Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.     

Effects of <Emphasis Type="Italic">Euphorbia hirta</Emphasis> plant leaf extract on growth performance,hematological and organosomatic indices of hybrid catfish, <Emphasis Type="Italic">Clarias macrocephalus </Emphasis>× <Emphasis Type="Italic">C. gariepinus</Emphasis>

This study was conducted to evaluate the effects of Euphorbia hirta leaf extract on the growth performance, hematological and organosomatic indices of hybrid catfish, Clarias macrocephalus?×?C. gariepinus. The fish were treated with 0 (control), 300, 500 and 800 mg/kg (ppm) for 90 days. The weight gain, average daily growth rate, and specific growth rate were at significantly higher levels in fish from all the treatment groups on days 75 and 90, while the feed efficiency and protein efficiency ratio were consistently higher in fish from all the treatment groups from day 60 up until day 90. The feed conversion rate significantly decreased from day 60 until day 90 in all treatment groups when compared with the control group, and the survival rate was significantly different from day 30 until day 90; a consistently higher rate was observed in fish fed 800 mg/kg. The highest viscerosomatic index and intraperitoneal fat were observed in the group fed 500 mg/kg (p?<?0.05). The hepatosomatic index was significantly increased alongside increasing levels of E. hirta extract. The total white blood cell count in the control group was significantly higher on day 30, but on day 90 all the treatment groups showed higher levels. Hematocrit percentage was significantly different on days 30 and 90. Lymphocyte, eosinophil and thrombocyte levels were shown to be significantly different (p?<?0.05) when different groups of fish were compared. In conclusion, 300 mg/kg of E. hirta leaf extract could improve growth performance, hematological and some organosomatic indices in hybrid catfish.     

Effect of nitrite exposure on oxygen-carrying capacity and gene expression of <Emphasis Type="Italic">NF-κB</Emphasis>/<Emphasis Type="Italic">HIF-1α</Emphasis> pathway in gill of bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>)

Nitrite (NO₂^?) contamination of water can severely impact the health of aquatic life and is a major concern for commercial aquaculture. In order to study the effect of nitrite on Aristichthys nobilis, we investigated the oxygen-carrying capacity, NF-κB/HIF-1α pathway, and the gill tissue structure under nitrite stress. In the current study, bighead carp (initial weight 180.05?±?0.092 g) were exposed to nitrite (48.634 mg/L) for 96 h and then for 96 h recovery test. After nitrite exposure for 6 h, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) mRNA expression increased significantly in the gill of bighead carp (P?<?0.05). After nitrite exposure for 12 h, hemoglobin (Hb) and methemoglobin reductase (MHBR) content in blood decreased significantly (P?<?0.05); TLR4 mRNA expression increased significantly (P?<?0.05). After nitrite exposure for 24 h, methemoglobin (MetHb) content increased significantly (P?<?0.05). After recovery test, all the indicators except TLR4 mRNA expression level recovered to initial level. In conclusion, nitrite exposure can affect hemoglobin dynamics, as oxidization of nitrite by hemoglobin results in the reduction of Hb to MetHb leading to hypoxia and nitrite exposure can also result into gill tissue damage. In the face of nitrite exposure, NF-κB and HIF-1α mRNA expression level increased immediately to protect the body from oxidative damage and eased hypoxic condition caused by nitrite. It was also observed that nitrite damage is recoverable in Aristichthys nobilis, but it may be need more than 96 h.     

Dietary ascorbic acid influences the intestinal morphology and hematology of hybrid sorubim catfish (<Emphasis Type="Italic">Pseudoplatystoma reticulatum</Emphasis> × <Emphasis Type="Italic">P. corruscans</Emphasis>)

