Molecular characterization and expression analysis of heat shock proteins 40, 70 and 90 from kuruma shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Heat shock proteins (HSPs) are proteins that are expressed more strongly when the cells are exposed to physiological and stressful conditions. In this study, the full-length cDNAs of heat shock proteins 40 (MjHSP40), 70 (MjHSP70) and 90 (MjHSP90) were cloned from kuruma shrimp Marsupenaeus japonicus. The open reading frames (ORFs) of the cDNA clones have lengths of 1,191, 1,959 and 2,172 bp and encode 396, 652 and 723 amino acid residues, respectively. The predicted MjHSP40 amino acid sequence contains a J domain, a glycine/phenylalanine-rich region, and a central domain containing four repeats of a CxxCxGxG motif, indicating that it is a type I HSP40 homolog. The signature sequences of the HSP70 and HSP90 gene families are conserved in the MjHSP70 and MjHSP90 amino acid sequences. The deduced amino acid sequences of MjHSP70 and MjHSP90 share high identity with previously reported shrimp HSP70s and HSP90s, respectively. The expression of MjHSP90 mRNA increased at 32°C. Additionally, the expressions of MjHSP40, MjHSP70 and MjHSP90 mRNAs increased in defense-related tissues (i.e., hemocytes and lymphoid organ) when the shrimp were challenged with white spot syndrome virus.     

Dietary supplementation with taurine improves ability to resist ammonia stress in hybrid snakehead (Channa maculatus♀ × Channa argus♂)

This study determined the effect of dietary supplementation with taurine on plasma biochemical indices, blood cell apoptosis rate, survival rate and expression of catalase (CAT), glutathione reductase (GR) and heat‐shock protein 70 (HSP70) gene in juvenile hybrid snakehead under ammonia stress. Six diets were formulated to contain 0, 3.0, 6.0, 9.0, 12.0 and 15.0 g/kg taurine. Each diet was fed to triplicate groups of fish in cylindrical tanks. After 8 weeks of feeding, 20 fish per tank were exposed to ammonia stress (total ammonia nitrogen = 200 mg/L) for 48 hr. The results showed that, after ammonia stress, plasma alkaline phosphatase (ALP), triacylglycerol (TG) levels and survival rate significantly decreased (p < 0.05) while plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), respiratory burst, blood cell apoptosis rate, hepatic CAT, GR and HSP70 mRNA expression levels significantly increased (p < 0.05). On the other hand, dietary supplementation with taurine significantly reduced levels of BUN, respiratory burst and blood cell apoptosis rate. Supplemented groups significantly increased relative mRNA expression of hepatic CAT, GR and HSP70 as well as increased survival rate (p < 0.05). These results indicated that dietary supplementation with taurine improved ability to resist ammonia stress in hybrid snakehead.     

HSP90抑制剂根赤壳菌素对斑马鱼胚胎发育的影响

为研究热休克蛋白90(heat shock protein 90,HSP90)在斑马鱼(Danio rerio)胚胎发育中的作用,本实验采用2μmol/L、5μmol/L、10μmol/L的HSP90抑制剂根赤壳菌素(radicicol)对斑马鱼发育期胚胎进行处理,监测斑马鱼胚胎不同发育时期两个HSP90功能抑制标志基因BAG3(BCL2-associated athanogene3)和HSPB1(heat shock protein beta-1)的mRNA的表达水平,并观察不同发育时期的胚胎发育状况。结果如下:(1)实时荧光定量PCR结果显示,BAG3和HSPB1 mRNA水平在根赤壳菌素处理胚胎12 hpf(hours post-fertilization,hpf)或24 hpf后显著增高,Western blot检测到5μmol/L根赤壳菌素处理胚胎24 hpf后HSP70表达上调,证明该实验条件下HSP90功能受到抑制;(2)根赤壳菌素处理后斑马鱼胚胎发育变缓,胚胎成活率统计显示:2μmol/L、5μmol/L、10μmol/L根赤壳菌素处理24 hpf胚胎成活率分别是95%、77%、35%,成活率随根赤壳菌素浓度的增高而降低;(3)5μmol/L根赤壳菌素处理72 hpf可见部分个体色素沉积减少、心包膜增大、肌肉萎缩等发育畸形。研究结果证明HSP90在斑马鱼胚胎发育过程中发挥重要的作用,根赤壳菌素在5μmol/L时已达到较佳抑制效果且成活率较高,而且胚胎发育出现了各种形态学变化,为后期研究HSP90调节斑马鱼胚胎发育奠定了基础。     

