Identification of sex‐specific markers and heterogametic XX/XY sex determination system by 2b‐RAD sequencing in redtail catfish (Mystus wyckioides)

Sex‐specific markers provide significant molecular basis for sex control breeding biotechnology to produce all‐male or all‐female fish in commercial breeding. Redtail catfish (Mystus wyckioides), one of the commercial bagrid catfishes distributed in Southeast Asian, which have a long sexual maturation period that can last 3–5 years and males have apparent growth advantage over females, but its sex determination system remains unknown. In this study, we first applied 2b‐RAD‐seq approach to identify three male‐specific 2b‐RAD‐tags and one male heterogametic SNP locus and validated by blast to the genome survey sequences and PCR amplification in both wild and breeding populations. To get longer sex‐specific region, we performed genome walking and obtained a 4,630 bp of Y‐specific sequence and 4,581 bp of X‐specific sequence from the 2b‐RAD‐tag ref189950 with 92.19% nucleotide identity between them. And 9,923 bp/3,935 bp of Y‐specific sequences and 8,491 bp/5,172 bp of X‐specific sequences were also identified with 77.49% and 57.07% nucleotide identity in ref208528 and ref210837, respectively. Subsequently, three different kinds of sex‐specific primers with different length products were designed based on the detected highly sex differentiated regions and could be used to distinguish males and females both in wild and artificially bred populations. What is more, the X‐specific fragment was discovered to produce the dosage effect association in females and in males. The data suggest that male heterogametic XX/XY sex determination system should exist in the redtail catfish. More significantly, the sex‐specific markers are of great value to protect wild resources and improve the efficiency of all‐male breeding practices for aquaculture in the redtail catfish.     

Molecular cloning and characterization of the cathepsin L gene in Pelodiscus sinensis and its expression in response to bacterial challenge

Cathepsin L is one of the most important lysosomal cysteine proteases for the initiation of protein degradation, and it is involved in the immune response in many vertebrates. However, its function in the innate immune system of turtles remains poorly understood. Here, we cloned the cathepsin L gene from the Chinese soft‐shelled turtle Pelodiscus sinensis. We then examined the mRNA expression in different tissues and at different time points after infection by Aeromonas hydrophila or Vibrio parahemolyticus. The full‐length cDNA sequence cloned from P. sinensis was 1,805 bp with a 1,071 bp open reading frame, which encodes a 40.39 kDa polypeptide of 356 amino acids. The deduced protein sequence contained an active triad of Cys, His and Asn, and conserved ERWNIN and GNFD motifs. Homology analysis showed that the deduced amino acid sequence shared 77%–96% identity with other known species. Sequence alignment, phylogenetic analysis and structural comparisons revealed that cathepsin L is a member of the cathepsin family. Real‐time PCR analyses showed that turtle cathepsin L mRNA is ubiquitously expressed in various tissues, and the expression level is higher in the liver than in other tissues. The expression level in the liver peaks 24 hr after A. hydrophila infection and at two times (12 and 72 hr) after V. parahemolyticus infection. These results suggest that the cathepsin L gene of P. sinensis is likely involved in the process of the antibacterial response to A. hydrophila and V. parahemolyticus. This study is of great importance for future work exploring the molecular mechanism of antibacterial immune responses in P. sinensis.     

Changes in activities and mRNA expression of lipoprotein lipase and fatty acid synthetase in large yellow croaker,Larimichthys crocea (Richardson), during fasting

