Response of facilitative glucose transporter 1 to salinity stress and dietary carbohydrate nutrition in white shrimp Litopenaeus vannamei

Facilitative glucose transporter 1 (GLUT1) is a transporter protein for glucose transport via the plasma membrane of the cells to provide energy through carbohydrate metabolism. GLUT1 cDNA from Litopenaeus vannamei was obtained and analysed in this study. Full‐length GLUT1 cDNA is 2062 bp long and contained a 1506‐bp ORF encoding a 502 amino acid protein, a 270‐bp 5′UTR and a 284‐bp 3′UTR. When shrimp were under acute low salinity stress, the expression in hepatopancreas, muscle, gill and eyestalk was all up‐regulated at 12 h (P < 0.05) and 96 h (P < 0.05), while the expression in the four tissues was all down‐regulated at 6 h (P < 0.05) and 48 h (P < 0.05) . The expression in the muscle of shrimp at water salinity of 3 was lower than that at water salinity of 30 independent of dietary carbohydrate levels, while expression in hepatopancreas, gill and eyestalk was up‐regulated at 200 and 300 g kg^?1 carbohydrate levels. The expression in all tissues fed glucose was up‐regulated when compared to the expression in shrimp held at a water salinity of 30. This study suggests that GLUT1 is a conserved protein in L. vannamei, and changes in expression due to environmental salinity and dietary carbohydrate level and source.     

大菱鲆水通道蛋白(AQP1、AQP3)以及离子通道蛋白(CFTR、NHE1)对低盐胁迫的响应

在鱼类适应环境盐度变化的过程中,鳃、肾、肠是主要的渗透调节器官,而水通道蛋白(Aquaporins, AQPs)、囊性纤维化跨膜传导调节因子(CFTR)、钠氢交换体(NHE)又是这些器官中重要的渗透调节基因。为研究AQP1、AQP3、CFTR、NHE1在大菱鲆(Scophthalmus maximus)低盐胁迫过程中的渗透调节功能,本研究采用荧光定量PCR技术,对4种基因在盐度5和盐度10下大菱鲆鳃、肾、肠中表达量随时间的变化进行检测。结果显示,AQP1表达量在鳃中极少(P<0.05),在肾和肠中较高,低盐胁迫下,盐度5组和盐度10组在鳃中的表达量无显著变化,在肾和肠中均显著上升(P<0.05)。AQP3表达量在肾中极少(P<0.05),在鳃中较高,在肠中较少,低盐胁迫下,盐度5组和盐度10组在肾中的表达量无显著变化,在鳃和肠中均显著上升(P<0.05)。CFTR表达量在肾中极少,在鳃中较高,在肠中较少,低盐胁迫下,盐度5组和盐度10组在肾中的表达量无显著变化,在鳃和肠中均显著下降(P<0.05)。NHE1在鳃和肠中表达量较少,在肾中较高,低盐胁迫下,盐度5组和盐度10组在鳃中的表达量无显著变化,在肾和肠中均显著上升(P<0.05)。这些结果表明,4种基因表达水平因组织、盐度和时间的不同而不同,反映了这4种基因的功能特异性;在低盐胁迫下,4种基因积极响应,表达量均发生不同程度的变化,表明AQP1、AQP3、CFTR和NHE1在大菱鲆低盐环境适应中可能具有潜在的重要作用。另外,本研究结果可为大菱鲆半咸水养殖和淡化养殖提供理论依据,同时为培育适应低盐环境大菱鲆良种提供理论和技术支撑。     

Molecular and functional characterization of Raptor in mTOR pathway from Litopenaeus vannamei