Haemato-biochemical responses and induction of HSP70 to dietary phosphorus in <Emphasis Type="Italic">Catla catla</Emphasis> (Hamilton) fingerlings

A feeding trial of 120 days was conducted to study the effect of graded levels of dietary phosphorus on haematology, serum protein concentrations and HSP70 expression in fingerlings of the Indian major carp, Catla (Catla catla). Eight isonitrogenous and isoenergetic purified diets were formulated to contain graded levels of dietary phosphorus (dP), i.e., T₁, 0.1%; T₂, 0.3%; T₃, 0.5%; T₄, 0.7%; T₅, 0.9%; T₆, 1.1%; T₇, 1.3%; or T₈, 1.5%. Four hundred and eighty fish (average weight 4.23 ± 0.016 g) were equally distributed into 24 tanks forming eight treatments with three replicates each. The fish were fed daily at the rate of 3.5% body weight in two instalments. At the end of feeding trial fish were sampled to study total RBC and WBC count, haemoglobin, serum lysozyme activity, serum total protein, albumin (A), globulin (G) concentration and HSP70 expression. Total RBC count, haemoglobin concentration and serum lysozyme activity did not vary significantly in response to different dietary phosphorus concentrations. Total WBC count was found to be significantly (P < 0.05) higher in T₁ relative to all other treatments. Serum albumin and A/G ratio was found to be significantly lower in fish of T₁ and T₂ in relation to T₇ group (P < 0.05). Serum globulin and total protein levels remained unaffected by variations in dietary phosphorus. HSP70 expression was observed in T₁ group (0.1% dP) in gills and brain tissue, but not in liver and muscle tissues. No HSP70 expression was observed in fish of T₄ (0.7% dP) and T₈ (1.5% dP) treatments. These prima facie results suggest that dietary phosphorus had only minor influence on the haemato-biochemical parameters studied; however dietary phosphorus deficiency caused organ specific induction of HSP70 in catla fingerlings.     

Pectin purified from pomelo (Citrus maxima) peel as a natural hatching agent for fish embryos

Pectin is a biodegradable polysaccharide, and it has been recently applied as a gene delivery, drug delivery, wound healing and tissue engineering agent. In this study, pectin was extracted from pomelo (Citrus maxima) peel and characterized. The extraction recovery of pectin form pomelo peel was 14.5%, and it had 72.56% degree of esterification, 1,245.56 equivalent weight, 7.82% methoxyl and 68.27% anhydrouronic acid contents. Use of pomelo pectin as a hatching enhancing agent for fish embryos and its effect on hatching enzyme 1 (ZHE1) was investigated. The pectin‐exposed zebrafish embryos (100 µg/ml) showed significantly (p < .05) higher hatching rate (96.6%) compared with untreated (control) embryos (66.6%) at 60 hpf. The mRNA expression of ZHE1 was also significantly (p < .05) elevated up to 55.6‐fold in pectin‐exposed embryos at 24 hpf. In situ hybridization results revealed remarkably strong expression of ZHE1 in pectin‐exposed embryos compared with the control group. In addition, considerably larger size of the hatching gland was observed in pectin‐exposed larvae than that of the unexposed larvae group. These results clearly indicate that pectin isolated from pomelo peel has an ability to enhance the hatching process of zebrafish embryos via upregulation of ZHE1.     

New type of heat shock protein 70 homologue gene abounds in the genomic sequence of kuruma shrimp Marsupenaeus japonicus

Heat shock proteins (HSPs) are a group of highly conserved molecular chaperones. Shrimp HSPs have recently been a topic of increasing interest because of their roles in shrimp immunity and homeostasis. In penaeid shrimp, HSP70s and the cognate forms, heat shock cognate (HSC) 70s, have been reported, but their responses towards various stimulations are different. We found a novel type HSP70 (MjHSP70-2) from the hyperexpansion of the large segmental duplication that is present in kuruma shrimp Marsupenaeus japonicus, which shows about 60 % identity with reported shrimp HSP70s. In a phylogenetic tree, MjHSP70-2 formed a sister clade with eukaryote HSP70 family while MjHSP70 was located close to the shrimp HSP70 and HSC70 group. MjHSP70-2 gene expression was not significantly increased by heat shock or pathogen challenge by Vibrio penaeicida, but it was significantly increased by infection with white spot syndrome virus. In contrast, MjHSP70 gene expression was increased by heat shock but decreased by infection with V. penaeicida. The kuruma shrimp genome was found to have 400-fold more copies of the MjHSP70-2 gene than the putative single-copy gene transglutaminase. In conclusion, our results reveal the presence of a novel type HSP70 gene, HSP70-2, from kuruma shrimp. There are multiple forms of HSP70 in crustaceans, and these HSP70s behave differently under various stressors.     