Over‐winter fasting and man‐made food deprivation to increase meat quality are common in the process of large yellow croaker (Larimichthys crocea) aquaculture. This study aimed to determine the changes in lipoprotein lipase (LPL) and fatty acid synthetase (FAS) activities and mRNA expressions, and the relationships between these changes and fat content in large yellow croaker liver and muscle tissues during fasting. A total of 2933 bp LPL cDNA, including an open reading frame of 1533 bp encoding 510 amino acids, and a 1233 bp fragment of FAS cDNA coding 411 amino acids were cloned. Expressions of both genes were ubiquitous. During a 35‐day fasting period, the hepatosomatic index and fat content in muscle and liver were significantly decreased (P < 0.05). mRNA levels of LPL increased significantly during the fasting period except the first 3 days, and FAS mRNA levels decreased significantly in both muscle and liver (P < 0.05) although there were some fluctuations. Muscle and liver LPL activities were significantly higher following fasting for 7 days, decreased to the initial value following fasting for 14 days, and elevating significantly afterwards (P < 0.05). FAS activities in muscle and liver maintained a significantly decreasing trend during the short‐term fasting (P < 0.05) and kept obviously rising thereafter (P < 0.05). Activities and mRNA levels of both LPL and FAS were not always consistent, which implied that both pre‐translational and post‐translational regulations existed during fasting. Our results suggest that the reasonable fasting time is 21 days before harvesting when fat content decreased significantly.     

Expression of four muscle proteins at different growth stages of Günther's walking catfish Clarias macrocephalus

The complete cDNA sequences of four contractile muscle genes of walking catfish Clarias macrocephalus were characterized by assembling partial EST sequences from a skeletal muscle cDNA library. The four genes were parvalbumin 4 (PV4) (670 bp), troponin C (TnC) (1065 bp), troponin I (TnI) (843 bp) and myosin light chain 3 (MLC3) (953 bp), leading to deduced amino acid sequences of 109, 160, 176 and 150 residues respectively. During the larval stage, TnC mRNA showed the highest levels of expression with a 1.4‐fold increase from day 1 to day 30 post hatch. At 90 days, the relative expression levels of PV4, TnC and MLC3 were the highest, with similar proportions in the skeletal muscle, corresponding to the highest relative growth rate of walking catfish. Expression of the three calcium‐binding proteins remained high in 6‐month‐old fish, with higher levels of PV4. The different proportions of muscle proteins expressed suggested the significance of their contributions to fish growth and appeared to be correlated with the functional properties of muscle cells, which were observed from changes in the swimming activity of the fish.     

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm‐raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive‐sequence‐mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep‐PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep‐PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.     

Molecular cloning,characterization, tissue expression and nutritional regulation of O‐GlcNAc transferase gene in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

O‐GlcNAc transferase gene (OGT) was considered as the sole rate‐limiting enzyme in the O‐GlcNAc modification. In the present study, the OGT gene of hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was cloned and characterized, and its expression in response to dietary carbohydrate level and acute glucose treatment was investigated. The full‐length of OGT (GenBank accession no. KY656469 ) was 4,063 bp, including a 302 bp 5′untranslated terminal region (UTR), a 3,165 bp coding region that encoded 1,054 amino acids residues and a 596 bp 3′ UTR. The highly conservation of OGT gene between fish and mammals was also observed through multiple sequences alignment and phylogenetic analysis. O‐GlcNAc transferase gene was ubiquitously expressed in all detected tissues with highest expressions in brain and liver, to a lesser degree, in eye, heart, kidney and intestine. The increasing dietary carbohydrate from 8.02% to 16.08% had no significant effect on the mRNA expression of OGT. However, the expression of OGT was slightly elevated at 6 hr post‐glucose injection, and the elevation became significant at 24 hr time‐point. These data may enhance our understanding on the nutritional regulation of OGT and O‐GlcNAc modification in fish species.     

Identification,annotation of Mucin genes in channel catfish (Ictalurus punctatus) and their expression after bacterial infections revealed by RNA‐Seq analysis

Mucins are large glycoproteins that cover epithelial surfaces of the body and play important roles in prevention of inflammatory and various infectious diseases. In this study, five membrane‐bound and seven secreted mucin genes in the channel catfish were identified. All these identified mucin genes possess at least one PTS, von Willebrand D (VWD) or SEA domains. The expression of the 12 mucin genes in channel catfish was first studied with infection of Edwardsiella ictaluri and Flavobacterium columnare. Expression difference in MUC13a, MUC13, MUC2 and MUC5b was found in the intestine after E. ictaluri infection. Eight mucin gene expressions (except MUC3a, MUC2, MUC4 and MUC5f) were up‐regulated at 4 hr and down‐regulated after 24 hr in the gill with F. columnare infection. Expression level of MUC2 gene was up‐regulated in the intestine with E. ictaluri infection without no significant change in the gill under the F. columnare infection, which indicate that MUC2 is tissue‐specific gene expression and has different immune respond to two bacterial challenge. Taken together, the study showed mucin from the gill by F. columnare challenge induced an obvious response than mucin from the intestine with E. ictaluri infection.     