Raptor, a member of the target of rapamycin complex 1 (TORC1), participates in the formation of complex proteins related to the mechanistic target of rapamycin (mTOR) signalling. In this study, a 5,020 bp cDNA of Raptor with an open reading frame (ORF) of 3,804 bp encoding for 1,267 amino acids was cloned from Litopenaeus vannamei. The protein contains three conserved domains: Raptor N, HEAT and WD40 domains. The expression of Raptor gene was detected by qRT‐PCR in different tissues of L. vannamei, including hepatopancreas, intestinal, stomach, eyestalk, gill and muscle. The mRNA expression profiles of Raptor in muscle were also analysed under suppression or stimulation of mTOR signalling pathway. The level of Raptor mRNA significantly increased either at 0.5–6 hr after an injection of rapamycin (RAPA) or after 3 days starvation. Leucine or arginine alleviated the up‐regulation of Raptor gene expression caused by RAPA or starvation. The Raptor gene was successfully suppressed using RNA interference (RNAi) technology, and the gene expression and the protein phosphorylation level of 4EBP1 and S6K were significantly decreased. The results of the study suggested that the expression of Raptor was sensitive to the immunology status of L. vannamei and participated in nutritional metabolism.     

iTRAQ proteomic analysis of salinity acclimation proteins in the gill of tropical marbled eel (<Emphasis Type="Italic">Anguilla marmorata</Emphasis>)

Osmoregulation plays an important role in the migration process of catadromous fish. The osmoregulatory mechanisms of tropical marbled eel (Anguilla marmorata), a typical catadromous fish, did not gain sufficient attention, especially at the molecular level. In order to enrich the protein database of A. marmorata, a proteomic analysis has been carried out by iTRAQ technique. Among 1937 identified proteins in gill of marbled eel, the expression of 1560 proteins (80 %) was quantified. Compared with the protein expression level in the gill of marbled eel in freshwater (salinity of 0 ‰), 336 proteins were up-regulated and 67 proteins were down-regulated in seawater (salinity of 25 ‰); 33 proteins were up-regulated and 32 proteins were down-regulated in brackish water (salinity of 10 ‰). These up-regulated proteins including Na⁺/K⁺-ATPase, V-type proton ATPase, sodium–potassium–chloride co-transporter and heat shock protein 90 were enriched in many KEGG-annotated pathways, which are related to different functions of the gill. The up-regulated oxidative phosphorylation and seleno-compound metabolism pathways involve the synthesis and consumption of ATP, which represents extra energy consumption. Another identified pathway is the ribosome pathway in which a large number of up-regulated proteins are involved. It is also more notable that tight junction and cardiac muscle contraction pathways may have correlation with ion transport in gill cells. This is the first report describing the proteome of A. marmorata for acclimating to the change of salinity. These results provide a functional database for migratory fish and point out some possible new interactions on osmoregulation in A. marmorata.     

Salinity increases total body prolactin and gill and skin prolactin receptor expression in the Chinese edible frog,Hoplobatrachus rugulosus,tadpole

Water salinity has effects on growth and metamorphosis of anuran species, including Hoplobatrachus rugulosus. Previously, we reported that cultured H. rugulosus tadpoles at low salinities (2–4‰) were of bigger size and grew faster than those in fresh water (FW). However, at a higher salinity level of 6‰, their sizes were reduced and the metamorphosis was delayed. It was therefore hypothesized that high salinity‐induced osmotic stress affected secretion of prolactin (PRL), which acts as osmoregulatory hormone and a regulator of metamorphosis in amphibians. In this study, transferring tadpoles into 4‰ and 6‰ brackish water increased the PRL levels by ~1.2‐ and ~twofold, respectively, as compared to FW group. These osmotic challenges also increased the total body fluid osmolality and levels of Na⁺, Cl^? and Ca²⁺. The contents of triiodothyronine (T₃) were significantly reduced in 4‰ and 6‰ groups, but not 2‰ group. Three sizes (49, 29 and 23 kDa) of H. rugulosus PRL receptors (PRLRs) were detected, and their protein expression was found in the skin, gill, tail fin, brain, intestine, heart, liver and kidney. The expression of PRLR‐49k protein was significantly higher in both skin and gills in 2–4‰ groups, whereas that of PRLR‐29k and PRLR‐23k were higher in gills and lower in skin of the 6‰ group than FW group. In conclusion, salinity challenge, particularly 4‰ and 6‰, increased the levels of PRL, while decreasing the T₃ levels, which could explain why salinity markedly modulated growth, metamorphosis and survival of tadpoles.     

