stress responses (HSF2), lipid metabolism (PPRC1), and MAP kinase signaling pathways associated with cell proliferation and differentiation (DUSP8). The effects of STX were more pronounced in young larvae than in embryos, indicating a stage-specific sensitivity to the toxin.
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Perciformes , Saxitoxina , Animais , Saxitoxina/toxicidade , Toxicogenética , Proliferação Nociva de Algas , Organismos Aquáticos , LarvaRESUMO
Developing sound breeding programs for aquaculture species may be challenging when matings cannot be controlled due to communal spawning. We developed a genotyping-by-sequencing marker panel of 300 SNPs for parentage testing and sex determination by using data from an in-house reference genome as well as a 90 K SNP genotyping array based on different populations of yellowtail kingfish (Seriola lalandi). The minimum and maximum distance between adjacent marker pairs were 0.7 Mb and 13 Mb, respectively, with an average marker spacing of 2 Mb. Weak evidence of the linkage disequilibrium between adjacent marker pairs was found. The results showed high panel performance for parental assignment, with probability exclusion values equaling 1. The rate of false positives when using cross-population data was null. A skewed distribution of genetic contributions by dominant females was observed, thus increasing the risk of higher rates of inbreeding in subsequent captive generations when no parentage data are used. All these results are discussed in the context of breeding program design, using this marker panel to increase the sustainability of this aquaculture resource.
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The buoyancy of eggs and embryos is associated with successful development in pelagic fish. Buoyancy is the result of oocyte hydration, which depends on the osmotic force exerted by free amino acids (FAA) generated by yolk proteolysis, and cathepsins are the main enzymes involved in this process. Seriola lalandi is a pelagic fish whose farming has been hampered by development failure that have been partially attributed to decreased buoyancy of embryos. Therefore, the aim of this study was to compare the mRNA expression and activity of cathepsins B, D, and L, as well as the FAA content in floating and low-floating embryos at different developmental stages. The chosen stages were eggs, morula, blastula, gastrula and 24 h embryos. Complementary assessments showed that there were no differences attributed to buoyancy status in embryo and oil droplet diameters, as well as the transcriptional status at any developmental stage. Cathepsin B did not show differences in mRNA expression or activity related to buoyancy at any stage. Cathepsin D displayed higher transcript and activity levels only in low-floating eggs compared with those floating. Cathepsin L showed higher expression in floating eggs and 24 h embryos compared with that of low-floating, but the activity of this enzyme was higher in floating eggs and morula. Total FAA content constantly decreased throughout development in floating embryos, but it was always higher than low-floating embryos until gastrula stage. In 24 h embryos floating and low-floating embryos share similar quantities of FAA. In summary, differences in the expression and activity of cathepsins between floating and low-floating embryos could be revealed at specific embryonic stages, suggesting different functions of these enzymes throughout development. Besides 24 h embryos, FAA content seems to be a decisive factor for buoyancy of embryos during early development of S. lalandi. Overall, considering the main role of cathepsins and FAA in buoyancy acquisition process and therefore in both embryo quality and viability, our study identifies good marker candidates to evaluate embryo quality in the farming of this species.
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We investigated a time-course larval transcriptional analysis (RNA-seq) in the longfin yellowtail Seriola rivoliana, from hatching to day four at 22 °C, without providing zooplankton as food. Larval starvation is a critical physiological stage that must be prevented to ensure survival. However, the transcriptional mechanisms to endure starvation have not been investigated in marine fish. Differential gene expression showed newly day-specific transcriptome events during larval development. On day 1 (yolk sac absorption), the predominant upregulated developmental processes were larval growth, muscle and vision development, cytoskeletal structure, protein synthesis, protein and fat digestion-absorption, and hormone biosynthesis, whereas the cell cycle was suppressed. On day 2 (yolk sac exhaustion), a new stage of energy regeneration (ATP) was supplied by the oil drop reserve, whereas protein digestion-absorption and growth were suppressed. On day 3 (mouth opening and starvation), stress signals and nutrition deprivation upregulated the p53 signal and triggered autophagy and the AMP-activated protein kinase (AMPK) pathways as an alternative catabolic pathway to enduring starvation, and the circadian rhythm was established. On day 4 (starving and weakened larvae condition), autophagy supported subsequent protein synthesis, activated the immune system, and promoted estrogen signaling and skeleton renovation. However, larvae suppressed muscle development, vision and carbohydrate, and fat digestion-absorption and became lethargic, evidencing limited physiological support by autophagy to maintain survival without exogenous nutrition in this species.
