The study of antioxidant enzymes and immune‐related genes expression in common carp (Cyprinus carpio) fingerlings fed different prebiotics

The aim of the present study was to study the effects of different prebiotics (galacto‐, fructooligosaccharide and inulin) on immune response and oxidative stress of common carp (Cyprinus carpio) fingerlings at the molecular level. A total number of 240 fish (13.85 ± 0.85 g) were supplied and randomly stocked in twelve fiberglass tanks (20 specimens per tank). Fish were fed a basal formulated diet (Control) or basal diet supplemented with equal level (2%) of different prebiotics (four treatments repeated in triplicated) for 8 weeks. At the end of feeding trial, the expression of immune‐related genes (interleukin 1 beta [IL‐1β], IL‐8, IL‐10, lysozyme [LYZ], tumour necrosis factor alpha [TNF‐α] and transforming growth factor beta [TGF‐β]) were determined in head kidney and intestine tissues and the expression of antioxidant‐related genes (glutathione S‐transferase [GST‐α], glutathione reductase [GR] and glutathione peroxidase genes [GPX]) were studied in intestine. The results revealed that dietary administration of prebiotics modulated the expression of immune‐related genes and the degree of expression was affected by the type of prebiotics and the organ that was used for analyses. Also, evaluation of antioxidant genes expression showed that GSTα and GR expression levels increased as a result of feeding common carp with the prebiotics. According to these findings, it can be concluded that feeding on different prebiotics had altered effects on the expression of immune and antioxidant‐related genes.     

Dietary Houttuynia cordata leaf extract and meal enhances the immunity and expression of immune genes in Labeo rohita (Hamilton, 1822)

A feeding trial was conducted to explore the effect of dietary Houttuynia cordata leaf extract (HCLE) and leaf meal (HCLM) on immunological responses and expression of interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) gene in Labeo rohita fingerlings. Six isonitrogenous (350 g/kg CP) and isocaloric (17 MJ/kg DE) purified experimental diets were formulated with Houttuynia cordata leaf extract and leaf meal comprising control, C (0 g/kg HCLE and HCLM), E2.5 (2.5 g/kg HCLE), E5 (5 g/kg HCLE), E10 (10 g/kg HCLE), M10 (10 g/kg HCLM) and M20 (20 g/kg HCLM). Labeo rohita fingerlings (3.37 ± 0.23 g) were distributed in six experimental groups in triplicates following the complete random distribution. Fish were fed twice daily with respective experimental diets for a period of 60 days. A significantly (p < .05) lower lactate dehydrogenase, malate dehydrogenase, superoxide dismutase and catalase activities were registered in supplemented groups compared with control group, while respiratory burst and lysozyme activities were significantly (p < .05) higher in E10 group compared with other experimental groups. Haemoglobin, total leucocyte count, total erythrocyte count and haematocrit values were significantly (p < .05) higher in E10 group. The expression of IFN‐γ and TNF‐α in both the kidney and liver was significantly up‐regulated in leaf extract and meal supplemented groups with the highest expression in the fish of E10 group. Overall, these results suggest that the dietary supplementation of ethanolic extract of the Houttuynia cordata leaf at 10 g/kg level can enhance the immune response of L. rohita fingerlings.     

Effect of dietary supplementation of palm fruit extracts on the transcriptomes of growth,antioxidant enzyme and immune‐related genes in common carp (Cyprinus carpio) fingerlings

This study investigates the effects of date palm extracts [DPE] (Phoenix dactylifera L. Arecaceae) on growth, immune function and antioxidant system in common carp fingerlings (Cyprinus carpio). One hundred and twenty fish (4.06 ± 0.13 g) were divided into six groups fed on control diet or diets containing 200 mL kg^?1 DPE for 8 weeks. At the end of feeding trial, the expression of different genes was measured. Selected genes were grouped into three categories: growth factor genes in brain and liver [growth hormone (GH), insulin‐like growth factors I and II (IGF‐I and IGF‐II), antioxidant‐related genes in liver [glutathione S‐transferase‐alpha (GST‐α), glutathione reductase (GR) and glutathione peroxidase genes (GPX)] and immune‐related genes in head kidney [interleukin‐8 (IL‐8), interleukin‐10 (IL‐10) and transforming growth factor‐beta (TGF‐β) genes]. The relative expression of the growth‐related genes in fish fed DPE showed no significant increase compared to control group (P > 0.05). On the other hand, DPE altered the expression of genes encoding antioxidants enzymes in liver of fingerlings which was statistically significant with respect to the control samples in case GPX (P < 0.05). Also, DPE caused remarkable increases in the expression of the immune‐related genes (IL‐8, IL‐10 and TGF‐β) analysed on head kidney of common carp fingerlings compared to the control group (P < 0.05). In conclusion, it can be suggested that administration of DPE in early stages of common carp culture can promote immune efficacy and increase the antioxidant activity.     

