Antioxidant and anti‐inflammatory activities of Pinus radiata bark extract in salmonid cell lines

A fish meal supply shortage is limiting aquaculture development. Currently, plant‐based proteins, such as soya bean meal, are being used as an alternative protein source, despite that such a diet can adversely affect fish, such as by inducing an inflammatory response. A possible solution is to include dietary additives in farm diets to counteract negative effects. One such solution originates from pine bark extracts, which present bioactive properties. In this study, the antioxidant and anti‐inflammatory properties of Pinus radiata bark extracts were evaluated for the first time in a salmonid cell line. This extract chemically demonstrated antioxidant activity through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH = 58.4 ± 1.1%) and ferric ion reducing antioxidant power (FRAP = 575 ± 17 mgEqFe(II)·g extract^?1) assays. Additionally, the extract showed high flavonoid and phenolic compound contents. Up to 100 mg mL^?1, the P. radiata extract showed no cytotoxicity in the CHSE‐214 salmonid embryo cell line. Moreover, the antioxidant activity of the extract (50 μg mL^?1) was evaluated by a dichlorofluorescein (DCFH) assay in the SHK‐1 salmon cell line challenged with an oxidant stimulus (H₂O₂), showing 58.9% activity. The extract also protected DNA from oxidative damage, as observed through a comet assay. When assessing anti‐inflammatory properties in an in vitro inflammation model, the extract significantly reduced the relative expression of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) and of the inducible cyclooxygenase‐2 (COX‐2) enzyme. These results suggest a potential application of P. radiata bark extract in functional foods in aquaculture.     

Immunomodulatory effect of Guavarine®, aqueous guava leaf extract,on ornamental Koi carp Cyprinus carpio var. koi L. 1758

The leaves and bark of guava (Psidium guajava) have a long history of medicinal uses. The immunomodulatory effect of Guavarine®, an aqueous guava leaf extract, was investigated on Cyprinus carpio var. koi L. The fish (8.33 ± 1.02 g) were injected with guava leaf extracts (10 or 100 µg/fish) intraperitoneally, and the innate immune parameters were monitored for 28 days. In fish injected with guava leaf extracts, the lymphocyte proliferation, nitric oxide production, and respiratory burst activity increased until the 14th day and decreased thereafter. The results indicated an elevation in the innate immune status of fish, possibly due to the presence of flavonoids in the leaf extracts.     

厚壳贻贝5-羟色胺2A受体(5-HT2AR)基因克隆和时空表达

为探究5-羟色胺2A受体（5-HT 2A receptor, 5-HT2AR）基因在海水贝类生长发育中的作用,本研究通过RACE技术和实时荧光定量PCR（qRT-PCR）克隆了厚壳贻贝5-HT2AR 基因的cDNA全长,并分析该基因的时空表达。结果显示,5-HT2AR基因全长2 636 bp,开放阅读框（ORF）2 124 bp,共编码707个氨基酸。序列分析结果显示该序列与人、小鼠、斑马鱼、长牡蛎和虾夷扇贝等物种分别具有45%、45%、48%、49%和67%的同源性。厚壳贻贝雌雄成体各组织和器官中均有5-HT2AR基因表达,雄性中的鳃表达量最高,而在雌性鳃、外套膜和性腺中的表达稍高于其他组织和器官;推测该基因可能与厚壳贻贝的摄食、对外界环境的感知及促进卵母细胞成熟有关。5-HT2AR基因在厚壳贻贝各发育阶段均有表达,且稚贝的表达量为眼点幼虫的1.4倍,推测5-HT2AR基因可能参与了调控厚壳贻贝幼虫的生长发育过程。本研究为进一步了解5-HT基因家族在双壳贝类中的功能奠定了基础。     

Effect of Dietary N‐acyl Homoserin Lactonase on the Immune Response and the Gut Microbiota of Zebrafish,Danio rerio,Infected with Aeromonas hydrophila

Quorum sensing interference is a novel strategy in bacterial disease control. Degradation of quorum sensing signal molecules, such as N‐acyl homoserine lactones (AHLs), by enzymes is a potential interfering method. In this study, the in vivo effect of an N‐acyl homoserine lactonase from Bacillus sp. B546, AiiA_B546, on the protection of zebrafish, Danio rerio, against Aeromonas hydrophila infection was first investigated by co‐feeding. The short‐term immune responses and the long‐term effects on the gut microbiota of zebrafish were analyzed by quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The results showed that the expression levels of some anti‐infectious immunity‐related factors, including interleukin 10 (IL‐10), toll‐like receptor 5b (TLR5b), and inducible nitric oxide synthase 2a (iNOS2a) that were related to A. hydrophila virulent factors under regulation of quorum sensing, were more susceptible to the addition of AiiA_B546. It indicated that AiiA_B546 may attenuate the infection of A. hydrophila by interfering with quorum sensing. A. hydrophila was not detected in the enzyme co‐feeding diet group, which might be due to its inability to adhere to the gut with enzyme interference. This study revealed that AiiA_B546 as a feed additive might represent a novel biocontrol agent in aquaculture; however, the topic merits further investigations using fish species of commercial interest.     