EDCs‐induced glucocorticoid receptor related genes expression of the river pufferfish,Takifugu obscurus

Endocrine disrupting chemicals (EDCs) are of substantial concern when contaminating waters. We studied gene expression of larvae from river pufferfish, Takifugu obscurus exposed to EDCs. We cloned the full‐ or partial‐length cDNA sequence of genes related to the cortisol pathway, such as the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), heat shock protein (HSP) 70 and 90, phosphoenolpyruvate carboxykinase (PEPCK) and the FK506‐binding protein 4 (FKBP52). The tissue and time‐dependent expression of mRNA was studied by real‐time PCR in T. obscurus exposed to two representative EDCs (bisphenol‐A and 4‐tert‐octylphenol). HSP70, HSP90, MR and FKBP52 mRNA expression level was much higher than GR and PEPCK expression. GR mRNA expression level was significantly upregulated after 48 h in both EDC‐treated fish groups compared with the control. Both groups also showed down‐regulation patterns after 48 h. The initial expression of PEPCK in both groups was down‐regulated after 24 h. In the bisphenol‐A treated group, the expression of FKBP52 showed substantially high up‐regulation from 6 to 48 h after exposure, and then strikingly reduced its expression level at 72 h. Our results provide insights into the EDCs metabolizing system of T. obscurus and offers baseline information for further research related to the possible use as a bio‐indicator of this commercially important aquaculture fish species.     

Physiological responses and HSP70 mRNA expression in GIFT tilapia juveniles,Oreochromis niloticus under short‐term crowding

Physiological responses and HSP70 mRNA expression to short‐term crowding were tested in juveniles genetically improved farmed tilapia (Oreochromis niloticus) (70.43 ± 4.43 g). Fish were kept at control group (5 g L^?1) and stress groups (low density 10 g L^?1, medium density 40 g L^?1 and high density 70 and 100 g L^?1) for 48 h. Each density was tested in triplicates. The effects of a short‐term exposure on the physiological responses of fish were determined before stress (0 h) and at 6, 12, 24 or 48 h post‐crowding. There was a significant increase in serum cortisol, total protein, lysozyme and aspartate aminotransferase (AST) activities in all stressed groups at 24 h post‐crowding (P < 0.05). Serum glucose and alanine aminotransferase (ALT) of 100 g L^?1 stressed group were lower than that of the control after 48 h of crowding stress (P < 0.05). The levels of serum triglyceride and cholesterol in crowding stress with high density were significantly decreased compared with the control group at 48 h. The mRNA expression data showed that hepatic HSP70 mRNA levels were markedly elevated at all stressed groups. HSP70 mRNA levels of 70 and 100 g L^?1 stressed groups decreased at 48 h compared with the 24 h post‐crowding. The protective ability of HSP70 was limited.     

Dietary oils influence ovary and carcass composition and embryonic development of zebrafish

To evaluate the effect of different dietary lipids on zebrafish reproduction, we examined parameters including ovary and carcass composition, oestradiol (E2) levels, eclosion rate and embryonic development. In our study, zebrafish were subjected to a 5‐month feeding trial whereby olive (OLV), linseed (LIN), fish (FIS) or corn (CRN) oil were used to compose four isonitrogenous and isoenergetic diets. A positive correlation was found between ovary EPA and whole‐body E2 concentration (P = 0.005). The developmental dynamics was affected by dietary lipids and embryos from CRN treatment females; which had higher percentage of ARA in ovary (1.48 ± 0.44%) (P = 0.015), developed faster at 8–9 h postfertilization (hpf) than the ones originated from females receiving other diets (P = 0.0069). This effect was not sustained during later observation periods, suggesting ARA may act as a modulator of developmental dynamics only during initial phases. There was no clear effect of dietary lipid source on ovary protein content (P = 0.304) and eclosion rates at 72 hpf (P = 0.0623). Dietary fatty acids play an important role in reproductive outcome; however, additional studies are necessary to elucidate the mechanisms by which HUFA affect embryonic development.     