This study evaluated the effect of graded levels of dietary ascorbic acid (AA) (12.47, 20.27, 115.44, 475.50, 737.72, and 850.70 mg kg^?1) on growth, hematology, intestinal morphometry, and phagocyte activity of hybrid sorubim Pseudoplatystoma reticulatum × Pseudoplatystoma corruscans. Fish (n = 420, 14.57 ± 2.71 g, 15.11 ± 0.90 cm) were distributed in 30 polyethylene tanks (80 l) (5 replicates per treatment with 14 fish per tank) and fed for 45 days. Dietary treatment did not have a significant effect on growth metrics (P > 0.05). Fish fed 737.72 mg AA kg^?1 had a higher villi height (289.80 ± 19.96 μm) (P < 0.05) than fish fed 850.70 mg AA kg^?1 (245.4 ± 18.25 μm). Hemoglobin in fish fed 850.70 mg AA kg^?1 (5.34 ± 0.96 g dl^?1) was higher (P < 0.05) than fish fed 12.47 mg AA kg^?1 (3.42 ± 0.55 g dl^?1) and 20.27 mg AA kg^?1 (3.06 ± 1.26 g dl^?1). The erythrocyte number of hybrid sorubim fed 115.40 mg AA kg^?1 (1.73 ± 0.27 × 10⁶ μl^?1) and 475.50 mg AA kg^?1 (1.70 ± 0.28 × 10⁶ μl^?1) were higher (P < 0.05) than in those fed diets containing 20.27 mg AA kg^?1 (1.11 ± 0.34 × 10⁶ μl^?1). There was no significant effect (P > 0.05) of dietary AA on leukocyte and thrombocyte and on phagocyte activity and phagocyte index. Inclusion of AA in feed seems to increase the integrity of the intestinal mucosa and stimulate erythropoiesis in hybrid sorubim catfish.     

Distribution patterns of settlement-stage juveniles of <Emphasis Type="Italic">Girella punctata</Emphasis> and <Emphasis Type="Italic">Girella leonina</Emphasis> on the rocky coast of the Kanto–Izu region,Japan

The early life history of girellid fishes in Japanese waters is unclear, and little is known about their species-specific reproductive strategies. We examined seasonal changes of distribution patterns for settlement-stage juveniles of Girella punctata and Girella leonina on the rocky shore in the regions of Kanto and Izu, Japan, to infer the influence of the Kuroshio Current on their reproduction. We collected 813 settlement-stage juveniles mainly in Sagami Bay and genetically identified the species. The juveniles of G. punctata were collected on the rocky shore in Sagami Bay during April to August, with the abundant catch in May and June. Thus, we infer that juvenile G. punctata ubiquitously inhabit the rocky shore in the area in spring and summer. By contrast, juveniles of G. leonina were rarely collected in Sagami Bay, with a total catch of only 66. Notably, no juveniles were collected during the wintertime in Sagami Bay, although an abundant catch of G. leonina had been previously reported for Sagami Nada off Sagami Bay during January to March. This clear-cut difference between the areas likely reflects the difference in proximity to the path of the Kuroshio Current. We expect that the Kuroshio Current strongly influences the reproductive success of G. leonina.     

Protective effect of squilla chitosan–silver nanoparticles for <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> larvae infected with <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

Antimicrobial nanoparticle therapy was proposed as an alternative strategy to reduce the use of antibiotics in larval-rearing systems. Antibacterial potential of the prepared squilla chitosan–silver nanoparticles and its protective effect on Dicentrarchus labrax (sea bass) larvae in the early stages were studied against Vibrio angularium. Different concentrations of squilla chitosan (Csq) and squilla chitosan–silver nanoparticles (Csq–AgNps) (1, 2, 5, 10, and 20 %) were, in vitro, tested against V.anguillarum and expressed as a role of Log₁₀ mean. Sea bass larvae were treated using: 10 % Csq and 5 % Csq–AgNps as effective inhibitory concentrations against the pathogen either encapsulated during the feeding regime or added directly to the model system via the water from the onset of 4 weeks. The long-term administration of Csq–AgNps through enriched food for both non-infected and infected systems had survival % of 74.5 ± 1.5 and 72.5 ± 2.5, respectively. Larval clinical observations using Csq–AgNps were studied compared with the two controls. The current study found that 5 % encapsulated Csq–AgNps was enough to suppress infection and considered as an alternative to antibiotics in controlling virulent fish pathogens.     