Organization and promoter analysis of a tiger shrimp Penaeus monodon single WAP domain-containing protein gene

The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb 3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone (GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish.     

Cloning and characterization of tryptophan 2,3-dioxygenase gene of Zhikong scallop Chlamys farreri (Jones and Preston 1904)

A Zhikong scallop (Chlamys farreri Jones and Preston 1904) tryptophan 2,3‐dioxygenase (TDO) gene fragment, down‐regulated by Vibrio anguillarum challenge, was isolated using mRNA differential display in our previous work. In this paper, the full‐length TDO gene was cloned by 5′‐RACE. Chlamys farreri TDO gene consists of 1292 nucleotides encoding an expected polypeptide of 383 amino acids with an estimated molecular weight of 44.8 kDa and an isoelectric point of 6.35. The deduced amino acid sequence is 54–61% homologous to TDOs from Caenorhabditis elegans, Mus musculus, Danio rerio, Homo sapiens and Drosophila melanogaster, and shares several histidine residues important for the enzyme function in other species. Chlamys farreri TDO is expressed in the mantle, gill, digestive gland, testis, adductor muscle and kidney. Immunohistochemical analysis showed that C. farreri TDO was located mainly in the cytoplasm of most cell types. The non‐specific distribution of C. farreri TDO suggests that it is involved in various cellular processes.     

Oral Administration of Hot‐water Extract of Tropical Brown Seaweed,Sargassum cristaefolium,to Enhance Immune Response,Stress Tolerance,and Resistance of White Shrimp,Litopenaeus vannamei,to Vibrio parahaemolyticus

The efficacy of hot‐water extract of tropical brown seaweed, Sargassum cristaefolium (SCE), supplemented in diets on immune response, stress tolerance, and disease resistance of Litopenaeus vannamei to Vibrio parahaemolyticus was evaluated. Shrimp were fed diets containing graded levels of SCE (0, 250, 500, 750, and 1000 mg/kg). The results showed that shrimp fed all diets containing SCE had significantly higher (P < 0.05) immune response in total hemocyte count (THC), differential hemocyte count (granular and hyaline cells), and phagocytic activity than those of shrimp fed the control diet. Similarly, in low dissolved oxygen stress tolerance test and the challenge test with V. parahaemolyticus, survival rates of shrimp fed all diets containing SCE were significantly higher (P < 0.05) (83–93% in stress test and 27–47% in challenge test) than those of shrimp fed the control diet (77 and 3.3%, respectively). These results suggest that oral administration of SCE at 500 and 750 mg/kg can be effectively used to enhance immune response, stress tolerance, and resistance of white shrimp, L. vannamei, against V. parahaemolyticus infection. These findings also confirm that using dietary SCE as immunostimulant is effective at increasing the nonspecific immune system in penaeid shrimp, L. vannamei.     

Essential oils to control ichthyophthiriasis in pacu,Piaractus mesopotamicus (Holmberg): special emphasis on treatment with Melaleuca alternifolia

In vitro effect of the Melaleuca alternifolia, Lavandula angustifolia and Mentha piperita essential oils (EOs) against Ichthyophthirius multifiliis and in vivo effect of M. alternifolia for treating ichthyophthiriasis in one of the most important South American fish, Piaractus mesopotamicus (Holmberg), were evaluated. The in vitro test consisted of three EOs, each at concentrations of 57 μL L^?1, 114 μL L ^?1, 227 μL L^?1 and 455 μL L ^?1, which were assessed once an hour for 4 h in microtitre plates (96 wells). The in vitro results demonstrated that all tested EOs showed a cytotoxic effect against I. multifiliis compared to control groups (P < 0.05). The in vivo treatment for white spot disease was performed in a bath for 2 h day^?1 for 5 days using the M. alternifolia EO (50 μL L ^?1). In this study, 53.33% of the fish severely infected by I. multifiliis survived after the treatment with M. alternifolia (50 μL L ^?1) and the parasitological analysis has shown an efficacy of nearly 100% in the skin and gills, while all the fish in the control group died. Furthermore, the potential positive effect of M. alternifolia EO against two emergent opportunistic bacteria in South America Edwardsiella tarda and Citrobacter freundii was discussed.     

