Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors(IGFs)are key regulators of development and growth.Here,we characterized the igf2 gene from olive flounder(Paralichthys olivaceus)and determined its temporal and spatial expression.We set up an in-vitro protein expression system in eukaryotic human embryonic kidney(HEK293 T)cells and explored its effects on cell proliferation.The flounder igf2 cDNA contained a 648-bp open reading frame(ORF)encoding a protein of 215 amino acids(aa),which spanned the complete signal peptide(47 aa),mature peptide(70 aa),and E domain(98 aa).In adult flounder,igf2 mRNA was detected in all selected tissues.In early development,igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos.In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region,floor plate,hypochord,otic vesicle,and pectoral fin during embryogenesis.Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells.In addition,the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma(HeLa)and HEK293T cells.These results provide new information about the structural and functional conservation,expression patterns,and biological activity of the igf2 in teleosts.

Dynamic Analysis of Hydrodynamic Behavior of A Flatfish Cage System Under Wave Conditions

This paper presents a simulation model based on the finite element method. The method is used to analyze the motion response and mooring line tension of the flatfish cage system in waves. The cage system consists of top frames, netting, mooring lines, bottom frames, and floats. A series of scaled physical model tests in regular waves are conducted to verify the numerical model. The comparison results show that the simulated and the experimental results agree well under the wave conditions, and the maximum pitch of the bottom frame with two orientations is about 12o. The motion process of the whole cage system in the wave can be described with the computer visualized technology. Then, the mooring line tensions and the motion of the bottom frame with three kinds of weight are calculated under different wave conditions. According to the numerical results, the differences in mooring line tensions of flatfish cages with three weight modes are indistinct. The maximum pitch of the bottom frame decreases with the increase of the bottom weight.

Numerical Simulation of Dynamic Response of A Net Cage for Flatfish in Waves

A numerical model of flatfish cage is built based on the lumped mass method and the principle of rigid body kinematics. To validate the numerical model, a series of physical model tests are conducted in the wave flume. The numerical results correspond well with the data sets from physical model test. The effect of weight of bottom frame, height of fish net and net shape on motion responses of fish cage and tension force on mooring lines is then analyzed. The results indicate that the vertical displacements of float collar and bottom frame decrease with the increase in the weight of bottom frame; the maximum tension force on mooring lines increases with the increasing weight of bottom frame. The inclination angles of float collar and bottom frame decrease with the increasing net height; the maximum tension force increases obviously with the increase of net height.

Morphological study of the asymmetrical buccal cavity of the flatfish common solea(Solea solea) and its relation to the type of feeding

Objective:To investigate the surface architecture of the asymmetrical buccal cavity of Solea solea which are considered one of the most important predators in benthic communities.Methods:Adult Solea solea were obtained from Mediterranean Sea near Damielta.The heads were removed and processed for scanning electron microscopy.Its buccal cavity is asymmetrical and divided into roof and floor and the tongue for histological studies.Results:The buccal cavity roof is formed from upper jaw,velum and the palate.The upper jaw has several wing like processes with teeth arranged in several rows which may help in cutting and pushing the food to the entrance of the digestive canal while the floor is formed from the lower jaw and the tongue.The tongue is divided into apex,body and root.There is a gradual decrease of goblet cells in the tongue from anterior to posterior.These goblet cells function in protection of the epithelium.CoDctusions:Teeth in the floor of the buccal cavity and taste buds can be considered adaptive changes of the oral cavity related to the feeding habits and was a source to identify new and better methods of nutrition in aquaculture of Solea solea.

Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

Ontogenetic changes in digestive enzyme activities and the amino acid(AA) profile of starry flounder,Platichthys stellatus,were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements.Larvae were collected at the egg stage and 0,2,4,7,12,17,24 days after hatching(DAH)for analysis.Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH.Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH.Pepsin activity increased sharply beginning 17 DAH.Lipase activity increased significantly 4 DAH and increased progressively with larval growth.Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease.High free amino acid(FAA) content was detected in starry flounder eggs(110.72mg/g dry weight).Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae.Most FAAs(except lysine and methionine) decreased >50%in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae.Changes in the protein amino acid(PAA) profile were much milder than those observed for FAAs.Most PAAs increased gradually during larval development,except lysine and phenylalanine.The percentages of free threonine,valine,isoleucine,and leucine decreased until the end of the trial,whereas the protein forms of these four AAs followed the opposite trend.A comparison of the essential AA composition of live food(rotifers,Artemia nauplii,and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA.These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food.Moreover,the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

Comparative Analysis of Mitochondrial Control Region Sequence from Three Flatfish Species(Pleuronectidae)

The 5’-end of the mitochondrial control region sequences of three flatfishes (Pleuronectiformes: Pleuronectidae) were amplified and sequenced. These sequences were compared with those of other three Pleuronectids species retrieved from GenBank. A phylogenetic tree was constructed based on the partial control region sequences. The results of phyloge- netic analysis are consistent with those of conventional systematics. Compared to previous studies, the structure of the 5’-end of mitochondrial control region was analyzed. The terminal associated sequence motif and its complementary motif were i- dentified at the 5’-end of the sequences. A conserved sequence block, named as CM5’d, was identified in the 5’-end of con- trol region sequences in all Pleuronectids. Another central conserved sequence block, named as CSB-F, was detected in the central conserved blocks.