盐碱胁迫对尼罗罗非鱼鳃Na+/HCO3-共转运子、碳酸酐酶基因表达的影响

为了探讨盐碱胁迫条件下鱼类渗透生理调节机制,以尼罗罗非鱼(Oreochromis niloticus)为实验材料, PCR扩增得到了Na+/3HCO-共转运子(NBCe1)基因cDNA部分序列,比较了单盐(盐度10、盐度15)、单碱(1.5 g/L、3 g/L NaHCO3)、盐碱混合(盐度10,碱度1.5 g/L;盐度15,碱度3 g/L)胁迫后不同时间(0 h、6 h、12 h、24 h、48 h、72 h、96 h)血清渗透压、离子浓度(Na+、K+、Cl–、Ca2+)以及鳃碳酸酐酶(CA)活性、CA与NBCe1基因mRNA表达变化。结果显示,不同胁迫条件下,血清渗透压、离子浓度、鳃组织 CA 酶活、CA 与 NBCe1基因 mRNA 表达变化均与胁迫强度呈正相关。随时间推移,血清渗透压、离子浓度呈现先上升后下降的变化趋势,单盐、盐碱混合组血清渗透压值较单碱组高。单盐、单碱、盐碱混合组中, NBCe1基因mRNA在鳃中均呈略微上调,但不显著(P>0.05)。单碱组和盐碱混合组鳃CA活性较单盐组高,低盐碱胁迫(盐度10,碱度1.5 g/L)下CA活性较晚达最高值;不同胁迫条件下, CA基因mRNA表达均表现上调,单碱、盐碱混合组更为显著(P<0.05),推测CA较NBCe1对体内3HCO-转运作用更为显著。研究结果为尼罗罗非鱼盐碱适应生理调节提供了基础资料。     

低盐胁迫下松江鲈ATP1A3和ATP2B1基因的表达变化规律

为了探究Na+/K+-ATP酶和Ca2+-ATP酶在松江鲈(Trachidermus fasciatus)应对低盐胁迫过程中的调节作用,本研究基于前期转录组数据,获取目标基因ATP1A3 (Na+/K+-ATP酶α3亚基基因)和ATP2B1 (Ca2+-ATP酶1基因)的序列信息并进行了系统进化分析。利用实时荧光定量PCR技术检测了松江鲈的鳃、肠、肾脏和肝脏组织中2个基因在2种低盐胁迫处理(盐度渐变处理,盐度变化速率为1.1/h;盐度骤变处理,盐度变化速率为27/h)下,不同时间点(0 h、12 h、24 h和48 h)的表达水平。系统进化分析结果表明,ATP1A3和ATP2B1基因分别聚类形成独立分支;在各基因分支中,松江鲈与已报道的鲈形目和鲽形目等鱼类共同聚在硬骨鱼类分支中。在2种低盐胁迫处理下,2个基因在鳃、肠、肾脏和肝脏组织中的表达量呈现不同的变化趋势。鳃组织中ATP1A3表达量在盐度渐变处理下先上升后下降,ATP2B1表达量仅在24 h显著升高;盐度骤变处理下,ATP1A3表达量显著下降,ATP2B1表达量显著上升。2种盐度渐变处理下,肠组织中ATP1A3表达量均在24 h显著下降;ATP2B1表达量在盐度渐变处理下显著上升,盐度骤变处理下在24 h显著上升。在盐度渐变处理下,肾脏组织中2个基因的表达量均在24 h显著上升至最大值;ATP1A3表达量在盐度骤变处理下显著上升,ATP2B1表达量在12 h和48 h显著上升。肝脏组织中2个基因的表达量在盐度渐变处理下均无显著变化;盐度骤变处理下,ATP1A3表达量持续显著上升,ATP2B1表达量在48 h显著上升。结果表明,低盐胁迫处理显著影响了ATP1A3和ATP2B1基因的表达水平,但2个基因的表达量变化规律存在显著性差异。上述结果为探讨Na+/K+-ATP酶和Ca2+-ATP酶在鱼类渗透压调节过程中的作用及洄游性鱼类适应盐度变化的分子调控机制提供了理论依据。     