Effects of dietary supplementation of glycyrrhizic acid on growth performance,survival, innate immune response and parasite resistance in juvenile large yellow croaker,Larimichthys crocea (Richardson)

An 8‐week feeding experiment was conducted to investigate the effects of dietary glycyrrhizic acid (GA) on growth, survival and immune response of juvenile large yellow croaker, in seawater floating net cages. GA was supplemented into the basal diet to formulate four isonitrogenous and isoenergetic practical diets containing 0.00% (the control diet), 0.01%, 0.02% and 0.04% GA of dry weight, respectively. Triplicate groups of 60 fish were fed to apparent satiation by hand twice daily. The results showed that the specific growth rate, survival rate and feed efficiency ratio revealed no significant differences among dietary treatments (P > 0.05). The phagocytic index of head kidney macrophage was significantly increased by the supplementation of 0.04% dietary GA compared to the control group (P < 0.05). Fish fed 0.04% dietary GA also showed significantly higher serum lysozyme activity than fish fed the control diet and diet with 0.01% GA (P < 0.05). The cumulative mortality rate after natural infestation of parasites (protozoan, Cryptocaryon irritans Brown) showed no significant differences among dietary treatments. These results suggested that dietary glycyrrhizic acid improved certain non‐specific immunological parameters of juvenile large yellow croaker. However, GA was not able to protect juvenile large yellow croaker effectively from protozoan infection.     

The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

Immune response of DNA vaccine against lymphocystis disease virus and expression analysis of immune‐related genes after vaccination

In this study, we found that an intramuscular injection of Japanese flounder (Paralichthys olivaceus, 60–80 g in weight and 15–20 mL in length) with 5 μg of a DNA vaccine (pEGFP‐N2‐LCDV‐cn‐MCP 0.6 kb, containing lymphocystis disease virus major capsid protein gene) induced a strong immune response. Subsequent real‐time polymerase chain reaction showed that the expression of immune‐related genes [e.g., major histocompatibility complex (MHC) class I α, MHC II α, T‐cell receptor (TCR), tumour necrosis factor (TNF), tumour necrosis factor receptor (TNFR), Mx, interleukin (IL)‐1β, CXC and IL‐8R] was significantly changed after DNA vaccination. The most remarkable alternation was the expression of MHC I α and MHC II α genes: MHC II α reached the maximum on day 8 in different tissues, and MHC I α on day 2 in the intestine and gills. The expression of TCR increased and reached a plateau in 2 days in the spleen, gills, kidney and liver after vaccination and then decreased after day 8. In contrast, the expression of TCR in the intestine increased and reached a plateau in 8 days. The expression of IL‐8R reached the maximum on day 2 in different tissues and then decreased on day 8. Mx increased in the gills, kidney, spleen and liver on days 2, 8, 2 and 2, but decreased in the intestine, gills, spleen and liver on days 2, 8, 8 and 8 respectively. The TNFR expression increased in the spleen, kidney and gills on days 2, 8 and 8, but decreased in intestine, liver and gills on days 2, 8 and 8 respectively. The expression of TNF, CXC and IL‐1β increased 2 and 8 days after the injection of DNA vaccine. However, the expression of TNF, CXC and IL‐1β altered on days 2 and 8 with different patterns in different tissues respectively. The fish responded to the DNA vaccine by yielding a specific immunoglobulin against lymphocystis disease virus (LCDV) as observed with indirect ELISA. The DNA vaccine induced a unique humoral response, suggesting that the DNA vaccine activated both cellular and humoral defences of the specific immune system of Japanese flounder.     

Dietary soya allergen β‐conglycinin induces intestinal inflammatory reactions,serum‐specific antibody response and growth reduction in a carnivorous fish species,turbot Scophthalmus maximus L.