Molecular cloning,characterization and expression analysis of the fatty acid‐binding protein (MnFABP), involved in dietary lipid sources response in oriental river prawn,Macrobrachium nipponense

A fatty acid‐binding protein (FABP) gene designated as MnFABP10 was cloned and characterized from the freshwater prawn Macrobrachium nipponense. The full‐length cDNA of MnFABP10 was 646 bp encoding a 130 amino acid. Real‐time quantitative RT‐PCR showed that the MnFABP10 gene was expressed in various tissues with the highest expression in the hepatopancreas. The MnFABP10 mRNA levels in the hepatopancreas and ovary of M. nipponense were dependent on the stages of ovarian development. Western blot results revealed a single immunoreactive band with an estimated molecular mass of approximate 14 kDa in the developmental ovary. Then, M. nipponense with an initial body weight of 0.090 ± 0.0010 g were fed with four isonitrogenous and isocaloric diets with different oils, that is, beef tallow (BT), soybean oil (SO), pollack fish oil (FO) and a mixture of fish oil and soybean oil (FO/SO 2 : 1 w/w) for 52 days. The mRNA levels of MnFABP10 in the hepatopancreas were influenced by different lipid sources, with a peak expression observed in prawns fed SO. This study suggests that MnFABP10 may have a putative function in ovary maturation, and its mRNA expression in the hepatopancreas can be regulated by the source of dietary lipids in M. nipponense.     

Dietary ascorbic acid requirement for growth of striped catfish,Pangasianodon hypophthalmus (Sauvage, 1878) juveniles

A sixty‐day feeding trial was conducted to determine the ascorbic acid (AA) requirement for growth of striped catfish, Pangasianodon hypophthalmus juveniles. Seven iso‐nitrogenous and iso‐energetic (370 g protein per kg and 19.6 MJ/kg) purified diets were prepared with different levels of ascorbic acid such as control (0), T₁ (17.5), T₂ (35), T₃ (70), T₄ (175), T₅ (350) and T₆ (700) mg ascorbic acid (L‐ascorbyl‐2‐polyphosphate) equivalent per kg diet. Fish with a mean body weight of 3.2–3.4 g were stocked (fifteen fish per tank) in triplicates following a completely randomized design. Each group was fed to satiation twice a day for 60 days. Significant differences were observed in growth, survival, body composition and metabolic enzymes activities with different dietary ascorbic acid levels. Maximum weight gain, specific growth rate (SGR) and protein efficiency ratio (PER) were found in fishes fed with 35 mg AA per kg diet, supported by best feed conversion. Fish fed a diet containing vitamin C had the highest activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) compared to those fed with vitamin C‐depleted diets. In this study, based on using broken‐line regression analysis, the dietary vitamin C requirement for growth of P. hypophthalmus juveniles was estimated to be in the range of 46–76 mg AA per kg, depending on the criterion used, growth and liver storage. Our results will be helpful for the formulation of cost‐effective ascorbic acid incorporated diets for striped catfish, P. hypophthalmus.     

