Abstract:To study the intestinal structure characteristics and the relationship with its growth pattern of Saurogobio dabryi, a total of 233 individuals were collected from the lower reaches of the Jialing River (Hechuan section) from 2015 to 2016. Morphological observation results showed that the intestinal structure of S. dabryi was Z-shaped, and belonged to carnivorous fish gut according to its relative short gut length (RGL) index (0.612 8). Based on its morphological structure, the intestine could be divided into three parts, the foreintestine, the midintestine and the hindintestine, with obvious difference in external morphology, internal structure and intestinal index. The growth pattern of S. dabryi intestine was allometry, with 2-year growth inflexion age and 145.07 mm corresponding fish total length (TL). Before and after the inflection point, there was different growth pattern of intestine, i.e., from the fast growth stage to slow growth stage. Meanwhile, the growth pattern of intestine was different between each part. In addition, the results of segmented analysis with R software showed that all of the inflexion points among three parts appeared at the age of 2 years, and their TL were 126.00 mm, 169.00 mm, 148.16 mm respectively; there were obvious differences in intestinal growth pattern among the three parts, with isokinetic growth in the foreintestine and the midintestine, but with significant allometric growth in the hindintestine, which had a fast growth stage before growth inflexion point and then changed to isokinetic growth pattern, based on the growth indices difference analysis between two stages. Thus, the difference of growth pattern of S. dabryi among the three parts of intestine might be related to their functional differentiation. Before the inflection point, the S. dabryi might improve their feeding frequency by the fast growth stage of the hindintestine, to meet the great demand for energy during the initial growth stage. The results obtained from this research can not only provide important scientific evidence for artificial cultivation of S. dabryi, but also offer a new example for studying the relationship between the growth pattern of fish intestine and its feeding habits.

Abstract:In order to reduce nutrient loss and the formation of formaldehyde in the fried squid, 2 g/L of chitosan solutions (high molecular weight chitosan and carboxymethyl chitosan) were used to treat fresh Dosidicus gigas before baking at high temperature. The results showed that the formaldehyde content in the control group (pure water immersion), chitosan group and carboxymethyl chitosan group was 45.56, 4.79, and 8.30 mg/kg, respectively. There was highly significant difference between the chitosan group and the control group. In addition, aspartic acid, serine, proline, cystine, lysine, twelve acid, pentadecylic acid, seventeen acid, octadecenic acid (n=9) and octadecenic acid (n=6), octadecadienoic acid (n=6), eicosapentaenoic acid (n=9), eicosadienoic acid, eicosatetraenoic acid, eicosapentaenoic acid(EPA), docosapentenoic acid (n=3) and elements Mg, P, Mn and vitamin C content were higher than the control group, and the other nutrients also have better keep. Squid aspartate, serine, leucine, methionine, lysine, histidine, mecilenic acid, pentadecoic aci, hexadecenoic acid (n=7), seventeen acid and eicosenoic acid (n=9), eicosadienoic acid, eicosatetraenoic acid, eicosapentaenoic acid (EPA) K, Mg, P and elements, Zn, Mn, Se and vitamin B in carboxymethyl chitosan group were higher than the control group, the other nutrients also have better keep. There was significant difference in nutrients between carboxymethyl chitosan group and control group. EPA, DHA, and amino acids can be maintained well in treated groups, especially better in high molecular chitosan group. It can be concluded that the two kinds of chitosan, especially the high molecular weight chitosan can significantly inhibit the formation of formaldehyde and can maintain the better quality and nutrients keep in squids during the baking process.

Abstract:In order to study the characteristics of fish bone cells, methods for isolating and culturing bone cells from caudal vertebrae and rib of blunt snout bream Megalobrama amblycephala were explored in the present study, and the biological characters of bone cells were identified. The bone cells were cultured with tissue culture and trypsin digestion respectively by using 3-month-old M. amblycephala caudal vertebrae and ribs from tail part. The optimal conditions were as follows:25 ng/mL epidermal growth factor (EGF) and 25 ng/mL basic fibroblast growth factor (bFGF) were added to the M-199 medium at 28℃ using tissue culture. The results of cell biology identification showed that the cultured cells had the biological activity of typical osteoblasts, with alkaline phosphatase staining, calcium nodules alizarin red staining and mineralized nodules von Kossa staining being positive. The osteocalcin content was 997.25 ng/L in the bone cells tested by the fish bone osteocalcin ELISA kit. The genes of runx2a, runx2b and osterix which regulated osteoblast differentiation, were expressed in bone cells, with the runx2b and osterix expression levels significantly higher than that in muscle cells. Results indicated that bone cells of fish have good proliferation and faster growth rate as well as stable cell biological characteristics, which is suitable for related studies in vitro.