This study was conducted to investigate the effects of dietary β‐conglycinin on the growth performance, digestion, gut morphology and immune responses of juvenile turbot (Scophthalmus maximus L.). Four diets were formulated to contain 0%, 2%, 4% and 8% purified β‐conglycinin. Triplicate groups of 30 fish were fed to apparent satiation twice daily for 12 weeks. Fish fed 4% and 8% dietary β‐conglycinin showed significantly reduced specific growth rate, feed efficiency ratio, apparent digestibility coefficient of nutrients and whole‐body lipid contents, as well as a profound infiltration of mixed leucocytes in the lamina propria and a significant decrease in the absorptive surface of distal intestine. The expression of pro‐inflammatory cytokines, TNF‐α and IL‐1β, in the distal intestine was significantly upregulated by 4% dietary β‐conglycinin, whereas a significantly lower expression level of IgM and anti‐inflammatory cytokine TGF‐β1 was observed in fish fed 8% dietary β‐conglycinin. Serum lysozyme and alternative complement pathway activity were first significantly enhanced by 2% dietary β‐conglycinin and then rapidly declined by 4% and 8% dietary β‐conglycinin. Respiratory burst activity of head kidney macrophages and serum superoxide dismutase activity were significantly suppressed by 4% and 8% dietary β‐conglycinin. Dietary β‐conglycinin (2–8%) significantly increased the level of specific antibody against β‐conglycinin in serum. Collectively, these results suggested that higher levels of dietary β‐conglycinin (4–8%) induced a variety of non‐specific and specific immune responses and intestinal mucosal lesions in turbot, resulting in inferior feed utilization and poor growth performance.     

Role of major histocompatibility complex II antigen-presentation pathway genes in orange-spotted grouper infected with Cryptocaryon irritans

Cryptocaryon irritans, a pathogen model for fish mucosal immunity, causes skin mucosal and systematic humoral immune response. Where and how MHC II antigen presentation occurs in fish infected with C. irritans remain unknown. In this study, the full-length cDNA of the grouper cysteine protease CTSS was cloned. The expression distributions of six genes (CTSB, CTSL, CTSS, GILT, MHC IIA and MHC IIB) involved in MHC II antigen presentation pathway were tested. These genes were highly expressed in systematic immune tissues and skin and gill mucosal-associated immune tissues. All six genes were upregulated in skin at most time points. Five genes expected CTSS was upregulated in spleen at most time points. CTSB, CTSL and MHC IIA were upregulated in the gill and head kidney at some time points. These results indicate that the presentation of MHC II antigen intensively occurred in local infected skin and gill. Spleen, not head kidney, had the most extensive systematic antigen presentation. In skin, six genes most likely peaked at day 2, earlier than in spleen (5–7 days), marking an earlier skin antibody peak than any recorded in serum previously. This significant and earlier mucosal antigen presentation indicates that specific immune response occurs in local mucosal tissues.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

Influence of dietary biotin levels on growth and non‐specific immune response in large yellow croaker,Larimichthys crocea R

A 9‐week feeding experiment was conducted to determine the effect of dietary biotin levels on growth performance and non‐specific immune response of large yellow croaker. Fish (6.16 ± 0.09 g) were fed twice daily to apparent satiation with diets containing 0.00 (as the basal diet), 0.01, 0.05, 0.25, 1.24 and 6.22 mg biotin kg^?1 diet. Results showed that fish fed the basal diet had the lowest survival rate, and fish fed 0.05 mg kg^?1 dietary biotin achieved significantly higher final weight and weight gain. Dietary biotin levels had no significant influence on carcass crude lipid, moisture and ash content, but significantly influenced the carcass crude protein. Liver biotin concentration significantly increased with the supplementation of biotin, but no tissue saturation was found within the supplementation scope of biotin. Broken‐line regression analysis of weight gain showed that juvenile large yellow croaker requires a minimum dietary biotin of 0.039 mg kg^?1 for maximal growth. The analyses of serum parameters showed that the moderate‐ (0.05 mg kg^?1) and high‐dose (6.22 mg kg^?1) dietary biotin significantly improved both lysozyme and alternative complement pathway activities, indicating dietary biotin within a certain range could improve the non‐specific immune response of large yellow croaker.     