Effects of dietary levels of essential oil (EO) extract from bitter lemon (Citrus limon) fruit peels on growth,biochemical, haemato‐immunological parameters and disease resistance in Juvenile Labeo victorianus fingerlings challenged with Aeromonas hydrophila

Essential oils (EOs) are used in the food industry because of their biological activity. We evaluated the effects of administration of essential oil (EO) extracted from bitter lemon (Citrus limon) fruit peels on the growth performance, biochemical, haemato‐immunological parameters and possible disease resistance in fingerlings (4 weeks old) Labeo victorianus challenged with Aeromonas hydrophila. Fish were divided into five groups and fed diets supplemented with C. limon fruit peels EO extract at 1%, 2%, 5% and 8% [as fed basis] and treatment compared with control group fed diet without C. limon fruit peels EO extract. The experiment was executed in triplicate. Concentration of plasma cortisol, glucose, triglyceride and cholesterol decreased while that of total protein and albumin increased as dietary inclusion of the EO extract of C. limon fruit peels was increased from 2% to 5%. Meanwhile haemato‐immunological parameters including red blood cell (RBC), white blood cell (WBC) counts, haematocrit (Htc), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and neutrophiles increased with increasing dietary inclusion from 1% to 5% inclusion of C. limon fruit peels EO extract. Serum immunoglobulins, lysozyme activity and respiratory burst increased with increasing dietary levels up to 5% inclusion of EO extract of C. limon fruit peels. We demonstrate that formulation of feeds by incorporating upto 5% the EO extract from C. limon fruit peels significantly improved biochemical, haematological and immunological response in juvenile fish resulting to lower mortality than the untreated groups and appear to be effective antibacterial against A. hydrophila.     

Effects of dietary Persian oak (Quercus brantii var. persica) fruit extract on survival,growth performance,haematological and immunological parameters in rainbow trout,Oncorhynchus mykiss,fingerlings

The aim of study was to evaluate the effects of jaft (internal layer of oak fruit) extract on growth, haematological and immunological parameters in rainbow trout fingerlings. A total of 360 fish (average weight 6.25 g, total length 7.75 cm) were randomly distributed in 12 fibreglass tanks (stocking density: 1.04 g L^?1) with flow rate of 8 L min^?1. The ethanolic extraction of jaft was taken, and, then, a basal diet was supplemented with 0 (control), 0.5, 1 and 2 g kg^?1 to formulate four experimental diets. After 8 weeks, blood sampling was performed for haematological and immunological parameters. While immunological parameters such as IgM and lysozyme showed their lowest level in control group, their highest amounts appeared in 2 g kg^?1 group. C3, C4 and alternative complement activity increased in fish fed 2 g kg^?1 extract compared to control (P ? 0.05). Haematological parameters such as white blood cell, red blood cell, haemoglobin and haematocrit were enhanced in experimental groups (P ? 0.05). Survival and growth indices did not show significant changes in experimental treatments (P ? 0.05). Results indicated feeding rainbow trout with Persian oak extract at 0.5, 1.0 and 2.0 g kg^?1 levels significantly enhance the immunological parameters.     

Characterization of spaC‐type Erysipelothrix sp. isolates causing systemic disease in ornamental fish

Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.     

Partial characterization of the gene encoding myoadenylate deaminase from the teleost fish <Emphasis Type="Italic">Platichthys flesus</Emphasis>

AMP-deaminase (AMPD, EC 3.5.4.6), which catalyzes the irreversible hydrolytic deamination of AMP to IMP and ammonia, is an important energy-related enzyme. The partial genomic sequence of the gene encoding myoadenylate deaminase (AMPD1) from the teleost fish Platichthys flesus was determined. The amino acid sequence of P. flesus AMPD1 shows 82% homology with that of the teleost fish Danio rerio. Comparison of genomic sequences of P. flesus and Rattus norvegicus reveals a high degree of conservation of both sequence and structural organization. A phylogenetic analysis of AMPD sequences shows that bony fish and mammalian AMPD1s arise by duplication of a common primordial gene.     