盐度胁迫对大鳞鲃抗氧化酶和血清皮质醇的影响

为阐明盐度胁迫对大鳞鲃(Luciobarbus capito)肝、肾和鳃组织抗氧化系统及血清皮质醇的影响,本研究设置4个NaCl盐度组(3、6、9和12 g/L)和1个淡水对照组,检测分析了不同盐度胁迫下曝露3、6、12、24、48、96 h和7 d大鳞鲃肝、肾和鳃组织中酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、谷胱甘肽过氧化物酶(GSH-Px)的活性和丙二醛(MDA)含量的变化,以及血清中皮质醇浓度的变化。结果显示,在相同盐度胁迫下,大鳞鲃的肝、肾和鳃组织中ACP、AKP、GSH-Px抗氧化酶的活力、MDA含量以及血清皮质醇含量随胁迫时间的延长均呈先上升后下降、随后趋于稳定的变化趋势,在胁迫开始24 h内各指标达到峰值,并在48 h开始逐渐趋于平稳;胁迫初期,相同曝露时间,大鳞鲃的肝、肾和鳃组织中3种抗氧化酶活力、MDA含量及血清皮质醇含量均与盐度呈显著正相关性。大鳞鲃在盐度胁迫过程中,ACP、AKP活力和MDA含量在肾组织的范围分别为1.42~2.15 U/g prot、1.01~1.87金氏单位/g prot和13.05~57.27 nmol/mg prot;肝组织中分别为1.27~1.96 U/g prot、0.31~0.86金氏单位/g prot和17.02~55.98 nmol/mg prot;鳃组织则为0.98~1.96 U/g prot、0.13~0.84金氏单位/g prot和8.33~53.93 nmol/mg prot,肾组织中ACP、AKP活力和MDA含量均高于肝、鳃组织;而GSH-Px的活力在肝、肾和鳃组织的范围分别为44.41~114.77、16.52~67.59和9.07~48.00活力单位,肝组织中GSH-Px活力显著高于肾和鳃组织。此外,血清皮质醇在盐度胁迫过程中的含量变化范围为197.00~355.50 ng/L。综上所述,在12 g/L的高盐胁迫下大鳞鲃通过自身调节,各项指标仍可恢复正常,表明其对盐度环境有较强的适应能力。     

Differential responses to low salinity on gene expression,physiological and biochemical indexes between the golden and brown noble scallops Chlamys nobilis

Low salinity is one of important environmental factors which often led to mass mortality of the noble scallop Chlamys nobilis cultivated in the South coast of China. It is well known that enzymic system and non‐enzymic system both play crucial roles in all living organisms against severe environments. To investigate how change about enzymic system and non‐enzymic system in the stenohaline marine bivalve under low salinity stress, an acute challenge lasting 48 hr was conducted using golden and brown noble scallops in the present study. The serine proteinase inhibitor from the noble scallop (CnSPI) was first cloned and expressed in different tissues. After low salinity stress, the gene expression levels were determined in haemocytes and compared between golden and brown scallops. Meanwhile, total carotenoids content (TCC) in adductor, superoxide dismutase (SOD) enzymatic activity and methylenedioxyamphetamine (MDA) content in gill and haemocytes were also determined and compared between the two colours scallops. Results showed that the CnSPI gene expression levels were significantly decreased after low salinity stress, and the golden scallops had higher gene expression levels than brown scallops (p < .05) at most times. Moreover, after low salinity stress, TCC, SOD enzymatic activity and MDA content also fluctuated, and the golden scallops contained higher TCC and SOD, but lower MDA than the brown ones. The present results indicated that enzymic system and non‐enzymic system were both changed under low salinity stress in the noble scallop and significantly different responses to the stress existed between golden and brown individuals. The SPI gene and carotenoids (CAR) both play a resistant role to low salinity stress in the noble scallop.     