Abstract:In order to ensure the accuracy and robustness of fish behavior data acquisition, an interference cancellation algorithm was proposed to optimize the 3D positioning algorithm of fish target.In this paper, the study was based on a singular phenomen-on caused by total reflection in this platform, the causes of the phenomenon were analyzed, and the influence of the phenomenon was eliminated. Firstly, based on the principle of geometrical optics and the principle of total reflection, the causes of interference phenomena were analyzed, and the characteristics of interference image were summarized to determine the elimination scheme.Using the digital image and video processing technology, the interference recognition and elimination algorithm was designed to optimize the original positioning algorithm.Based on the experiment of red crucian carp and simulated fish, the results showed that the proposed algorithm is accurate and confirmed that this algorithm has high precision and robustness.

Abstract:Using dynamic method and the static method, we studied the effects of acclimation temperature (14.0, 18.0, 24.0, 30.8℃) and temperature-rising rates (0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 9.0, 12.0, 15.0℃/h) on the thermal tolerance of five common marine animals (Siganus fuscessens, Sebastiscus marmoratus, Epinephelus bruneus, Oratosquilla oratoria and Charybdis japonica) in Huangmao Sea, the South China Sea. The results showed that both acclimation temperature and temperature-rising rate significantly affected the critical thermal maximum (CTM) and the 24 h upper incipient lethal temperature (24 h UILT₅₀) of the experimental animals. The CTM of each experimental species was positively related to acclimation temperature. The patterns in which temperature-rising rate affected the thermal tolerance of the animals varied among species, depending on acclimation temperature. In the static experiments, 24 h UILT₅₀ of the experimental animals was species-specific and was positively related to acclimation temperature. The 24 h UILT₅₀ of S. fuscessens, S. marmoratus, E. bruneus, O. oratoria and C. japonica increased from 28.1, 28.9, 30.3, 28.4, 36.3℃ to 34.6, 36.1, 36.6, 35.1, 38.2℃ with the increasing acclimation temperature (14.0℃ to 30.8℃). In terms of thermal tolerance, the five experimental animals showed a ranking of C. japonica>E. bruneus>S. marmoratus>O. oratoria>S. fuscessens.

Abstract:In order to explore the influence of allantoin on monospores formation and release of Pyropia chauhanii, we used the wild-type strain (PC-WT) which could release monospore and its induced strain (PC-Y1) which could not release monospore as materials, then different strains blade discs of apical, middle and basal sections were cultured in medium adjusted with different concentration of allantoin. The research results showed that, in 6 days after treatment, the order of the release number of the different parts of each disc was middle>apical>basal; With the increasing of allantoin concentration, the numbers of release monospores of PC-WT strain blade discs showed a trend of increasing first and then decreasing, and 10 mmol/L was the optimal allantoin concentration of promoting monospore release. But allantoin could not promote PC-Y1 release monospore. We used 20 mmol/L allantoin of medium to culture the discs of PC-Y1 blade for 8 days, and single somatic cell was obtained using enzymatic hydrolysis from the discs of different section, and continue culturing in vitro. We found that the percentage of single cells developed into conchocelis was 66.1% in treatment group; and the percentage of single cells developed into conchocelis was only 15.2% in control group. The result showed that allantoin could promote differentiation from somatic cell to germ cell of blade in Pyropia.