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Privação de Alimentos , Perciformes/crescimento & desenvolvimento , Perciformes/metabolismo , Animais , Autofagia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Perciformes/genética , TranscriptomaRESUMO
In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.
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The Seriola genus includes species of worldwide commercial importance due to its rapid growth and easy adaptability to confinement conditions. However, like other fish species, large mortalities occur during their early life stages, where the main problems are caused by opportunistic bacteria. Disease control strategies are thus urgently needed. The present study aimed to evaluate the efficacy of phage vB_Pd_PDCC-1 during the early development of longfin yellowtail (Seriola rivoliana), as well as its effect on microbial communities. This broad-host-range phage was added to the culture every 3 days starting from the egg-stage until 12 days after hatching (DAH) at a concentration of 1.41×1010 plaque-forming units (PFU) per mL and at a multiplicity of infection (MOI) of 1. The results showed positive effects (p<0.05) on egg hatching, survival, growth, and pigmentation area in treated larvae. Moreover, high-throughput sequencing analysis of 16S rRNA genes showed that phage administration did not produce significant changes (p>0.05) in the composition and structure of the associated microbiota. However, sequences affiliated to the Gammaproteobacteria class were displaced by those belonging to the Alphaproteobacteria class over time regardless of the treatment received. At the family level, there was a decrease in Rhodobacteraceae, Pseudoalteromonadaceae, and Flavobacteriaceae in both groups over time. To our best knowledge, this study represents the first attempt to evaluate the effect of a phage as a biological control agent during ontogenetic development of longfin yellowtail larvae. KEY POINTS: ⢠Phages can be used against proliferation of Vibrio in fish cultures. ⢠Seriola includes several important commercial fish species due to its rapid growth. ⢠Phages do not cause significant changes in the associated microbiota.
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Bacteriófagos , Vibrio , Animais , Bacteriófagos/genética , Peixes , Myoviridae , RNA Ribossômico 16S/genéticaRESUMO
Seriola rivoliana intestinal microbiota (IM) was characterised under aquaculture conditions through 16S rRNA amplicon sequencing. Specimens of 30 days after hatching (DAH) were maintained in three tanks and fed under the same environmental conditions for characterisation 15 days prior to sampling. Three fish were randomly taken from each tank; total DNA extraction of the gut microbiota was performed to characterise microbial composition and its metabolic prediction. The V3 hypervariable region of the 16S rRNA was amplified and sequenced with Illumina pair-end technology. The prokaryotic components in the S. rivoliana intestine were dominated mainly by the phyla Proteobacteria, Firmicutes, Bacteroidetes, Cyanobacteria and Actinobacteria. No significant differences in beta diversity were detected in the three samples (tanks). However in alpha diversity, they were detected in juveniles of the same cohort within the same group, as exemplified by enrichment of certain bacterial groups, mainly of the Clostridia class, which were specific in each fish within the same tank. The metabolic prediction analyses suggested that S. rivoliana IM contribute to the metabolism of amino acids, carbohydrates, lipids, and immune system. This study provides the first IM characterisation under rearing conditions of S. rivoliana-a species with broad economic potential-and contributes to novel information for potential use of probiotics in future trials.
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Actinobacteria/metabolismo , Bacteroidetes/metabolismo , Cianobactérias/metabolismo , Firmicutes/metabolismo , Perciformes/microbiologia , Proteobactérias/metabolismo , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Aminoácidos/imunologia , Aminoácidos/metabolismo , Animais , Aquicultura , Bacteroidetes/classificação , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Metabolismo dos Carboidratos , Carboidratos/imunologia , Cianobactérias/classificação , Cianobactérias/genética , Cianobactérias/isolamento & purificação , DNA Bacteriano/genética , Firmicutes/classificação , Firmicutes/genética , Firmicutes/isolamento & purificação , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/imunologia , Imunidade Inata , Metabolismo dos Lipídeos , Lipídeos/imunologia , Perciformes/imunologia , Perciformes/metabolismo , Proteobactérias/classificação , Proteobactérias/genética , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/genética , Simbiose/imunologiaRESUMO
AIMS: This study describes the effect of phage therapy on hatching of longfin yellowtail (Seriola rivoliana) eggs challenged with Photobacterium damselae subsp. damselae. METHODS AND RESULTS: A lytic phage (vB_Pd_PDCC-1) against P. damselae subsp. damselae was isolated and characterized. The use of phage vB_Pd_PDCC-1 increased the hatching rate of eggs, and reduced presumptive Vibrio species to non-detectable numbers, even in non-disinfected eggs. High-throughput 16S rRNA gene sequencing analysis revealed that phage vB_Pd_PDCC-1 caused significant changes in the composition and structure of the associated microbiota, allowing that members (e.g. those belonging to the family Vibrionaceae) of the class Gammaproteobacteria to be displaced by members of the class Alphaproteobacteria. CONCLUSIONS: To the best of our knowledge, this represents the first study evaluating phage therapy to control potential negative effects of P. damselae subsp. damselae during hatching of longfin yellowtail eggs. SIGNIFICANCE AND IMPACT OF THE STUDY: The Seriola genus includes several important commercial fish species due to its rapid growth and easy adaptability to confinement conditions. However, bacterial infections (especially those caused by Vibrio and Photobacterium species) are among the main limiting factors for the intensification of marine fish aquaculture, particularly during early development stages. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.