急性盐度胁迫对摄食不同镁水平饲料鲈血清渗透压和离子水平以及鳃丝ATP酶活力的影响

为研究饲喂不同镁水平饲料后,鲈血清渗透压、离子水平和鳃丝ATP酶活力对急性盐度胁迫的反应,实验用镁水平为0.413 g/kg(D1,对照组)、1.042 g/kg(D2)、1.577 g/kg(D3)、1.991 g/kg(D4)的4种实验饲料,分别投喂初始平均体质量为(30±0.5)g的鲈。实验鱼在淡水循环养殖系统中饲养50 d后立即转入海水循环养殖系统,并在转入前(0 h)和转入后1、3、6、12、24 h分别采集鲈鳃丝和血液样本。结果显示:盐度胁迫前饲料镁水平对鲈血清渗透压、Na+、K+、Cl-、Mg2+含量和鳃丝中Na+/K+-ATP(NKA)与Ca2+/Mg2+-ATP(CMA)酶的活力有显著影响(P<0.05),其中血清渗透压、Mg2+含量和NKA酶活力随饲料镁水平的增加而升高。D4组的血清渗透压、Mg2+含量、NKA和CMA酶活力显著高于其他组(P<0.05),表明该组鲈处于高强度的渗透压调节的生理状态。急性盐度胁迫中,血清Na+和Cl-含量随胁迫时间的延长呈逐渐上升趋势,血清渗透压、K+、Ca2+、Mg2+和鳃丝ATP酶活力在0～24 h内呈波动性变化。D1组的NKA和CMA酶活力在胁迫1 h时显著低于其他组(P<0.05),表明长期摄食低镁水平的饲料会降低鲈鳃丝NKA和CMA酶对环境盐度刺激的敏感度。研究表明,淡水养殖鲈的饲料中镁水平应低于1.991 g/kg,以减少维持鳃丝NKA与CMA酶高活力而导致的能量损失,而在环境由低盐向高盐的快速转变过程中,较高的饲料镁水平可通过快速提高的鳃丝NKA与CMA酶活力而有利于鲈快速适应高盐环境。     

Dietary supplementation with biofloc promotes growth,improves immune and antioxidant status,and upregulates NF‐κB/Nrf2 signalling molecules and stress resistance in Rhynchocypris lagowskii Dybowski

Bioflocs are rich in a variety of probiotics and bioactive compounds, which have been documented to promote growth, regulate antioxidant status and enhance the immune system. A 60‐day experiment was conducted to investigate the effects on growth, antioxidant and immune status, NF‐κB/Nrf2 signalling molecules and stress resistance in juvenile Rhynchocypris lagowskii Dybowski fed different dietary biofloc supplementations. Four hundred fifty R. lagowskii were fed five experimental diets containing graded levels of biofloc from 0 to 160 g/kg (referred to as B0, B4, B8, B12 and B16, respectively). The results showed that biofloc supplementation markedly promoted growth performance (weight gain rate, specific growth rate); improved amylase, lipase and protease activity; and enhanced the activities of glutathione peroxidase, catalase, total superoxide dismutase, total antioxidant capacity, lysozyme, complement C3, complement C4 and immunoglobulin M in the gill, hepatopancreas, kidney, gut and serum. The malondialdehyde content was significantly reduced with treatment B12. In addition, the levels of heat shock protein 70, heat shock protein 90, interferon‐γ and insulin‐like growth factor I were substantially increased with treatment B12 compared to those with the control treatment. Furthermore, biofloc assisted in regulating the expression of NF‐κB/Nrf2 signalling molecule genes, including NF‐κB, TNF‐α, IL‐1β, IL‐8, IL‐10, TGF‐β, Nrf2, Keap1, Maf, HO‐1, CAT, GPX, GCLC and CuZn‐SOD. Overall, our results suggest that dietary supplementation with biofloc can promote growth, improve immune and antioxidant status, and enhance NF‐κB/Nrf2 signalling molecule expression and stress resistance in juvenile R. lagowskii. A suitable supplementation level of approximately 120 g/kg biofloc is recommended in the present study.     