Abstract:Lingshui Bay is a lagoon harbor located in the southeast of Hainan Island, and it is the focus of the marine development zone in Hainan Province, China. In recent years, with the development of coastal marine aquaculture, offshore coastal waters ecological environment has been influenced to a certain extent so that the community structure and diversity of fisheries resources have changed. How to effectively monitor the fisheries resources in species composition and quantity change has become an important problem to be solved. In order to know the background conditions of fishery resources in Lingshui Bay, Hainan Province, four acoustic investigations were conducted with portable split beam scientific echosounder (Simrad EY60, Norway) from November 2014 to January 2016 for various seasons. The structure composition of fisheries resources, number density, biomass density and spatial distribution in the survey sea area were investigated and estimated according to the use of echo integration method and trawl sampling. As a result, a total of captured nectons and benthic invertebrates were 86 species, of which the average number density and biomass density of 55 kinds of acoustic assessment species were 9.34×10⁵ ind/km² and 5.08 t/km² respectively in November 2014. The sum of captured nectons and benthic invertebrates were 114 kinds, of which the average number density and biomass density of 63 kinds of acoustic assessment species were 1.12×10⁵ ind/km²and 0.93 t/km² respectively in August 2015. A total of captured nectons and benthic invertebrates were 105 species, and the average number density and biomass density of 55 kinds of acoustic estimation species were 0.16×10⁵ ind/km²and 0.32 t/km² respectively in January 2016. In May 2015, the sum of captured nectons and benthic invertebrates were 56 species, including 34 kinds of acoustic assessment species. Fish echo was dominantly distributed in 30 m shallow depth layer in November 2014 and August 2015, and mainly focused on 10 m to 20 m layer in May 2015. In January 2016, fish echo was mainly distributed in 20 m shallow depth layer and the echo in 20 m to 30 m water layer is slightly greater than half of 0 m to 10 m layer or 10 m to 20 m layer. Single target strength in the survey area was mainly less than -58 dB of small individual fish. Single target strength tended to increase with the increasing water depth, and the individuals whose target strength were greater than -50 dB were entirely distributed under 10 m depth layer.

Abstract:Northern pike (Esox lucius) is a very popular edible fish with high quality and an economically important fish. In China, northern pik is only distributed in the Ertixche River in northern Xinjiang. Northern pike females grow faster than males. To investigate and develop the feminization induction method on northern pike, 750 fries of this fish were divided into five groups in this study. Among them, four groups were used as experimental groups, and fed with Tubifex hattai, which had been soaked with 20 mg/L 17β-estradiol, starting from different developmental time (10 d, 20 d, 30 d and 40 d, respectively after hatching). The other one group was used as control group, and fed with normal T. hattai. All fish in experimental groups were fed with 17β-estradiol for 30 d, and then all the fish in the experimental groups and the control group were fed normally. On 206 d, survival situations of the fish in all groups were counted, and then all the fish were dissected, gonads were removed to perform histological observation. The results showed that feeding with T. hattai, which had been soaked with 20 mg/L 17β-estradiol, had very obvious lethal effect on northern pike fries, and made the survival rate decreased from 43.5% in the control group to 14.3%±8.3%. Higher ratios of female to male were obtained in 30 d treated group and 40 d treated group, especially the highest ratio, which reached 15:7, was obtained in the 30 d treated group, so it can be concluded that the key time point of gonad differentiation of northern pike may be in the period of 30-40 d after hatching. There were high proportions of hermaphroditism in both experimental and control groups, that is, a large number of primary oocytes (phase Ⅱ) and spermatocytes appeared simultaneously in the same gland, and moreover, these oocytes showed marked degeneration. It is presumed that there was a protogynous juvenile hermaphroditism in the process of sexual differentiation of northern pike. This study will provide certain basis for production of all female northern pike fries using hormone induction techniques and further research on sex determining mechanism of northern pike.