Optimum time for weaning South African <Emphasis Type="Italic">Scylla serrata</Emphasis> (Forskål) larvae from rotifers to <Emphasis Type="Italic">Artemia</Emphasis>

To determine the optimum time at which to wean Scylla serrata larvae from rotifers onto Artemia two experiments were conducted, approximately 1 month apart, using larvae from two different female crabs. In the first experiment, the larvae in three treatment groups, with nine replicates each, were fed rotifers for the first 8 days after hatching. Artemia were introduced on days after hatch (DAH) 0 – during the first zoeal instar (treatment R + A); on DAH 4 – during the second zoeal instar (treatment R4A); on DAH 8 – during the third zoeal instar (treatment R8A). In a control (ROT) larvae were fed with rotifers exclusively for 18 days until the completion of metamorphosis to megalopa. In the second experiment, the same four feeding schedules as in experiment 1 were used with an additional group of larvae (treatment AC) that were fed only on Artemia throughout the rearing period. Similar results were recorded in the two experiments. Larvae in treatments R + A and R4A performed significantly better than those in treatments R8A, ROT and AC. This was particularly evident when examining the proportion of zoeae which successfully completed metamorphosis to megalopa. Poor performance of larvae in treatments AC and ROT implied that rotifers are needed as a first food, but that rotifers alone do not fill the nutritional requirements of S. serrata larvae. Poor performance of larvae in treatment R8A suggested that the diet should be supplemented with Artemia before the end of the zoea 3 stage.     

Species differences and effects of soft coral extracts from <Emphasis Type="Italic">Sinnularia maximus</Emphasis> on the expression of cytochrome P4501A and 2N in butterflyfishes (<Emphasis Type="Italic">Chaetodon</Emphasis> spp.)

Cytochrome P450 (CYP) has been shown to confer resistance in numerous terrestrial insects that consume potentially toxic secondary metabolites in plants, but fewer studies have examined the role of critical biotransformation enzymes in allowing marine organisms to consume chemically defended foods. This study examined the expression of CYP1A and CYP2N mRNAs in several butterflyfish species, which can feed on numerous chemically defended soft and hard corals. In addition, the effect of an extract from a soft coral (Sinnularia maxima) on expression of hepatic CYP1A and CYP2 mRNAs was also examined. Fish were fed extracts on days 1, 3 and 5, and expression was examined on day 5. Phylogenetic analyses of the CYP1A cDNA from 12 species of butterflyfish (DNA, amino acid) indicate well-separated groupings according to their feeding strategies. The non-coralline feeding Chaetodon xanthurus exhibited a 7-fold higher basal expression of CYP2N8 relative to the other species studied. Although induction of CYP2N7 expression was observed in C. punctatofasciatus, CYP1A and CYP2N was largely unaffected or diminished by extract treatment in the other species of butterflyfish. These results indicated groupings of feeding strategy with CYP1A phylogeny in Chaetodon, but generally unaltered expression of CYP1A and CYP2N following dietary treatment with an extract from a chemically defended soft coral suggesting an inconclusive role of these isoforms in the detoxification of chemicals in these extracts.     

Experimental infection of <Emphasis Type="Italic">Betanodavirus</Emphasis> in freshwater fish <Emphasis Type="Italic">Gambusia affinis</Emphasis> (Baird and Girard, 1853)—a potential infection model for viral encephalopathy and retinopathy

Betanodaviruses are the causative agents of the disease known as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in a variety of marine and freshwater fish species. The aim of this study was to demonstrate experimental infection of an isolate of betanodavirus (RGNNV genotype) in freshwater fish, Gambusia affinis, for elucidation of transmission mechanism and potential use as a laboratory model. Morbidity and mortality rate was significantly higher by injection route of infection as compared to immersion by bath and resembled the natural infection of juvenile marine fish. The fish in disease affected group showed severe neurological disorders accompanied by extensive vacuolar degeneration and mild to moderate neuronal necrosis of the brain in comparison to control. Amplification of ~?427 bp product in the variable region of the coat protein gene of betanodavirus was achieved by RT-PCR with 100% sequence homology to RGNNV genotype.     

Hematologic and serum biochemical values for jundiá (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>)

The determination of hematologic parameters for jundiá Rhamdia quelencultivated in ponds is an easy and useful tool for the control of their sanitary and nutritional conditions. In the present work hematologic indices (RBC, WBC, Ht, Hb, MCV, MCH, MCHC), electrolytes (Cl, Na, K, Ca, Mg, P), metabolic products (creatinine, urea, ammonia, glucose, total protein, triglycerides, cholesterol, bilirubin) and enzymes (alanine and aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase) were measured in a population of jundiá. The mean ± SEM and range were established.     