Ex situ and in situ measurements of juvenile yellowfin tuna <Emphasis Type="Italic">Thunnus albacares</Emphasis> target strength

To provide target strength (TS) information for estimating the body length of yellowfin tuna Thunnus albacares and its abundance around fish aggregating devices, TS was measured ex situ and in situ. In the ex situ TS measurements, two cameras synchronized with a 200 kHz echosounder were used to obtain the precise orientation of the yellowfin tuna under free swimming conditions. The ex situ TS (dB re 1 m²)–fork length (FL, cm) regression was: TS = 27.06 log (FL) − 85.04. Ex situ TS was found to reach its maximum in the tilt angle range of −15° to −20° after excluding TS samples with insignificant correlation to the tilt angle. The angle between the vertebra and the swim bladder was approximately 25° according to X-ray images, supporting the above tilt range. The relationship between the swim bladder volume (V _SB, ml) and the fork length was: V _SB = 0.000213 FL³. The results from the in situ TS measurements indicated that the tilt angle was highly concentrated between −10° and 15°. The results from a calculation using the ex situ TS–FL equation with the fork length from biological sampling agreed strongly with the average in situ TS.     

Critical role of connexin43 in zebrafish late primitive and definitive hematopoiesis

In vitro studies have suggested that connexin43 (cx43) expression is of particular importance during establishment and regeneration of the mammalian hematopoietic system. However, little is known about its in vivo functions during hematopoiesis due to the embryonic lethality of mammalian knockout models. In this study, we observed that zebrafish cx43 is not only expressed in the eyes, cerebellum, heart, and vasculature, but also expressed, albeit at low levels, in intermediate cell mass (ICM, the primitive hematopoietic site). Knockdown of cx43 leads to vacuolization in the wedge of the ICM and an apparent reduction in the number of circulating blood cells, but does not affect their cellular morphology. Whole-mount in situ hybridization analysis revealed that the hemangioblastic marker flk-1 and the primitive hematopoietic markers lmo2 and scl are basically maintained at normal levels in cx43 morphant embryos at 12–13 h postfertilization (hpf) compared with the con-MO injected embryos. However, subsequent expression of the definitive hematopoietic stem cell (HSC) marker c-myb was severely downregulated in the ventral wall of the dorsal aorta of cx43-depleted embryos at 36 hpf. Furthermore, we confirmed this phenotype by injection of cx43-MO into Tg(gata1:EGFP) embryos. Together, our results show that cx43 contributes to late primitive and definitive hematopoiesis in zebrafish embryos.     

Effects of dietary proline on swim bladder collagen synthesis and its possible regulation by the TGF‐β/Smad pathway in spotted drum,Nibea diacanthus

Swim bladder is an ideal source of collagen production in fish for improved human health. Proline (Pro) is the main proteinogenic amino acid needed for collagen production. However, the effects of Pro supplementation on the swim bladder collagen synthesis have rarely been evaluated in fish. We determined the effects of dietary Pro supplementation on swim bladder collagen synthesis and its possible signalling pathway in spotted drum, Nibea diacanthus. A total of 450 N. diacanthus (100 ± 3.05 g) were randomly assigned into six treatments and fed with diets supplemented with different levels of Pro (0, 2.5, 5, 7.5, 10 and 12.5 g/kg of dry diet, hereafter P0, P1, P2, P3, P4 and P5, respectively) for 8 weeks. At the end, we evaluated collagen synthesis in swim bladder and the expression of genes related to TGF‐β/Smad pathway in the fish. Dietary Pro levels increased significantly the contents of crude protein, total collagen (TC) and the levels of some amino acids in swim bladder than the control diet (p < .05). The optimum amount of dietary Pro inclusion in diets for swim bladder collagen synthesis in N. diacanthus was 7.6 and 7.5 g/kg based on crude protein and TC in swim bladder, respectively. Dietary Pro levels increased significantly the proline 4‐hydroxylase (P4H) content in fish serum, swim bladder, muscle and liver tissues than control (p < .05). The relative expression of collagen type I alpha 1 (COL1A1), alpha 2 (COL1A2) and mothers against decapentaplegic homolog 2 (Smad2) genes in liver and swim bladder initially increased significantly as the concentration of Pro and later decreased (p < .05). Similarly, the relative expression of transforming growth factor beta (TGF‐β), P4Ha2 and P4Ha3 genes in the swim bladder increased significantly as dietary Pro levels increased (p < .05). Using K‐means clustering analysis, dietary proline partly promoted collagen accumulation in swim bladder through upregulation of Smad2 and TGF‐βRT genes. Taken together, Pro affected the collagen metabolism in swim bladder, probably by regulating the TGF‐β/Smad pathway, most likely via transient overexpression of Smad2 gene.