Abstract:In recent years, the study on ribosomal genes has always been a hot spot. However, only a few reports on ribosomal genes of fish were reported, especially about the ITS1 (internal transcribed spacer 1) pseudogene. In order to investigate the applicability of ITS1 as a molecular marker in species identification and the phylogenetic analysis in fishes, 11 species from five families, belonging to Perciformes, were selected as the representatives, including Latidae, Toxotidae, Rachycentridae, Xiphiidae and Carangidae. A total of 348 ITS1 sequences were obtained, and the length of these sequences ranged from 442 to 661 bp. Compared with the genetic characteristics, such as the length differences, number of variable sites, GC content, nucleotide diversity and haplotype diversity, the ITS1 sequences of 11 species showed obvious polymorphism within intra-species and inter-species. The unusual case was that a shorter sequence with 55 bp deletion was detected among the 71 clones of Rachycentron canadum. Based on the data of GC content, secondary structure and minimum free energy of the shorter sequence, this sequence was inferred as pseudogene. The neighbor joining tree was constructed based on ITS1 sequences of 11 species from five families with Remora australis and Echeneis naucrates as outgroup. The result showed that the ITS1 sequences of different individuals from each species gathered together, and the species from each genus also grouped to one clade. These indicated the ITS1could be used as the molecular marker for phylogenetic analysis at the genus level. At the family level, the Carangidae was divided into two clades, which was similar to the result of phylogenetic analysis based on the mitochondrial marker, but both were contradictory with the classification based on morphological characteristics. To a certain extent, the ITS1 plays a role of molecular marker for phylogenetic analysis at the family level. These results provide useful scientific information for further studies on ITS1 in fishes and contribute to polymorphism analyses of ribosomal RNA genes in teleostean fishes.

Abstract:A trial was conducted to determine nutrient composition and heavy metal content of dried Daphnia magna from artificial-cultivation pond in spring, summer, autumn, winter and eutrophic lake sewage in summer, aiming to assess potential utilization value in aqua-feed. The results showed that there was significant seasonal difference in the moisture of artificial cultivated D.magna, and the highest was 5.14% in winter, and the lowest was 2.51% in summer. The moisture content (9.12%) of D.magna in summer sewage was significantly higher than that of artificial cultivation. The content of protein of artificially cultivated D.magna was 30.93% - 50.21% with significant differences in different seasons, and in winter and summer sewage was significantly higher than that of other groups. The total content of 15 amino acids determined markedly differed among groups, with the highest (36.65%) in winter and the lowest in summer (24.74%).In summer sewage, the amino acid content was 41.79%, and 9 essential amino acids accounted for 22.15%, lysine content reached 3.4%, methionine 1.14%, and threonine 2.45%. The fat content was also significantly different with seasonal variation, the highest fat content was observed in winter (18.88%), while the lowest in autumn (4.19%), and no significant difference on the fat content was found in winter and summer sewage. The essential fatty acids were the highest in autumn and the summer sewage (17%),and the arachidonic acid and linolenic acid were not detected in all samples. The highest levels of heavy metal chromium, copper, zinc and lead appeared in summer sewage and artificial-cultivation pond in autumn, spring and summer, respectively, and no lead was detected in summer sewage and winter, no cadmium in all groups. Except for chromium, the lowest concentrations of copper, lead, arsenic and zinc were found in the summer sewage. All the results indicated that the D.magna was rich in protein and fat with lower potential toxicity of heavy metals, and both the essential amino acid and essential fatty acid nutrient values in summer sewage were generally higher than those of artificial cultivation, so it is more feasible to use D.magna in summer sewage as the feed protein source.

Abstract:To investigate the cause of sudden death of the koi (Cyprinus carpio), the moribund koi that showed typical symptoms including sunken eyes and head, rotted gill and haemorrhages on the skin were examined. No bacteria were isolated from spleen, liver and kindey of diseased fish by bacteriological examination, but a large number of viral nucleocapsid were found in gill tissue by electron microscopy observation. After filtration treatment, the gills, brain, liver, spleen, and kidney tissue suspension was inoculated to carp fin cells, Cytopathic effects (CPE) were presented 7 days post co-cultivation. In artificial infection test, the death rate of fish by infecting tissue homogenate and culture liquid of infected cell reached 93.3% and 86.7% respectively. Histopathological analysis showed that the target organs included the gill, liver and kidney. Positive results were obtained from diseased koi according to a PCR diagnosis of koi herpesvirus (KHV) proposed by Office International des Epizooties (OIE), and high concentration of KHV was detected in gill and kidney tissue by fluorescence quantitative PCR. Based on neighbor-joining analyses of the TK gene sequences, phylogenetic tree was constructed and the result indicated that the KHV isolated in our study was Asian genotype isolate. Furthermore, according to the sequence alignment of Marker I and Marker Ⅱ regions of KHV, the KHV detected in this study belongs to I⁺⁺Ⅱ^-genotype, and was named KHV-TJ1601. The present study demonstrated that the I⁺⁺Ⅱ^-genotype KHVwas isolated from the naturally infected fish in North China for the first time, and provided important material for virus genesis, evolution, classification, and vaccine preparation.