Dietary lipid concentrations influence growth,liver oxidative stress,and serum metabolites of juvenile hybrid snakehead (<Emphasis Type="Italic">Channa argus</Emphasis> × <Emphasis Type="Italic">Channa maculata</Emphasis>)

The study was conducted to evaluate the effect of dietary lipid levels on growth, liver oxidative stress, and serum metabolites of juvenile hybrid snakehead (Channa argus × Channa maculata). Five isonitrogenous (crude protein 420 g kg^?1) practical diets containing 58, 87, 115, 144, and 173 g kg^?1 crude lipid (named L58, L87, L115, L144, and L173, respectively) were fed to triplicate groups of 30 fish (mean initial weight 24 g) for 8 weeks. The results showed that the final body weight (58.68–78.81 g), specific growth rate (1.41–1.75 % day^?1), and protein efficiency ratio (1.66–2.64) increased significantly with the increasing dietary lipid levels. Liver lipid contents (71.65–101.80 g kg^?1) and crude lipid (52.10–83.63 g kg^?1) of whole body increased with increasing dietary lipid levels and reached the highest values in fish of L173. Fish of L173 showed lower alkaline phosphatase (23.81 King Unit gprot^?1) and catalase activities (4.44 U mgprot^?1) but higher malondialdehyde content (0.69 nmol mgprot^?1) in liver than the other groups. Higher alanine transaminase activity (8.20 U L^?1), aspartate transaminase activity (63.65 U L^?1), and triglyceride (0.29 mmol L^?1) in serum were observed in fish of L173 compared to the other treatments. Fish of L144 showed higher superoxide dismutase activity and glutathione peroxidase activities in liver than that of fish fed diet L58. Fish fed diet L58 showed lower total cholesterol (3.61 mmol L^?1), high-density lipoprotein cholesterol (1.39 mmol L^?1), and low-density lipoprotein cholesterol (0.46 mmol L^?1) in serum. These results suggested that juvenile snakehead (Channa argus × Channa maculata) achieved good growth performance with dietary lipid level 173 g kg^?1. Diet with 143 g kg^?1 lipid was more conductive to liver health. The appropriate dietary lipid supplementation needs to be determined in further studies.     

A comparison of growth and biochemical composition of <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> (Lmk.) and <Emphasis Type="Italic">Mytilus edulis</Emphasis> (L.) on the West coast of Cotentin,Normandy, France 1999–2000

The blue mussel, Mytilus edulis has been reared along the Normandy coast line since the 1960s. The gonadal cycle of this mussel species shows a sharp decrease in meat quality during the winter period after spawning. This decline in meat quality is so severe that sales have to be suspended from December to July. Another species of mussel, Mytilus galloprovincialis, which is reared in the lagoons along the French shore of the Mediterranean Sea has a different spawning cycle. An experiment was undertaken to study the meat quality of M. Galloprovincialis throughout the year after the spat were transferred from the Mediterranean Sea to the Normandy coast. This species showed an immediate adaptation to the gonadal cycle of M. edulis. Despite suggestions from researchers, no interest was expressed to consider such transfers of M. galloprovincialis in the future.     

Comparative analysis of mitochondrial control region in polyploid hybrids of red crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>) × blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>)

The entire sequences of the mitochondrial (mt)DNA control region (CR) and portions of its flanking genes in the red crucian carp (RC) and blunt snout bream (BSB) as well as their polyploid hybrids (3nRB, 4nRB and 5nRB) were determined and subjected to a comparative analysis. The mtDNA-CRs of these five fish species ranged from 923 to 937 bp in length, they had the same flanking gene arrangement as other vertebrates and the pattern of nucleotide substitution bias was also similar to that in other vertebrates. Our data are consistent with the viewpoint of three domains [extended terminal associated sequence (ETAS domain), central conserved sequence block domain and conserved sequence block (CSB) domain] within the mtDNA-CR of mammals. On the basis our comparative analysis of the mtDNA-CRs of these five fish species, we were able to identify the consensus sequences of functional conserved units, including the ETAS, CSB-F, CSB-D, CSB-E, CSB1, CSB2 and CSB3 and putative promoter. The percentage of variable nucleotide positions (41.98%) in the central domain was lower than those in the ETAS and conserved domain (71.70 and 47.12%, respectively), suggesting that the central domain was the most conserved part of the mtDNA-CR. These results provide useful and important information for the further study of mtDNA-CR structure in fish. The sequence similarities of mtDNA-CR among the 3nRB, 4nRB, 5nRB hybrids and their respective female parents were higher than those among the 3nRB, 4nRB, 5nRB hybrids and their respective male parents, providing the direct evidence of stringent maternal inheritance of mtDNA-CR in the 3nRB, 4nRB and 5nRB hybrids.     