低盐胁迫下金乌贼转录组学分析

为探讨低盐胁迫对金乌贼影响的分子机制,通过转录组测序技术,测定了正常盐度(30)培养和低盐胁迫(15)6 h的孵出30 d、体质量(1.3±0.3)g金乌贼幼体的转录组数据。测序共获得87326026条序列,经过质量剪切和从头拼接得到575171条转录本和513053条Unigenes。分别在NR、Swiss-Prot、KEGG、String和Pfam数据库对Unigenes进行功能注释,共获得62485条注释结果。Unigenes包含数目较多的KEGG通路有嘌呤、嘧啶和碳代谢,PI3K-AKT、cAMP和Rap1信号通路,内吞作用,RNA转运,局灶性黏附,赖氨酸降解和泛素介导的蛋白水解等。低盐胁迫产生1923条差异表达基因,GO功能富集分析显示,一些可能与低盐胁迫相关的生物学过程如α-氨基酸、羧酸、氧乙酸、有机酸和RNA等代谢过程得到了显著富集。GO可视化分析发现,低盐胁迫对金属离子、阴离子及核苷酸结合,α-氨基酸代谢和水解酶活性等过程影响显著。KEGG通路富集分析显示,低盐胁迫6 h后差异表达基因主要富集到雌激素和心肌细胞肾上腺素信号通路,类固醇生物合成,抗原加工与表达,脂肪和蛋白质消化与吸收,甘油脂质、花生四烯酸和酪氨酸代谢等信号通路上。本研究中获得的通路及基因信息可为今后开展金乌贼低盐胁迫生理机制的探讨、分子标记的挖掘和关键基因的克隆等提供技术支撑。     

盐度对条石鲷幼鱼Na+/K+-ATP酶活力的影响

研究了盐度变化对条石鲷幼鱼鳃、肾脏和肝脏中Na⁺/K⁺-ATP酶活力的影响。经不同盐度(8、18、28、38、48)的处理,条石鲷幼鱼3种组织Na⁺/K⁺-ATP酶活力均受到不同程度的影响。经低盐度(8和18)处理的幼鱼鳃Na⁺/K⁺-ATP酶活力在前6 h略微增加,然后逐渐降低,在处理24 h时下降到最低,之后又开始增加。经高盐度(38和48)处理时,鳃中Na⁺/K⁺-ATP酶活力在前6 h有所降低,然后迅速升高,并在处理24 h时达到最大,之后酶活力逐渐降低,并在处理96 h后与对照组无显著性差异(P>0.05)。所有盐度处理组幼鱼肾脏Na⁺/K⁺-ATP酶活力在处理开始6 h均稍有增加,而从处理6 h开始降低,在处理24 h下降到最低,此后酶活力又呈现增加的趋势。在盐度为8的处理组中,肝脏Na⁺/K⁺-ATP酶活力与肾脏中变化趋势相似,而其它3组则逐渐降低,在处理24 h时达到最低,之后又逐渐增加。结果表明,条石鲷幼鱼适盐范围广,具有较强的渗透压调节能力。3种组织的Na⁺/K⁺-ATP酶活力酶活性在盐度为18～38的范围内变化不明显,而在8和48的盐度下变化较大,最终酶活力均高于对照组。与肾脏相比,盐度变化对鳃和肝脏Na⁺/K⁺-ATP酶活力的影响较大。     

Effects of salinity on the physiological responses of the large yellow croaker Pseudosciaena crocea under indoor culture conditions

The physiological responses to different environmental salinities were assessed in juveniles of large yellow croaker Pseudosciaena crocea. This species shows a good capacity to adapt to comparatively low environmental salinities by evaluating some physiological responses, i.e. superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LZM) in the liver, spleen, gill and kidney respectively. Growth and survival at salinity 5‰, 10‰ and 25‰ were better than those at salinity 15‰ and 20‰. No significant differences in ACP, AKP, SOD and LZM in the liver were observed among different salinity treatments; SOD, AKP and LZM in the spleen among different treatments only showed significant differences at the beginning or the 2nd week; in the gill, no significant difference of AKP and LZM were observed during the whole experiment, SOD among different treatments showed significant difference at the beginning and the 8th week, and ACP only showed significant difference at the end of the experiment; in the kidney, significant differences in ACP, AKP and SOD among different salinities were merely observed at the end of experiment, and LZM showed significant difference among different treatments at the 2nd week. Overall, some slight stress responses were observed, but few significant differences were observed between low salinity and normal salinity, especially the growth and physiological functions were not influenced by low salinity, i.e. 5‰ and 10‰. We conclude that juvenile large yellow croaker is tolerant to low salinity and shows a potential for low salinity culture.     