Abstract:To understand the sequence features and phylogeny of interleukin 17 (IL-17) in sweet fish (Plecoglossus altivelis), and its correlation to Listonella anguillarum infection, the cDNA sequence of IL-17C was cloned from P. altivelis by RACE-PCR, and the expression changes of different tissues were detected after L. anguillarum infection by real-time quantitative PCR (qRT-PCR). The full length of P. altivelis IL-17C cDNA was 1 087 bp including a 534 bp ORF and seven 3'UTR instability motifs (ATTTA), and it encoded 177 amino acids with eight conservation cysteine (Cys) and a 19 amino acids N-terminal signal peptide. Phylogenetic tree analysis showed that the same IL-17 subtype homologue from different species gathered together, and the P. altivelis IL-17C was clustered in one group with other vertebrate IL-17C. Multiple sequence alignment and phylogenetic analysis showed that P. altivelis IL-17C has higher similarity to Oncorhynchus mykiss IL-17C1 (40.72%) and O. mykiss IL-17C2 (34.04%). In healthy P. altivelis, IL-17C mRNA expression was detected in heart, liver, spleen, head kidney, gill, brain, muscle and intestine, high expression was shown in gill and head kidney. After L. anguillarum infection, the expression of IL-17C increased 9.17 folds in liver at 8 hpi (hours post infection) and 6.69 folds in spleen at 4 hpi compared to the control group. IL-17C gene is closely related to the pathogenic bacteria infection of P. altivelis, and it may play an important role in inflammatory immune response.

Abstract:In the present study, full length cDNA encoding the serine/threonine kinases Akt from Eriocheir sinensis (EsAkt) was cloned by using 3'RACE and 5'RACE techniques, and the sequence and structural analysis of the EsAkt was conducted with bioinformatics methods. The results showed that the full length cDNA encoding EsAkt consisted of 2 200 bp nucleic acids in length, including a 5'-UTR of 159 bp, a 3'-UTR of 496 bp and an open reading frame (ORF) of 1 545 bp encoding 514 amino acids. Analysis of the protein domain features showed that the deduced polypeptides contained three conservative domains characteristic of Serine/Threonine protein kinases family. Multiple sequence alignment revealed that the amnio acids sequences of EsAkt have the 0.889 identity with Fenneropenaeus chinensis and Litopenaeus vannamei. The phylogenetic analysis showed that the EsAkt was arranged in the same clade with Akts from other arthropods. The tissue distribution of EsAkt mRNA in sexual maturity individuals and different muscle groups during molt cycle in juvenile crabs were analyzed by quantitative real-time PCR (qRT-PCR). In sexual maturity crabs, the EsAkt transcript was detected in eyestalk, claw muscle, ovary, heart, hepatopancreas, epidermis, testis, gill and triangular membrane, and the expression level was relatively high in ovary, eyestalk and testis, and was low in hepatopancreas. In juvenile crabs, the EsAkt transcript in different muscle groups was different depending on the molt stages. In walking leg muscles, the EsAkt expression level has no obvious change. In abdominal muscles, the EsAkt expression level was much higher in later pre-molt D₃−D₄ stage than post-molt A-B stage and inter-molt C stage. In claw muscles, the EsAkt expression level was decreased rapidly in pre-molt D₃−D₄ stage and increased in post-molt A-B stage, and lasted to inter-molt C stage. These results suggested that the expression of EsAkt transcript was related with the molt stage of E. sinensis, and it is possible that the EsAkt is involved in the muscle atrophy, growth and rebuilding during the molt cycle of E. sinensis.