Are coping styles consistent in the teleost fish <Emphasis Type="Italic">Sparus aurata</Emphasis> through sexual maturation and sex reversal?

Individual differences in behaviour and physiological responses to stress are associated with evolutionary adaptive variation and thus raw material for evolution. In farmed animals, the interest in consistent trait associations, i.e. coping styles, has increased dramatically over the last years. However, one of limitations of the available knowledge, regarding the temporal consistency, is that it refers always to short-term consistency (usually few weeks). The present study used an escape response during a net restraining test, previously shown to be an indicative of coping styles in seabream, to investigate long-term consistency of coping styles both over time and during different life history stages. Results showed both short-term (14 days) consistency and long-term (8 months) consistency of escape response. However, we did not found consistency in the same behaviour after sexual maturation when the restraining test was repeated 16, 22 and 23 months after the first test was performed. In conclusion, this study showed consistent behaviour traits in seabream when juveniles, and a loss of this behavioural traits when adults. Therefore, these results underline that adding a life story approach to data interpretation as an essential step forward towards coping styles foreground. Furthermore, a fine-tuning of aquaculture rearing strategies to adapt to different coping strategies may need to be adjusted differently at early stages of development and adults to improve the welfare of farmed fish.     

Effects of dietary riboflavin levels on antioxidant defense of the juvenile grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

This study was conducted to evaluate the effects of dietary riboflavin on antioxidant defense in the juvenile grouper Epinephelus coioides. Graded levels of riboflavin (0.9, 1.6, 4.4, 6.7, 12.9 and 19.4 mg kg⁻¹ dry diet) were fed to grouper juveniles (mean weight: 14.90 ± 0.46 g) for 12 weeks. Higher levels of liver thiobarbituric acid reactive substances (TBARS) content were observed in grouper fed low doses (0.9 and 1.6 mg kg⁻¹ diet) of riboflavin. Both liver glutathione reductase (GR) activity and its activation coefficient (GR-AC) poorly responded to riboflavin deficiency. In addition, other indices of the glutathione-dependent defense system, including the activities of glutathione peroxidase (GSH-PX) and glutathione-S-transferase (GST), and the content of glutathione (GSH), were also non-significantly affected by dietary riboflavin levels. However, the activities of liver superoxide dismutase (SOD) and catalase (CAT) were significantly lower in fish fed 0.9 mg kg⁻¹ diet, with a positive correlation between the different groups. In conclusion, the present study indicated that the juvenile grouper fed the riboflavin-unsupplemented diet was susceptible to lipid peroxidation (LPO), with lower SOD and CAT activities in the liver. However, the glutathione-dependent defense system of grouper was not affected by dietary riboflavin levels.     

Maturation and reproductive cycle of female armorhead <Emphasis Type="Italic">Pseudopentaceros wheeleri</Emphasis> from the southern Emperor—northern Hawaiian Ridge Seamounts

ABSTRACT:   The stage of ovarian development and annual reproductive cycle of female armorhead Pseudopentaceros wheeleri collected from seamounts of the southern Emperor–northern Hawaiian Ridge in 1993 and 1995–1996 was investigated through use of the gonadal somatic index ( GSI ) and ovarian histology. The stages of oocyte development were classified into eight developmental stages (perinucleolus, cortical alveolus, oil droplet, early yolk globule, late yolk globule, migratory nucleolus, prematuration, and maturation) based on histological characteristics. Oocytes of different size and developmental stage were present within ovaries during the spawning season. Oocyte development appears to be asynchronous and armorhead apparently spawn several times during the spawning season. The stage of the most developed oocyte was used to classify the stage of ovarian maturation. The GSI increased gradually with ovarian development. Oocytes in the perinucleolus, cortical alveolus, and oil droplet stages were present in ovaries throughout the year. Early and late yolk globule stages appeared during August–September indicating that yolk formation begins during late summer. The migratory nucleolus through maturation stages were detected during November–January. Post ovulatory follicles were present during November–March, and GSI values rapidly decreased in February. Results indicate that spawning occurs during November–February and peaks during December–January.     

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker <Emphasis Type="Italic">Miichthys miiuy</Emphasis> larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.