Effects of dietary astaxanthin on growth,blood biochemistry,antioxidant, immune and inflammatory response in lipopolysaccharide‐challenged Channa argus

The present study investigated the effects of dietary astaxanthin on the growth, blood biochemical, antioxidant, immune and inflammatory response in lipopolysaccharide‐challenged Channa argus. A total of 0, 50, 100 and 200 mg/kg of astaxanthin were added to the basal die for 56 days. After the feeding experiment, each group was subjected to a lipopolysaccharide challenge (except for the Control group). The results showed that adding astaxanthin to the diet can significantly increase the weight gain and specific growth rate and decrease the feed conversion ratio of C. argus; the highest weight gain, specific growth rate and minimum feed conversion ratio occurred in the 100 mg/kg group. Furthermore, dietary astaxanthin supplementation can alleviate the negative effects of lipopolysaccharides by increasing the levels of alkaline phosphatase, lysozyme, complement 3, complement 4, total serum protein, albumin, globulin, superoxide dismutase, catalase and glutathione peroxidase and decrease the serum cortisol, aspartate aminotransferase, alanine aminotransferase, glutathione peroxidase, and malondialdehyde. Dietary astaxanthin supplementation also can decrease the relative expression of inflammatory genes (nuclear factor κB, interleukin‐1, interleukin‐8 and tumour necrosis factor‐α) in the liver, spleen, kidney and intestine. To summarise, dietary astaxanthin addition can improve the growth performance and attenuate the negative effects of lipopolysaccharide challenge in C. argus. The optimal amount of astaxanthin is 100 mg/kg.     

The effects of Coriandrum sativum L. as feed additive on mucosal immune parameters,antioxidant defence and,immune‐related genes expression in zebrafish (Danio rerio)

This study investigates the effects of Coriander (C. sativum) on the growth, antioxidant, and immune‐associated genes and serum and mucosal immune parameters in zebrafish (D. rerio). The experimental fish were treated with 0 [control], 5, 10 and 20 g/kg coriander supplemented diets for 8 weeks. The results revealed that coriander remarkably increased mucosal immune parameters (the total Immunoglobulin, protease and lysozyme activity). The highest levels of the measured parameters were noticed in 20 g/kg CP treatment. Also, the zebrafish fed 20 g/kg coriander powder showed significantly higher expression gene for lysozyme and TNF‐alpha. The same results were noticed in case of IL‐1B gene expression. In case of sod gene expression there was no significant difference between treatments. However, regarding cat gene expression, significant difference was noticed between 20 g/kg CP treatment and other groups. In addition, no significant changes were observed between coriander fed zebrafish and control treatments regarding GH gene expression level, although IGF‐I remarkable up‐regulate in 20 g/kg coriander powder treatment compared other groups. In conclusion, it can be proposed that dietary Coriander powder can improve mucosal immune parameters and immune and antioxidant genes expression.     

Dietary histidine affects intestinal antioxidant enzyme activities,antioxidant gene expressions and inflammatory factors in juvenile blunt snout bream (Megalobrama amblycephala)

Six semipurified diets of graded histidine levels (from 4.1 to 14.2 g/kg) were fed to fish for 8 weeks. The results showed that the intestinal content of malondialdehyde (MDA), activities of total superoxide dismutase (T‐SOD) and glutathione peroxidase (GPx) were noticeably lower in 8.0 g/kg diet compared to control group. While the lowest activities of copper–zinc superoxide dismutase (Cu/Zn‐SOD) and manganese superoxide dismutase (Mn‐SOD) were observed in 9.9 g/kg diet, the intestinal activities of total antioxidant capacity (T‐AOC) and catalase (CAT) in 14.2 g/kg diet were noticeably higher than those in 8.0 and 9.9 g/kg diets. Plasma biochemical indexes were not significantly affected by dietary histidine levels. In the intestine, the Kelch‐like ECH‐associated protein 1 (Keap1) mRNA levels were increased in 8.0 g/kg diet, which suppressed the nuclear translocation of nuclear factor E2‐related factor 2 (Nrf2), and subsequently decreased CAT, GPx1, Cu/Zn‐SOD and Mn‐SOD expression levels. The lowest mRNA levels of interleukin 8 (IL‐8) and tumour necrosis factor‐α (TNF‐α) were observed in 8.0 g/kg diet, whereas the highest mRNA levels of transforming growth factor‐β (TGF‐β) and interleukin 10 (IL‐10) were observed in 8.0 g/kg diet. These results indicated that dietary histidine plays a major role in maintaining intestinal health in juvenile blunt snout bream.     