Abstract:To acquire the data of genotype and diversity upon the clinical dominant strains Streptococcus iniae of golden pompano (Tranchinotus ovatus) in Guangxi, a total of 17 bacterial isolates were analyzed by molecular typing, antibiogram type and virulence-related gene which were collected from diseased golden pompano in 7 farms located in Guangxi Province during 2015-2016. By using the random amplified polymorphic DNA (RAPD) and repetitive primer polymerase chain reaction (rep-PCR) method, we successfully differentiated all 17 isolates into two distinct genotypes. Seven virulence-related genes were detected by PCR using specific primer, and the results revealed that the virulence-related genotype of these strains was simA⁺scpI⁺pdi⁺sagA⁺cpsD⁺pgmA⁺cfi⁺. All isolates seemed to show a high virulence. In addition, the antibiotic resistance profiles of 17 strains were tested with 20 common antibiotics using Kirby-Bauer disc diffusion methodology. The antibiogram of the isolates belonging to genetype 1 in rep-PCR analysis was AZT, while the antibiograms of genetype 2 were SIZ/T/S/PEN/AZT/SPE/CAZ/PB, SIZ/T/S/PEN/AZT/SPE/CAZ/CR and SIZ/T/S/PEN/AZT/SPE/CAZ/PB/RIF, respectively. Our research revealed that the two genotypes display significant differences of two antibiogram types and the same genetype isolates have similar antibiogram types. Therefore, a correlation between the genotype and the antibiogram types of S. iniae isolates has been identified. This study provides theoretical foundation and technical support for epidemiological study, vaccine development and disease control of S. iniae in golden pompano.

Abstract:This experiment was conducted to investigate the protective effects of different concentration of Bacillus subtilis peptidoglycan on the β-conglycinin-induced inflammatory lesions in intestinal epithelial cells of juvenile carp (Cyprinus carpio). In 24-cell microplates, the intestinal epithelial cells (IECs) of juvenile carp were primarily cultured for 72 h at 26℃ and 6% CO₂, and then the IECs were randomly divided into 6 groups with 4 replicates per group. One of the 6 groups was set as negative control group, and the other groups were all supplemented with 1.0 mg/mL β-conglycinin in culture medium in order to establish inflammatory lesions. After 24 h further culture in the same conditions, the culture medium was changed into the B. subtilis peptidoglycan culture medium with 0 (positive control group), 0.15, 0.30, 0.45 and 0.60 mg/mL, respectively. The samples were collected to measure the antioxidant and anti-inflammatory indices after 12, 24 and 36 h B. subtilis peptidoglycan culture. The results showed as follows:β-conglycinin exposure significantly decreased the activity of ASA, AHR, SOD, CAT, GPx, and increased the content of PC and the mRNA expression of inflammatory cytokines (IL-1β, IL-8, TNF-α₁, IL-10 and TGF-β). The 12, 24 and 36 hours after the peptidoglycan were given, the activity of ASA, AHR, SOD, CAT, GPx and the content of PC in the cells were decreased in a dose-dependent manner; and the mRNA levels of IL-1β, IL-8 and TNF-α1 were down-regulated and IL-10 and TGF-β were up-regulated. Different concentration of B. subtilis peptidoglycan could significantly improve the anti-oxidative activity, and increase the mRNA levels of anti-inflammation cytokine, and decrease the pro-inflammation cytokine, and inhibit the inflammatory response of the carp IECs induced by β-conglycinin.

Abstract:In order to investigate the differentiation change of tissue proteins in fish after infection with Vibrio mimicus, Carassius auratus were artificially infected by V. mimicus 04-14 isolates. Differential proteome analysis in spleen and intestinal mucosa tissues before and after infection was performed by using two-dimensional polyacrylamide gel electrophoresis (2-DE) combined with matrix assisted laser desorption/ionization time-of-flight mass spectrometry techniques (MALDI-TOF-MS). The results showed that there were 11 and 12 significantly differentially expressed proteins in spleen and intestinal mucosa tissues after infection, respectively. A total of 19 significantly differentially expressed proteins were identified by MALDI-TOF-MS, with 10 up-regulated and 9 down-regulated proteins. Amylase, apolipoprotein A-I, α-actin and peroxiredoxin 2 were the common significantly differentially proteins both in spleen and intestinal mucosa tissues. Gene Ontology (GO) annotation indicated that significant differentially expressed proteins were located in extracellular region, cytoplasm, mitochondrion and reticulum, respectively. The molecular functions of these proteins included catalytic activity, binding, transport, immune, antioxidant, cytoskeleton and membrane skeleton formation, involved in eight biological processes of substance and energy metabolism, cell membrane transport, complement activation, response to stimulus, cytoskeleton formation, protein folding, redox and iron ion transport. These results might provide a basis for further research of V. mimicus pathogenic mechanism and bacteria- host interactions.