Production and Purification of Antioxidant Peptides from Flatfish Skin Protein Hydrolysates

Antioxidant peptides of flatfish skin protein hydrolyzed by four enzymes （Papain, Pepsin, Trypsin and Neutrase, respectively）were investigated. The Trypsin hydrotysate obtained by hydrolysis exhibited the highest 1,1- dipheny-l-2-picrylhydrazyl（DPPH） radical scavenging activity （DRSA） compared with other hydrolysates. Re- sponse surface method ology （RSM）, based on Box-Behnken design, was used to study the influence of hydrolysis conditions on the DRSA. The optimal hydrolysis conditions were as follows： pH 7.38, temperature 48.2~C and en- zyme/substrate （E/S）ratio 2 840 U/g. Under these conditions, the maximum DRSA was （22.85 ＋ 0.57）%. The experimental values agreed with the value （23.09%） predicted by the model within a 95% confidence interval. By using gel filtration chromatography and reversed-phase high performance liquid chromatography （RP-HPLC）, anti- oxidant peptide （D2-P）was isolated from flatfish skin protein hydrolysates （FSPH）and could exhibit a （54.28 ± 1.37） % scavenging activity on DPPH radical at the concentration of 5 mg/mL. This is the first report of a scientific basis for the preparation of antioxidant peptides from flatfish skin. The results suggested that the antioxidant pep- tides can be exploited into functional foods or used as a novel source of nutraceuticals.

Cryptic Behaviour of Juvenile Turbot Psetta maxima L.and European Flounder Platichthys flesus L.

The aim of this study was to examine the burying behaviour of hatchery-reared European flounder Platichthys flesus and turbot Psetta maxima, and whether conditioning on a sandy substrate would improve burying efficiency. Both species buried shortly after release on a sandy substrate. However, the study revealed interspecies differences;the flounder buried immediately after release, while the turbot buried gradually. No significant difference in burying efficiency was observed between na&iumlve and conditioned flounder and turbot. An effect of size on burial efficiency was observed for both flounder and turbot with a tendency for larger fish to bury more efficiently than smaller fish, despite previous conditioning. Size at settlement was found to be >2 cm for flounder and >3 cm for turbot.

Investigation of hydrolysis conditions and properties on protein hydrolysates from flatfish skin

Response surface method （RSM）, based on Box-Behnken design, was used to optimize the enzymatic hydrolysis conditions of flatfish skin protein hydrolysates （FSPH）. Among the tested proteases, the combination of nutrase and trypsin was selected. The optimal hydrolysis conditions were as follows： pH 7.3, temperature 51.8℃, and the enzyme/substrate （E/S） ratio 2.5; under these conditions, the maximum peptide yield （PY） was 69.41 =1：0.43%. The physiochemical analysis showed that the amino acids （His, Asp and Glu） of FSPH accounted for 18.15%, and FSPH was a mixture of polypeptides mostly distributed among 900-2000 Da. FSPH could exhibit a 93% chelating effect on ferrous ion at a concentration of 400 gg/mL, and also a notable reducing power. This study showed bioprocess for the production of FSPH for the first time, which had a good potential for valuable ingredients in the food, cosmetic and medicine industries.

Transcriptome analysis and candidate gene identification reveals insights into the molecular mechanisms of hypermelanosis in Chinese tongue sole(Cynoglossus semilaevis)

Blind-side hypermelanosis has emerged as a major concern in flatfish aquaculture worldwide,including tongue sole(Cynoglossus semilaevis)in China.The causative gene and the molecular basis are still unclear.In this study,comparative transcriptome analyses were performed using different skin tissues of tongue sole:ocular-side normal(pigmented)skin,blind-side normal(non-pigmented)skin and blind-side hypermelanotic(pigmented)skin.Finally,60 key hypermelanosis-related genes were mined,providing potential candidate gene resources involved in blind-side hypermelanosis.These genes were selected based on the log2(FoldChange)and false discovery rate(FDR)values(with corresponding P-Values<0.05),and they were verified in other species to assess if they were directly or indirectly related to melanogenesis.The protein-protein interaction network of these 60 genes and the relationship between tyr and other key hypermelanosis-related genes were illustrated.The qRT-PCR validation of 16 differentially expressed genes(DEGs)showed that the data of qRT-PCR were consistent with those of RNA-seq.Further analyses revealed that the selected DEGs were significantly overrepresented in several pigment metabolic processes and in the melanogenesis pathway.Our results may imply that blind-side hypermelanosis is a pattern of environmental regulation of gene expression and adaptation in flatfish.Membrane transport proteins(such as OCA2 and SLC45A2)may serve as a“switch”for melanogenesis in tongue sole.Overall,this study provided novel insights into the molecular mechanism of hypermelanosis in flatfish species and will facilitate future selective breeding of tongue sole for this market-favoured trait in aquaculture.

Eye location, cranial asymmetry, and swimming behavior of different variants of Solea senegalensis

Many flatfish species,such as Scophthalmus maximus,Paralichthys olivaceus,Solea senegalensis,are extensively cultured worldwide.In flatfishes,eye migration varies among different species and even within the same species.The genetic mechanism underlying eye migration is still unclear,and it is the key to improve metamorphosis in flatfish aquaculture and understand flatfish evolution.In this study,the high frequency of eye location variants in an artificial S.senegalensis population allowed us to investigate the eye preference during migration;completeness of the eye migration;and other related left-right asymmetric characteristics,such as asymmetrical skull remodeling and lying down on one side.The different frequencies of eye preference during migration,completeness of the eye migration,and settling side suggest that they are determined by different genotypes and imply independent evolutionary events during flatfish evolution.We found that the swimming time may be related to the completeness of eye migration,and further the migration of the eye,the more time the fish spent at the sea bottom.The left-right asymmetric degrees of not only the frontal bones and lateral ethmoids but also the jaw bone,including the premaxillary,maxillary,and dentary bones,depend on the completeness of eye migration.Finally,we proposed a hypothesis for the asymmetric origin of flatfish that provides reasonable explanations for the selective advantages of primitive flatfish species.