Possible effect of lesser galangal (Alpinia officinarum) extracts encapsulated into mesoporous silica nanoparticles on the immune status of rainbow trout (Oncorhynchus n)

Using plant extracts as immunostimulants in aquaculture has prove effectiv in resisting infectious disease, in addition to being safe and inexpensive. The present study is concerned about the prospective mode of action of using lesser galangal extract (Alpinia officinarum) encapsulated into amine surface functionalized mesoporous silica nanoparticles (MSN) in order to elevate the immune status of rainbow trout (Oncorhynchus mykiss). Fish were randomly divided into 6 groups and fed for 4 weeks with commercial diets supplemented with 0% (control), 0.5% and 1.5% of lesser galangal extract, and with previous concentrations encapsulated with MSN (0.5% + MSN, 1.5% + MSN) and MSN (without extract). The effects of the feeding trial on the innate humoural immune parameters (total protein, myeloperoxidase content, lysozyme antiproteases and bactericidal activities) in rainbow trout were examined. Also, the influence of dietary supplement on some immune‐related genes in rainbow trout head kidney (TNF, IL‐8, IL‐1b, LYZ2 and CD4) after challenge with Yersinia ruckeri was examined. The results revealed enhancement in immune parameters in all treatment groups compared to the control, especially in the fish group fed with 1.5% + MSN which showed the highest significant difference (p < .05) in total protein, lysozyme and antiproteases activities. Also, feeding lesser galangal extract encapsulated into MSN led to an increase in myeloperoxidase content and bactericidal activity. An improvement in the expression of immune‐related genes has been recorded in fish groups fed doses of lesser galangal extract and lesser galangal extract encapsulated into MSN compared to the control or to the group fed with MSN only. Particularly, the group fed with 0.5% + MSN showed a significant up‐regulation in most of the immune‐related genes. The current investigation supports using lesser galangal extract encapsulated into MSN in fish diets as a supplement to enhance the immune response of rainbow trout and elevate its resistance to infectious diseases.     

Effects of glutamate in low‐phosphorus diets on growth performance,antioxidant enzyme activity,immune‐related gene expression and resistance to Aeromonas hydrophila of juvenile mirror carp (Cyprinus carpio)

This study investigated the effects of glutamate (Glu) in low‐phosphorus diets on growth performance, haematological indices, antioxidant enzyme activity, immune‐related gene expression and resistance to Aeromonas hydrophila in juvenile mirror carp (Cyprinus carpio) (5.07 ± 0.02 g). Fish were fed either graded levels of Glu (0 g/kg, 5 g/kg,  10 g/kg and 20 g/kg, named G0, G0.5, G1 and G2, respectively) in a low‐phosphorus diet (15 g/kg NaH₂PO_4, 0.49), or a normal phosphorus diet ( 20 g/kg NaH₂PO_4, 0.61) without added Glu (C), for 8 weeks. At the end of the feeding trial, the fish were challenged with A. hydrophila. Compared with G0 group, 10 g/kg and 20 g/kg Glu supplementation of the low‐phosphorus diet significantly improved the final weight, WGR, SGR and PER, and decreased FCR (p < .05). Glu supplementation of the low‐phosphorus diet significantly enhanced the T‐AOC, SOD activity and GSH content in intestine (p < .05). Glu supplementation significantly reduced MDA content in foregut and midgut and increased CAT activity in midgut and hindgut (p < .05). Regarding immune‐related gene expression, Glu supplementation significantly diminished the up‐regulation of intestinal TNF‐α, IL‐1β and IL‐8 mRNA levels induced by phosphorus deficiency (p < .05). The survival rate of the G1 group was significantly higher than that of the G0 group (p < .05). In conclusion, 10 g/kg Glu supplementation in low‐phosphorus diets can improve the growth performance, enhance the activity of intestinal antioxidant enzymes and strengthen the immune function of juvenile mirror carp.     

The effects of dietary Eucommia ulmoides Oliver on growth,feed utilization,antioxidant activity and immune responses of turbot (Scophthalmus maximus L.)