Non‐specific immune responses and intestinal immunity of common carp (Cyprinus carpio) fed Jujube (Ziziphus jujube) fruit extract

This study investigated the effects of Jujube (Ziziphus jujube) fruit extract (JFE) as an excellent source of nutrients and phytochemicals on the innate immune responses and expression of genes involved in intestinal immunity of common carp (Cyprinus carpio) fingerlings. Three hundred healthy carps (10.78 ± 0.05 g) fingerlings were randomly allocated to 12 fibre glass tanks (300L; 15 fish per tank) and fed for 8 weeks with experimental diets which contained different levels of JFE (0%, 0.25%, 0.5%, or 1%). Thereafter, serum non‐specific immune parameters [total immunoglobulin (Ig) as well as alternative complement (ACH50) and lysozyme activity (SL)] and expression of genes involved in intestinal immunity (lyz, tnf‐alpha, il1b, il8, il10, and tgfb) were measured. The results revealed a significant elevation of Ig level in the JFE‐fed fish (p < 0.05) over the control fish. However, there was no significant difference among treated groups in case of serum total Ig. The highest levels of SL and ACH50 were observed in fish fed 1% of JFE. Nonetheless, no significant difference was noticed regarding SL and ACH50 of control and other supplemented groups (0.25% and 0.5% JFE). Also, expression of lyz, tnf‐alpha, and il1b genes increased significantly (p < 0.05) in JFE‐fed fish, in a dose‐dependent manner. On the other hand, significant (p < 0.05) down‐regulation of il110 and tgfb were noticed in treated groups. The present findings suggested that extraction of Jujube, Z. jujube fruit, possesses beneficial effects on immune responses of common carp.     

Embryonic development and morphology of eggs and newly hatched larvae of Pacific herring <Emphasis Type="Italic">Clupea pallasii</Emphasis>

The embryonic development and morphology of eggs and newly hatched larvae of the Pacific herring Clupea pallasii were described using laboratory-reared specimens originating from the Miyako Bay stock. The eggs were almost spherical in shape, 1.33–1.46 mm (mean: 1.38 mm) in diameter, and had a thick adherent chorion. They had a segmented pale yellow yolk, no oil globule, and a relatively wide perivitelline space. A subgerminal cavity was observed during the gastrula period, whereas the blastocoel did not appear. Mass hatching occurred by 271 h 45 min after fertilization, and the newly hatched larvae were 7.1–7.7 mm (mean: 7.5 mm) in total length with 53–56 myomeres at 9.6°C. The embryonic development of Pacific herring was substantially similar to that of zebrafish Danio rerio, American shad Alosa sapidissima, as well as Atlantic herring Clupea harengus, and generally followed the basic developmental pattern of teleosts. However, Pacific herring larvae hatched at a more developed stage than some other clupeoids, such as Japanese sardine Sardinops melanostictus, and the progressed developmental stage at hatching could be interpreted as an advanced adaptation.     

Transmission of a common intestinal neoplasm in zebrafish by cohabitation

Intestinal neoplasms are common in zebrafish (Danio rerio) research facilities. These tumours are most often seen in older fish and are classified as small cell carcinomas or adenocarcinomas. Affected fish populations always contain subpopulations with preneoplastic lesions, characterized by epithelial hyperplasia or inflammation. Previous observations indicated that these tumours are unlikely caused by diet, water quality or genetic background, suggesting an infectious aetiology. We performed five transmission experiments by exposure of naïve fish to affected donor fish by cohabitation or exposure to tank effluent water. Intestinal lesions were observed in recipient fish in all exposure groups, including transmissions from previous recipient fish, and moribund fish exhibited a higher prevalence of neoplasms. We found a single 16S rRNA sequence, most similar to Mycoplasma penetrans, to be highly enriched in the donors and exposed recipients compared to unexposed control fish. We further tracked the presence of the Mycoplasma sp. using a targeted PCR test on individual dissected intestines or faeces or tank faeces. Original donor and exposed fish populations were positive for Mycoplasma, while corresponding unexposed control fish were negative. This study indicates an infectious aetiology for these transmissible tumours of zebrafish and suggests a possible candidate agent of a Mycoplasma species.     