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Bacteriófagos/fisiologia , Agentes de Controle Biológico/farmacologia , Doenças dos Peixes/terapia , Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Photobacterium/efeitos dos fármacos , Animais , Aquicultura , Doenças dos Peixes/microbiologia , Peixes/fisiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/terapia , Microbiota/efeitos dos fármacos , Óvulo/microbiologia , Óvulo/fisiologia , Terapia por Fagos , Photobacterium/crescimento & desenvolvimentoRESUMO
Seriola lalandi is an economically important species that is globally distributed in temperate and subtropical marine waters. Aquaculture production of this species has had problems associated with intensive fish farming, such as disease outbreaks or nutritional deficiencies causing high mortality. Intestinal microbiota are involved in many processes that benefit a host, such as disease control, stimulation of the immune response, and the promotion of nutrient metabolism. The aim of this study is to evaluate the in vitro probiotic properties of bacteria isolated from the intestinal content of wild Seriola lalandi. The probiotic potential was evaluated in terms of (i) the antimicrobial activity against vibrios causing outbreaks in farmed fish; (ii) the ability to stimulate genes related to an innate immune response in fish; and (iii) antibiotic resistance. Nineteen isolates identified as Pseudomonas, Shewanella, Psychrobacter, and Acinetobacter showed antimicrobial activity and significant relative expression of cytokines, serum amyloid A protein (SAA), hepcidin, and lysozyme. A positive correlation was observed between the levels of expression and the bacterial load after 24 h of exposure. Pseudomonas isolates showed a level of antibiotic resistance. In conclusion, isolates of the genera Shewanella, Psychrobacter, and Acinetobacter could serve as potential probiotics in S. lalandi culture.
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Bactérias , Peixes/microbiologia , Microbioma Gastrointestinal , Probióticos/isolamento & purificação , Animais , Aquicultura , Bactérias/imunologia , Bactérias/isolamento & purificação , Farmacorresistência BacterianaRESUMO
Seriola rivoliana cultivated in Mexico are infected by Neobenedenia sp. (Monogenea: Capsalidae), resulting in dermal ulceration and subsequent bacterial invasion that can cause fish death. This study assesses the effects of temperature over hatching success, oncomiracidia longevity, and infection success. The experimental design consisted of culturing the parasite at temperatures ranging between 16 and 32 °C. The oncomiracidia infection success, time to sexual maturity, and size at sexual maturity of Neobenedenia sp. were examined only at three temperatures (20 °C, 24 °C, and 30 °C). Experiments were conducted under controlled conditions in the laboratory. The oncomiracidia development was found to be faster at warmer temperatures (4-5 days between 24 and 30 °C) than in colder treatments (7-11 days between 18 and 20 °C). Hatching success and oncomiracidia longevity were higher at 24 °C and 26 °C. At 20 °C, 24 °C, and 30 °C, infection success was greater than 90%. Additionally, the laid eggs were observed at 9, 12, and 15 days at 30 °C, 24 °C, and 30 °C, respectively. The results of this study will allow for improving the temporal schedule of applications of treatments against Neobenedenia sp. by the function of temperatures. In conclusion, it is recommended to treat fish more frequently if the temperature in cultures is higher than 24 °C, because Neobenedenia sp. development is faster. As an alternative, the fish could be moved to deeper and cooler waters.