Effect of dietary Yucca schidigera extract on growth,total ammonia–nitrogen excretion and haematological parameters of juvenile striped catfish Pangasianodon hypophthalmus

A feeding trial with striped catfish, Pangasianodon hypophthalmus was performed to determine the effect of Yucca schidigera in practical diet on the growth, feed utilization, body composition, total ammonia–nitrogen (TAN) excretion and haematological parameters. A diet with fish meal as the main protein source without yucca extract was used as the control diet (Diet 1). Four diets were formulated with 0.075 (Diet 2), 0.1 (Diet 3) and 0.15 (Diet 4)% of yucca extract respectively. Fifteen fish per tank (initial mean weight 1.78 ± 0.05 g) were randomly allocated to 15 fibreglass tanks (80‐L) connected to a freshwater closed recirculation system (temperature 29.7 ± 1.0°C). The experimental diets were tested in triplicates for 12 weeks. The specific growth rate of fish fed Diet 4 was significantly higher when compared with fish fed Diet 1. The growth of fish fed diets Diet 2 and Diet 3 were not significantly different compared with fish fed the Diet 1. Striped catfish fed Diet 4 had significantly lower feed conversion ratio compared with fish fed Diet 1 and Diet 2 (P < 0.05).The incorporation of high level Yucca schidigera extract in the diets reduced TAN compared with Diet 1. Dietary inclusion of Yucca extract levels did not significantly affect the biometric parameters or whole body proximate composition of the striped catfish (P > 0.05). The PCV (%) in fish significantly increased with high levels of Yucca inclusion (Diet 4) compared with control diet. Fish fed Diet 4 showed significantly higher haemoglobin levels than Diet 1 (P < 0.05). The results indicate that dietary inclusion of Yucca schidigera extract is promising as a feed additive that could improve growth performance and some haematological parameters and the best Yucca schidigera level was 0.15%.     

Effects of dietary Nutrafito Plus on growth,haemotological parameters and total ammonia‐nitrogen excretion of juvenile striped catfish Pangasianodon hypophthalmus

A feeding trial was performed to determine the effect of a commercial source of Yucca schidigera and Quillaja saponaria extracts (Nutrafito Plus^®), in practical diets, on striped catfish Pangasianodon hypophthalmus growth, feed utilization, body composition, total ammonia‐nitrogen (TAN) excretion and haematological parameters. Four experimental diets were supplemented with Nutrafito Plus^® at 0%, 0.01%, 0.02% and 0.03% (diets: control, N01, N02 and N03 respectively). Three replicate groups of striped catfish, with initial mean weight of 1.78 ± 0.05 g, were fed one of the four diets for 12 weeks. The specific growth rate (SGR) and final weight of fish fed diet N03 was significantly higher (P < 0.05) than fish fed the other dietary treatments. The growth performance of fish fed diets N01 and N02 were not significantly different compared to fish fed the control diet. Striped catfish fed diet N03 had improved feed conversion ratio (FCR), net protein utilization (NPU) and protein efficiency ratio (PER) than the control (P < 0.05). The inclusion of the high dietary level of Yucca schidigera and Quillaja saponaria, diet N03, reduced TAN compared to all groups. Dietary inclusion of Yucca schidigera and Quillaja saponaria at all levels investigated did not affect the whole body proximate composition of the striped catfish (P > 0.05). The packed cell volume (PCV) and haemoglobin level in fish fed diet N03 was significantly higher than in the fish fed the control diet. The present study demonstrates that dietary inclusion of Yucca schidigera and Quillaja saponaria induced positive effects on growth performance and haematological parameters and decreased TAN excretion in striped catfish.     

Molecular cloning of sea perch (Lateolabrax japonicus) TLR1 and analysis of its expression pattern after stimulation with various bacteria