Molecular characterization,inductive expression and mechanism of interleukin‐10 gene induction in the Indian major carp,catla (Catla catla)

Interleukin‐10 (IL‐10) is a multifunctional cytokine and plays an important role in diseases. In this study, IL‐10 gene was cloned and characterized from catla (Catla catla), which is a highly commercially important fish species in the Indian subcontinent. The result indicated that the full‐length catla IL‐10 (cIL‐10) gene had five exons and four introns with an open reading frame of 540 nucleotides encoding a polypeptide of 179 amino acids. A phylogenetic analysis of cIL‐10 gene sequence showed that cIL‐10 clustered with freshwater carps group as expected. Quantitative real‐time polymerase chain reaction analysis showed that cIL‐10 was expressed in gill, liver, kidney, intestine, skin and heart and its expression profile was up‐regulated in bacterial infection and LPS treatment. A close relationship of high cIL‐10 expression and low pro‐inflammatory cytokine IL‐1β expression was observed in the treated group of fish, which might reveal the role of cIL‐10 as an anti‐inflammatory cytokine. Mechanism of cIL‐10 induction was investigated by blocking nuclear factor (NF)‐κB ‐signalling with BAY 11‐7082 in catla kidney cell culture. Blocking NF‐κB suppressed IL‐10 induction by LPS, and thus it revealed that cIL‐10 was induced through NF‐κB signalling. These data could be helpful to understand the function of IL‐10 in fish in response to vaccinations, probiotics and various diseases.     

Effects of low salinity stress on immune response and evaluating indicators of the swimming crab Portunus trituberculatus

The purpose of the present study was to investigate the effects of salinity stress on immune responses and evaluating indicators in swimming crab Portunus trituberculatus. The crabs (150 ± 8.5 g in body weight) were exposed to different salinities as 21, 26 and 31‰ (control) for 6 days. The results showed the total haemocyte counts (THC) and prophenoloxidase (proPO) activity in the haemocytes decreased significantly in the treatment groups after 6 hr, and reached the lowest levels at 12 hr. The phenoloxidase (PO) activity in the plasma increased significantly and peaked at 12 hr, then recovered to control level after 24 hr. The phagocytic per cent of haemocyte, antibacterial and bacteriolytic activities in the plasma decreased significantly in the treatment groups, and reached the lowest level at 12 hr, then recovered to control level after 72 hr. The dopamine (DA) and 5‐hydroxytryptamine (5‐HT) contents in the plasma increased significantly and peaked at 12 hr, then recovered to control level, while the noradrenaline (NE) content in the plasma had no significant change throughout the duration of the experiment. The DA and 5‐HT receptors were significantly up‐regulated in the treatment groups. The highest value of mRNA expression of DA and 5‐HT receptors occurred at 12 hr and recovered to control level after 24 hr. In addition, the cAMP and protein kinase A (PKA) contents in the haemocytes increased significantly and peaked at 12 hr, then recovered to control level after 72 hr. The phospholipase C (PLC) and protein kinase C (PKC) contents in the haemocytes increased significantly and peaked at 12 hr, then resumed to control level after 24 hr. These results speculated that biogenic amine (DA and 5‐HT) is likely to play an important role in immune modulation via cAMP/PKA signalling pathway or PLC/PKC signalling pathway when P. trituberculatus is exposed to low salinity and these results will provide scientific data for immune evaluation.     