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Estágios do Ciclo de Vida , Perciformes/parasitologia , Temperatura , Trematódeos/crescimento & desenvolvimento , Animais , Doenças dos Peixes/parasitologia , MéxicoRESUMO
The purpose of this study was to characterize the TLR9 gene from yellowtail (Seriola lalandi) and evaluate its functional activity using the class B Cytosine-phosphate-guanine-oligodeoxynucleotide2006 (CpG-ODN2006) in an in vivo experiment after one-week immunostimulation. The gene expressions of TLR9, Immunoglobulin M (IgM), antimicrobial peptides and cytokines were evaluated by real time PCR, and humoral immune parameters were analyzed in serum. The TLR9 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. The yellowtail full-length cDNA sequence of SlTLR9 was 3789 bp in length, including a 66-bp 5'-untranslated region (UTR), a 3'-UTR of 528 bp, and an open reading frame (ORF) of 3192 bp translatable to 1064 amino acid showing a high degree of similarity with the counterparts of other fish species and sharing common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR9 mRNA in all analyzed tissues; the highest levels were observed in intestine, liver and spleen where they play an important role in the fish immune system. The expression levels of TLR9 after B class CpG-ODN2006 (the main TLR9-agonist) was significantly up-regulated in all analyzed tissues, with the high expression observed in spleen followed by intestine and skin. The CpG-B has been shown as a potent B cell mitogen, and interestingly, IgM mRNA transcript was up-regulated in spleen and intestine, which was highly correlated with TLR9 after CpG-ODN2006 stimulation. The antimicrobial peptides, piscidin and NK-lysine, were up-regulated in spleen and gill after CpG-ODN2006 injection with a high correlation (râ¯≥â¯0.82) with TLR9 gene expression. Cytokine genes were up-regulated in spleen, intestine and skin after CpG-ODN was compared with the control group. No significant correlation was observed between TLR9 and IL-1ß, TNF-α and Mx gene expressions. The results showed that CpG-ODN2006 intraperitoneal injection enhanced lysozyme, peroxidase and superoxide dismutase activities in serum and demonstrated that CpG-ODN2006 can induce a specific immune response via TLR9 in which IgM and antimicrobial peptides must have an important role in the defense mechanisms against infections in yellowtail.
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Imunidade Humoral/genética , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Transdução de Sinais/imunologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/imunologia , Imunoglobulina M/imunologia , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/imunologia , Filogenia , Transdução de Sinais/genética , Receptor Toll-Like 9/químicaRESUMO
Seriola lalandi is an economically important species that is globally distributed in temperate and subtropical marine waters. Aquaculture production of this species has had problems associated with intensive fish farming, such as disease outbreaks or nutritional deficiencies causing high mortalities. Intestinal microbiota has been involved in many processes that benefit the host, such as disease control, stimulation of the immune response, and the promotion of nutrient metabolism, among others. However, little is known about the potential functionality of the microbiota and the differences in the composition between wild and aquacultured fish. Here, we assayed the V4-region of the 16S rRNA gene using high-throughput sequencing. Our results showed that there are significant differences between S. lalandi of wild and aquaculture origin (ANOSIM and PERMANOVA, P < 0.05). At the genus level, a total of 13 genera were differentially represented between the two groups, all of which have been described as beneficial microorganisms that have an antagonistic effect against pathogenic bacteria, improve immunological parameters and growth performance, and contribute to nutrition. Additionally, the changes in the presumptive functions of the intestinal microbiota of yellowtail were examined by predicting the metagenomes using PICRUSt. The most abundant functional categories were those corresponding to the metabolism of cofactors and vitamins, amino acid metabolism and carbohydrate metabolism, revealing differences in the contribution of the microbiota depending on the origin of the animals. To our knowledge, this is the first study to characterize and compare the intestinal microbiota of S. lalandi of wild and aquaculture origin using high-throughput sequencing.