Toll‐like receptors (TLRs) play an indispensable role in fish immunity, being involved in pathogen recognition and the triggering of immune reactions. Here, a member of the TLR family, TLR1, from Lateolabrax japonicus was characterized and its expression pattern and intracellular localization were analysed. The full‐length LjTLR1 cDNA (2,755 bp) was found to encode a polypeptide of 827 amino acids. The deduced amino acid sequence contained three main structural domains: an extracellular leucine‐rich repeat domain, a transmembrane domain and a Toll/IL‐1 receptor domain. Tissue distribution analysis indicated that LjTLR1 was expressed in all of the examined tissues to varying degrees, with the highest levels being measured in the head kidney. In order to assess the antibacterial functions of LjTLR1 during infection, the pathogenic bacteria Vibrio harveyi and Streptococcus agalactiae were used. LjTLR1 was significantly upregulated in the three immune organs (the head kidney, spleen and liver) following bacterial stimulation, and its expression was detected 6 hr after initial exposure. In mRNA in situ hybridization experiments, positive signals were more numerous in the treatment group than the control group, verifying the expression patterns observed. Assessment of the intracellular localization of LjTLR1 revealed it to be present in the cytoplasm. These results indicate the potential role of LjTLR1 in immune responses to bacterial infection. This study enriches our knowledge of L. japonicus immune genes and provides a theoretical basis for further research concerning the antibacterial functions of fish TLRs during infection.     

Functional linseed fibres and their impacts on silver catfish (Rhamdia quelen) nutrition

This study was conducted with the objective of evaluating the combination of different ratios of soluble and insoluble linseed fibre for silver catfish (Rhamdia quelen). For this, the soluble and insoluble fractions were concentrated separately and combined in four ratios (1:0.5, 1:1, 1:2, 1:4), which were added to silver catfish diets and evaluated in a bioassay. After 45 days of receiving the experimental diets, the animals were submitted to biometry to collect data and tissues. The experimental design was completely randomized, with five treatments and four replications and the means were compared by Tukey's test (p < .05). Diets 1:2 and 1:4 provided higher weight gain, specific growth rate and crude protein deposition to the fish, whereas only the 1:4 diet reflected higher crude body protein. The 1:0.5 diet altered the trypsin activity in the intestine, and, together with the 1:4 diet, it provided a higher intestinal villi height. Body yield, somatic and digestive parameters, chymotrypsin activity and glucose, glycogen and liver protein were not altered, regardless of the experimental diets. In conclusion, the results indicate that linseed fibre acts effectively as a growth promoter in silver catfish diets, with the use of 1:2 and 1:4 ratios optimizing its prebiotic action.     

Improved vitellogenesis,gonad development and egg diameter in catfish (Pangasianodon hypophthalmus) supplemented with turmeric (Curcuma longa) powder

An experiment was conducted to study the effect of supplementing catfish (Pangasianodon hypophthalmus) with turmeric (Curcuma longa) on the vitellogenic capacity of the liver as a new method to improve reproductive performance in oviparous animals. The experimental catfish were assigned to a completely randomized design consisting of four doses of supplemental turmeric (0, 1.2, 2.4 and 4.8 g/kg feed) with 10 catfish with body weights ranging from 2 to 4 kg in each group. Blood samples were collected on days 0, 14, 28, 42 and 56 of turmeric supplementation to measure the concentrations of plasma oestradiol‐17ß and vitellogenin. The weights of the body, gonads and liver were measured on days 0, 28 and 56. The fecundities of the catfish were measured on days 28 and 56 of turmeric supplementation, and egg diameters were measured on days 28 and 42. The results showed that catfish supplemented with turmeric at a dose of 2.4 or 4.8 g/kg feed had higher growth rates, higher plasma oestradiol‐17β and vitellogenin concentrations, and higher gonad somatic index values and egg diameters. This is the first report demonstrating that the hepatoprotective activity of the curcumin in turmeric could be used to improve both vitellogenin synthesis, which improves nutrient deposition in the ovulating eggs, and the reproductive performance of teleost fish and oviparous animals.     