三疣梭子蟹PtDNMT1基因的克隆及其 在低盐适应中的表达分析

甲基转移酶是维持基因组甲基化状态的重要基因,本研究利用SMART-RACE技术克隆了三疣梭子蟹(Portunus trituberculatus)甲基转移酶基因(PtDNMT1)。PtDNMT1基因cDNA序列全长5919 bp,包括4832 bp的开放阅读框,编码1610个氨基酸,预测分子量为148.15 kDa,理论等电点为4.68。结构预测发现,PtDNMT1有2个特殊的结构域,分别是锌指结构域(zf-CXXC)和甲基转移酶家族特有的Dcm结构域。进化树分析显示,PtDNMT1基因与昆虫类的DNMT基因聚为一支。组织表达分析发现,PtDNMT1基因在肝胰腺、鳃、卵巢、肌肉、胃、心脏、血液中均有表达,其中在肝胰腺中表达最高,卵巢和鳃次之。进一步研究了低盐胁迫后PtDNMT1基因在鳃、肝胰腺和肌肉组织中的表达变化规律为胁迫6 h时鳃组织中PtDNMT1基因的表达即达到峰值(5.3倍),并一直持续到12 h (4倍),随后逐步下降,在72 h时仍显著高于对照组(2.3倍);PtDNMT1基因在肝胰腺中的表达规律类似于鳃,然而其达到峰值的时间稍晚于鳃(24 h),且上调倍数高于鳃(8倍);低盐胁迫后PtDNMT1基因在肌肉中的表达最初呈现下调趋势,之后(24 h)上调表达至峰值(2.2倍),且一直上调表达至72 h。本研究首次克隆了PtDNMT1基因,根据其在各组织中的表达分布特征以及盐度胁迫后的表达变化情况,推测DNA甲基化在三疣梭子蟹低盐适应中发挥了重要作用。     

盐度对点篮子鱼的存活、生长及抗氧化防御系统的影响

在容积为500 L的圆锥形塑料缸中分别用对照(自然海水)、配制的盐度为20、10、5的海水和淡水(地下水)养殖点篮子鱼［(67.76?26.12) g］,研究了不同盐度对点篮子鱼存活、生长和抗氧化酶活性的影响。结果显示,淡水组第9天出现死亡,至第27天时死亡率达100%,其余各组未出现异常,死亡率为0%。各盐度组鱼的特定生长率未表现出显著性差异,但盐度10组鱼体重显著高于盐度20和盐度5组,与对照组无显著性差异;盐度5组全长显著低于其余各盐度组。驯化40 d后,对各组鱼鳃、肝脏、肾脏和肌肉中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗超氧阴离子自由基、羟自由基活性检测结果显示,除盐度5组外,盐度10、20组点篮子鱼鳃、肝脏、肾脏、肌肉中SOD、CAT、抗超氧阴离子自由基和羟自由基活性在驯化40 d后均恢复到对照组水平,组间无显著性差异;盐度5组中鱼鳃的SOD和抗超氧阴离子自由基活性显著高于盐度10和盐度20组,肝脏、肌肉和肾脏中SOD、CAT、抗超氧阴离子自由基和羟自由基活性均恢复到对照组水平。点篮子鱼不同组织中SOD和CAT酶活力在不同盐度下均以肝脏中最高,肾脏和鳃次之,肌肉中最低;抗超氧阴离子自由基活力以肝脏中最高,极显著高于其余各组织中抗超氧阴离子自由基活力(P<0.01),肌肉次之,肾脏和鳃最低;羟自由基活力以肾脏中最高,显著高于其余各组织中羟自由基活力(P<0.05);鳃次之,肝脏和肌肉中最低,结果表明盐度能影响抗氧化酶活力大小,但并未影响鱼体内酶的分布。     

Fish protein hydrolysate affected amino acid absorption and related gene expressions of IGF‐1/AKT pathways in turbot (Scophthalmus maximus)

The effects of fish protein hydrolysate (FPH) on growth, peptide and amino acid (AA) transporters, postprandial free AA and related gene expression of IGF‐1/AKT pathway were evaluated in turbot (Scophthalmus maximus). Three diets were formulated to contain the same low level of fishmeal; meanwhile 0, 45 and 180 g/kg FPH were, respectively, supplemented to the FF (FPH‐free), FL (FPH‐Low) and FH (FPH‐High) diets. Fish fed the FH diet improved the growth compared with the other groups. For peptide and AA transporters, PepT1, B⁰AT1, CAT1 and PAT1 mRNA levels in proximal or distal intestine decreased in fish fed the FH diet. The concentration of free total essential AAs in serum was higher in the FH treatment than that in the FF treatment at 2 and 6 hr after feeding. For IGF‐1/AKT pathway in muscle, IGF‐1, 4E‐BP1 and FoxO1 mRNA levels were the highest in the FH group, whereas IGF‐1R mRNA levels were the highest expression level in the FF group. In conclusion, down‐regulated AAs transport, alleviated the delayed postprandial peak of serum‐free AAs and increased muscle protein synthesis were observed to improve the growth when turbot was fed high FPH level diets containing a high plant protein.