Isolation, cloning, sequencing of brain type aromatase and its expression in male and female Wrasse, Pseudolabrus sieboldi

Pseudolabrus sieboldi, wrasse being a diurnal spawner provides a good opportunity to study the endocrine mechanism of estrogen formation in brain and gonads. Moreover, an extremely large amount of E2 was produced in serum and testis of wrasse. It is assumed that the presence of E2 may play a major role in diurnal gametogenesis in male fish. In this study brain type aromatase have been isolated, cloned and sequenced from the brain of wrasse. Further, the expression pattern of brain type aromatase in gonads and adult tissue of male and female fish have been analyzed. In addition, the diurnal expression pattern of brain type aromatase in both male and female fish brain during spawning season have been analyzed. The P450arom (br) was isolated, cloned and sequenced from both male and female bamboleaf wrasse. The P450arom (br) gene (1877 sequenced nucleotide) contains an ORF of 1470 bp, a 5′-UTR of 18 bp and at least 407 bp in 3′-UTR. The amino acid sequence homology in the coding region of wrasse P450arom (br) is high compared to that of medaka, Oryzias latipes (80%), rainbow trout type 2, Oncorhynchu mykiss (78.2%), fugu, Takifugu ribripes (78%) rainbow trout type 1, (76%), goldfish, Carassius auratus (66.8%) and zebrafish, Danio rerio (66.2%). Expression study reveals that P450arom (br) mRNA were most abundant in brains of both male and female fish throughout the day during the spawning season. RT-PCR study revealed that P450arom (br) was expressed in skin, anal fin and tail fin of both male and female wrasse. P450arom (br) was not detected at any time of the spawning day in either ovary or testis of wrasse.     

Skin‐injured Zebrafish,Danio rerio,are more Susceptible to Vibrio anguillarum Infection

Vibrio anguillarum, which is part of normal microflora on fish, is the causative agent of vibriosis in aquaculture. It is speculated that V. anguillarum does not affect the host in most situations, but can cause a severe disease once the host is compromised. In the study reported herein, skin‐injured and intestine‐injured zebrafish, Danio rerio, were established as a model to mimic the natural infection caused by V. anguillarum when fish suffered an injury to a mucosal surface. Our results showed the lethal dose to 50% of the population (LD₅₀) of skin‐injured zebrafish was 6.8 × 10³ colony‐forming unit (CFU)/mL, which was much lower than intestine‐injured zebrafish (1.9 × 10⁶ CFU/mL) or non‐injured zebrafish (5.5 × 10⁶ CFU/mL). With the quantitative polymerase chain reaction and immunohistochemical analysis, we found that V. anguillarum proliferated rapidly in the skin and muscle after the bacteria entered into the host via the skin injury. The bacteria were subsequently transported to the immune organs and then caused a systemic infection in the fish. However, mortality of skin‐injured zebrafish significantly decreased if the fish were allowed to heal. These results indicate that minimizing injury to the mucosal surfaces of fish, especially the skin, will reduce infections caused by V. anguillarum.     

黄河鲤酪氨酸酶基因的克隆、表达模式及定位分析

为探究Tyrosinase(Tyr)基因在黄河鲤体色形成中的作用,利用生物显微镜对黄河鲤黑色鳞片、银色鳞片、鳍条色素细胞的组成进行观察,并利用RACE克隆黄河鲤Tyr基因全长cDNA序列,荧光定量PCR分析其在不同组织中的表达情况,Western blot印迹和免疫组织化学方法检测其蛋白的表达定位。结果显示,黄河鲤具有黑色素细胞、黄色素细胞和虹彩细胞3种色素细胞,3种色素细胞的数量、种类和分布在不同组织中存在较大差异。黄河鲤Tyr基因cDNA全长1717 bp(GenBank ID:No.KY305667),其中ORF长1605 bp,编码535个氨基酸,5′-UTR区41 bp,3′-UTR区71 bp。蛋白同源分析发现,黄河鲤Tyr与鲤科鱼类相似性最高,与哺乳类及鸟类等脊椎动物相似性最低。系统进化分析显示,黄河鲤Tyr与鲤、瓯江彩鲤、锦鲤和斑马鱼等鲤科鱼类的亲缘关系最近。实时定量PCR分析表明,Tyr基因在黄河鲤不同组织中均有表达,肌肉中的表达量最高,其次是黑色皮肤,另外在黄色皮肤、臀鳍和尾鳍等黑色素细胞存在的组织中也有较高表达。Western blot印迹结果显示,Tyr基因在黑色皮肤中表达最高,其次是臀鳍和尾鳍,银色皮肤中没有表达。免疫组织化学结果显示,Tyr基因在黑色皮肤的黑色素细胞和黏液细胞中有表达。研究表明,Tyr基因表达水平与黑色素细胞数量和分布具有一定相关性,参与黄河鲤体色的形成。