SNP discovery and gene annotation in the surf clam Mesodesma donacium

The main objective of this research was to identify single‐nucleotide polymorphisms (SNPs) from an Expressed Sequences Tags (EST) data set generated by 454 pyrosequencing in the soft clam Mesodesma donacium. A total of 180 159 ESTs were yielded from a M. donacium cDNA library. De novo assembly was performed using stringent calling parameters, producing 10 178 contigs and 41 765 singletons. Here, a total of 2594 SNPs were discovered related to 613 consensus sequences, achieving a frequency of 1 SNPs per 260 bp. SNP variants showed that A/G, A/T and C/T were the most abundant among the identified polymorphisms. We validated a total of 12 SNPs loci by HRMA for annotated genes such as heat shock protein‐70 and the translation elongation factor 1‐alpha. The Gene Ontology analysis regarding molecular function level revealed that sequences with SNPs were mainly classified to protein and nucleotide binding, as well hydrolase activity, ion binding and oxidoreductase activity. Further, biological processes like cellular and metabolic process, biogenesis, localization and biological regulation were highly annotated. The most expressed genes were related to the mitochondrial electron transport chain, senescence‐associated protein, ubiquitin and actin. Interestingly, some relevant genes related to immune response and biomineralization showed a high abundance, such as tumor necrosis factor (TNF)‐alpha‐receptor‐like protein, serine protease inhibitor, heat shock protein, aragonite‐binding protein and ferritin. This study contributes to relevant genes associated with functional polymorphisms and gives an overview for future genetic investigations.     

Evaluation of immune response and performance of silver catfish fed functional linseed fibres in response to hypoxia stress

This study was conducted to evaluate the immunostimulating activity and growth promoting of diets supplemented with different rations of soluble and insoluble linseed fibre to Rhamdia quelen under hypoxia‐induced acute stress. For this reason, soluble and insoluble fractions of linseed fibre were concentrated separately and combined into four ratios (1:0.5; 1:1; 1:2 and 1:4), which were added to the diets of silver catfish (6.43 ± 0.12 g) and evaluated in a biological assay, along with a control diet (without addition of linseed fibre). After being fed the experimental diets for 45 days, specimens of silver catfish were submitted to hypoxia‐induced acute stress. They were kept out of water for 60 s. Immediately afterwards, blood and cutaneous mucus were collected for subsequent determination of immunological indicators and stress. After stress, the fish were weighed and measured for performance parameter calculation. The experimental design was completely randomized with five treatments and four replications. The data underwent analysis of variance and the means were compared by Tukey's test (p < .05). The fish fed diets containing the 1:2 and 1:4 soluble:insoluble fibre ratios, showed higher total protein content, globulin, plasma alkaline phosphatase activity, and performance in addition to higher mucoprotein content in the cutaneous mucus of the fish. Regardless of their ratio in the diet, linseed fibre provided higher plasma levels of total immunoglobulins and reduction in cortisol levels. The 1:1, 1:2 and 1:4 diets led to higher levels of total immunoglobulins and alkaline phosphatase activity in cutaneous mucus. The results indicate that linseed fibre has a stress‐ reduction, immunostimulant and a growth promoter effect on silver catfish. The 1:2 or 1:4 soluble: insoluble fibre ratios provided greater stimulation of the target immunological indicators and performance.     

Dietary supplementation of autolysed yeast enhances growth,liver functionality and intestinal morphology in African catfish

A feeding trial was conducted to evaluate the potential of dietary supplementation of autolysed brewer's yeast (AY) on African catfish. The catfish (22.5 ± 1.15 g/fish, 20 fish 33 L/tank) were fed with either of diets (390 g/kg crude protein, 140 g/kg lipid) supplemented with 0, 3, 6 or 10 g/kg AY (n = 3). After 49 days of feeding, the final body weight and metabolic growth rate of the catfish fed 3 g/kg AY (3‐AY) diet were higher than those fed the control diet (p < .05). The lowest level (p < .05) of alanine transaminase was detected in the blood of the catfish fed 3‐AY diet. The mid‐intestinal histology of the catfish revealed no significant difference (p > .05) in intestinal perimeter ratio. However, an elevated (p < .05) abundance of goblet cells and intraepithelial leucocytes were found in the intestine of catfish fed 3, 6 and 10 g/kg AY diets, with the highest level of abundance recorded in the mid‐intestine of the catfish fed 3‐AY diet. The results suggest that dietary 3 g/kg autolysed brewer's yeast supplementation improves growth performance of African catfish without deleterious effect on liver functionality and gut morphology.