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This work contributes basic knowledge on larval development of Seriola rivoliana. A histological study describes the development of the digestive tract and accessory glands in S. rivoliana larvae reared under laboratory conditions at 24 °C from hatching to 30 days post-hatching (DPH). At hatching (2.6 ± 0.12 mm), larvae had an undifferentiated digestive tract with a closed straight tube and a large yolk sac with an oil globule. The liver and pancreas were observed at 1 and 2 days, and the mouth and anus opened at day 2. Enriched rotifers were visible in their digestive tract. At the beginning of the pre-flexion stage, a mixed nutritional period was observed. At day 3, exogenous feeding began; the digestive tract became differentiated into the buccopharynx, esophagus, an undifferentiated stomach, and the intestines. Zymogen granules were visible in the exocrine pancreas. At day 4, supranuclear vacuoles were present in the posterior intestine, indicating the beginning of intracellular digestion. At day 5, goblet cells were present in the esophagus and became functional at day 7 in the esophagus and intestine. The buccopharynx goblet cells developed at day 15. The presence of gastric glands and differentiation of the stomach in the fundic, cardiac, and pyloric regions during the post-flexion stage occurred at day 20. This was the onset of the juvenile period and the beginning of weaning; however, a long co-feeding phase is recommended. Pyloric caeca were observed at day 30 (13.6 ± 1.6 mm). These results provide valuable information on S. rivoliana larvae biology and digestive physiology, which should be useful to improve cultivation techniques and identify ecological features involved in ontogeny.
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Peixes/anatomia & histologia , Peixes/crescimento & desenvolvimento , Trato Gastrointestinal/crescimento & desenvolvimento , Animais , Trato Gastrointestinal/anatomia & histologia , Larva/anatomia & histologia , Larva/crescimento & desenvolvimentoRESUMO
In the context of global climate change where harmful algal blooms (HABs) might become more frequent and more severe, several studies have been conducted on the perturbation of embryonic development of marine animals by microalgal toxins. Okadaic acid (OA) and analogs (DSP toxins) produced by dinoflagellates of the genera Dynophysis and Prorocentrum are known to disturb embryogenesis. This study investigated the impact of dissolved DSP toxin (OA and Dinophysistoxin 1, DTX-1) exposure on embryo development of Longfin yellowtail Seriola rivoliana. Eggs were exposed to different concentrations of dissolved DSP toxins (low treatment: at 120µgl-1 OA eq; high treatment 175µgl-1 OA eq.). The first objective was to study the global toxic effect of DSP toxins with hatching percentages. Secondly, the effect of these toxins was investigated at molecular and functional level by measuring expression of responsible genes for bone morphogenetic protein (BMP) and proliferating cell nuclear antigen (PCNA) measuring phosphatase enzyme (serine/threonine and alkaline phosphatases) activities. Our results showed drastic mortalities induced by DSP toxins in both low and high concentration treatments. Activities of both protein and alkaline phosphatases were significantly inhibited by DSP toxin treatments, whose effects on gene expression were less evident, but levels of BMP expression in eggs treated with the lowest toxin concentration were significantly different from that in the control treatment. This work revealed an embryotoxic effect of DSP toxins resulting in high mortality of eggs. Phosphatase inhibition could have participated in part in these global effects by perturbing the regulation of pathways related to embryogenesis and resulting in a perturbation of gene expression.
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Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Peixes , Ácido Okadáico/toxicidade , Piranos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Mudança Climática , Dinoflagellida/metabolismo , Inibidores Enzimáticos , Peixes/embriologia , Proliferação Nociva de AlgasRESUMO
In pelagic species such as Seriola lalandi, survival of both the eggs and embryos depends on yolk processing during oocyte maturation and embryo development. The main enzymes involved in these processes are the cathepsins, which are essential for the hydration process, acquiring buoyancy and nutrition of the embryo before hatching. This study aimed to investigate the mRNA expression profiles of cathepsins B, D and L (catb, catd and catl) and the activity of these enzymes during early development in S. lalandi. We included previtellogenic oocytes (PO). All three enzymes were highly expressed in PO, but the expression was reduced throughout development. Between PO and recently spawned eggs (E1) the transcript to catb and catd decreased, unlike catl. Cathepsin B activity, showed stable levels between PO until blastula stage (E4). High activities levels of cathepsins D and L were observed in E1 in comparison with later developmental stages. Cathepsin L activity remained constant until E1, consistent with observations in other pelagic spawners, where its participation in a second protolithic cleavage of the yolk proteins, has been proposed for this enzyme. Their profiles of both mRNA expression and enzymatic activity indicate the importance of these enzymes during early development and suggest different roles in egg yolk processing for the hydration process and nutrition in early embryos in this species.