The present study was conducted to evaluate the effects of dietary Eucommia ulmoides (EU) on growth, feed utilization, antioxidant activity and immune responses of turbot fed a basal diet (CON) or EU‐supplemented diets with 5.0 g/kg (EU1), 10.0 g/kg (EU2) and 20.0 g/kg (EU3) EU leaf powder. After 70‐day trial, EU supplementation did not affect nutrient utilization, but reduced feed intake (FI) and specific growth rate (SGR) of fish at doses above 5.0 g/kg. Superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T‐AOC) activities in the EU‐supplemented groups were significantly higher than those in the control group at a dose of 20.0 g/kg. Significantly reduced malondialdehyde (MDA) contents were observed in EU‐supplemented groups at doses over 5.0 g/kg. Furthermore, fish fed 20.0 g/kg EU showed the highest lysozyme (LZM) activity among groups. The EU‐supplemented diets with doses above 5.0 g/kg significantly enhanced the mRNA expressions of cytokines. The expression level of major histocompatibility complex II alpha (MHC IIα) was significantly upregulated compared to that of the control fish when the supplemental level was at 20.0 g/kg. Taken together, the present study indicated that the EU could remarkably enhance the antioxidant activity, non‐specific immunity and maintain an active immune response in turbot.     

大黄鱼ATG5基因的分子特征及促进病毒增殖的作用研究

自噬是维持真核细胞稳态的重要过程，在细胞分化、发育、免疫等生理过程中发挥作用。目前，人们对于自噬相关基因（Autophagy related gene，ATG）在鱼类免疫应答中的功能仍知之甚少。本研究从大黄鱼（Larimichthys crocea）中克隆得到了自噬相关基因ATG5（LcATG5），其开放阅读框（ORF）全长828个核苷酸，编码275个氨基酸的蛋白质，预测的分子量为32.3 kD，等电点为5.7。氨基酸序列比对和系统进化分析结果显示，LcATG5与其他物种ATG5之间的序列一致性较高，含有1个高度保守的APG5结构域，并且与棘头梅童鱼（Collichthys lucidus）ATG5的亲缘关系最近。荧光定量PCR结果表明，LcATG5在所检测的11个组织或器官中均有表达，在血液中表达量最高，在脾脏中表达量最低；LcATG5在来源于大黄鱼头肾组织的原代粒细胞、淋巴细胞和巨噬细胞以及细胞系LYCK细胞中也均有表达，在原代粒细胞中表达量相对较高，而在巨噬细胞中相对较低；病毒类似物poly(I:C)刺激后，这4种免疫细胞中LcATG5的表达水平都显著上调，其在LYCK细胞中变化最为显著，刺激后12 h上调了3.93倍。过表达LcATG5的鲤上皮瘤（Epithelioma papulosum cyprinid, EPC）细胞受鲤春病毒血症病毒（Spring Viremia of Carp Virus, SVCV）感染48 h后，细胞病变效应（Cytopathic effects, CPE）明显高于对照组，细胞培养上清中SVCV的滴度为1013.82 TCID50/Ml，高于对照组109.27 TCID50/mL，同时细胞内SVCV标志基因SVCV-G、SVCV-M和SVCV-P的表达量分别上调了13.77倍、15.72倍和11.39倍，表明LcATG5过表达促进了EPC细胞中SVCV病毒增殖，这些结果为深入研究自噬和自噬相关基因在鱼类病毒感染过程中的作用及机制奠定了基础。     

大黄鱼碱性磷酸酶基因的克隆及表达特征分析

碱性磷酸酶(alkaline phosphatase,ALP)是一种广泛分布的单脂磷酸水解酶,是动物生物代谢过程中重要的调控酶。实验首次克隆了大黄鱼ALP基因cDNA全序列,命名为LcALP,其全长为2 345 bp,开放阅读框为1 629 bp,编码543个氨基酸。实时荧光定量PCR检测发现,LcALP在大黄鱼雌雄各组织中均有表达,其中在眼和头肾的表达量最高,雌性鳃和脑中LcALP表达量显著高于雄性,而在性腺和脾脏中的表达量则显著低于雄性;胚胎发育过程中,多细胞期、囊胚期和原肠期LcALP基因表达水平相对较低,在卵黄栓形成期明显提高,至孵出期达到峰值,而初孵仔鱼期则显著下调;大黄鱼肌肉细胞系经LPS处理后6 h LcALP表达量降低,12 h显著下调,而poly I:C处理后表达水平持续上升,24 h显著上调至峰值。研究表明,LcALP在大黄鱼胚胎发育调控以及机体免疫防御中发挥重要作用。