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Catepsinas/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Perciformes/embriologia , RNA Mensageiro/genética , Animais , Catepsinas/genética , Desenvolvimento Embrionário/fisiologiaRESUMO
The purpose of this study was to characterize the TLR21 gene from yellowtail (Seriola lalandi) and its functional activity using TLR agonist stimulation and Aeromonas antigens. The TLR21 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. Basal TLR21 gene expression was analyzed in several tissues. Subsequently, the gene expression of TLR21 and cytokines IL-1ß and TNF-α was evaluated in TLR agonist (CpG-ODN2006, LPS, and Poly I:C) exposing head kidney leucocytes, which were then subjected to Aeromonas antigen stimulation. The yellowtail full-length cDNA sequence of SlTLR21 was 3615 bp (980 aa) showing a high degree of similarity with the counterparts of other fish species and sharing the common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR21 mRNA in all the analyzed tissues; the highest levels were observed in spleen and head kidney where they play an important role in the fish immune system. Transcripts of TLR21 and the downstream IL-1ß and TNF-α cytokine genes were most strongly up-regulated after exposure to the TLR agonists following Aeromonas antigen stimulation, suggesting they are involved in immune response. The results indicated that TLR agonists, in combination with Aeromonas antigens in head kidney leucocytes, synergistically enhance TLR21 and cytokines in yellowtail.
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Aeromonas/imunologia , Proteínas de Peixes/metabolismo , Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Rim Cefálico/metabolismo , Baço/metabolismo , Receptores Toll-Like/metabolismo , Animais , Antígenos de Bactérias/imunologia , Clonagem Molecular , Proteínas de Peixes/genética , Imunidade Inata , Leucócitos/imunologia , Lipopolissacarídeos/imunologia , Oligodesoxirribonucleotídeos/imunologia , Filogenia , Poli I-C/imunologia , Análise de Sequência de DNA , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , TranscriptomaRESUMO
Seriola lalandi is an ecologically and economically important species that is globally distributed in temperate and subtropical marine waters. The aim of this study was to identify large numbers of genic single nucleotide polymorphisms (SNPs) and differential gene expression (DGE) related to the early development of normal and deformed S. lalandi larvae using high-throughput RNA-seq data. A de novo assembly of reads generated 40,066 genes ranging from 300 bases to 64,799 bases with an N90 of 788 bases. Homology search and protein signature recognition assigned gene ontology (GO) terms to a total of 15,744 (39.34%) genes. A search against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) retrieved 6808 KEGG orthology (KO) identifiers for 10,520 genes (26.25%), and mapping of KO identifiers generated 337 KEGG pathways. Comparisons of annotated genes revealed that 1262 genes were downregulated and 1047 genes were upregulated in the deformed larvae group compared to the normal group of larvae. Additionally, we identified 6989 high-quality SNPs from the assembled transcriptome. These putative SNPs contain 4415 transitions and 2574 transversions, which will be useful for further ecological studies of S. lalandi. This is the first study to use a global transcriptomic approach in S. lalandi, and the resources generated can be used further for investigation of gene expression of marine teleosts to investigate larval developmental biology. The results of the GO enrichment analysis highlight the crucial role of the extracellular matrix in normal skeleton development, which could be important for future studies of skeletal deformities in S. lalandi and other marine species.
Assuntos
Perciformes/genética , Transcriptoma , Animais , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Perciformes/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo ÚnicoRESUMO
TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. In this study, we cloned the full-length cDNA sequence of yellowtail (Seriola lalandi) TLR22 (SlTLR22). The complete cDNA sequence of SlTLR22 was 4208 bp and encodes a polypeptide of 961 amino acids. Analysis of the deduced amino acid sequence indicated that SlTLR22 has typical structural features of proteins belonging to the TLR family. These included 17 LRR domains (residues 91-633) and one C-terminal LRR domain (LRR-CT, residues 693-744) in the extracellular region, and a TIR domain (residues 800-943) in the cytoplasmic region. Comparison with homologous proteins showed that the deduced SlTLR22 has the highest sequence identity to turbot TLR22 (76%). Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of SlTLR22 mRNA in all examined tissues with higher levels in the head kidney, intestine, skin and spleen. Further, SlTLR22 expression was significantly up-regulated following TLR ligands injection with lipopolysaccharide (LPS), CpG ODN2006 and polyinosinic: polycytidylic acid (poly I:C) in spleen and liver. Amyloodinium ocellatum infection also induced a high expression of SlTLR22 in spleen, intestine, muscle, skin and gill, with maximum increases ranging from 1000 to 100 fold upon different ligands and organs. Finally, histological examination in gill tissue confirmed infection by the parasite and histopathological lesion was observed also in spleen and skin. These findings suggest a possible role of SlTLR22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and parasites.