Ecological influences on the difference in δ15N and δ13C values between fish tissues: implications for studies of temporal diet variation

Temporal diet changes can be detected by comparing δ¹³C and δ¹⁵N values between tissues with different isotopic turnover times. However, other factors contribute to disparities in δ¹³C and δ¹⁵N signatures between tissues and could confound the interpretation of stable isotope data. We examined the effects of ecological factors on differences in muscle and liver δ¹³C and δ¹⁵N signatures of Arctic charr [Salvelinus alpinus, (L.)] to determine whether their effects were large enough to influence studies of diet change. In addition, we examined whether differences in lipid content accounted for a significant portion of the differences in δ¹³C values between tissues. Results indicated that life history, diet, reproductive status and gender had significant effects on the differences in δ¹³C values between tissues, while gender and diet significantly affected differences in δ¹⁵N values. Differences in % lipid content between tissues also explained approximately 74% of the observed variation in the difference in δ¹³C values between tissues. The differences in δ¹³C values observed between muscle and liver tissues may be partially explained by physiological differences in lipid storage and use between tissues associated with ecological factor effects. These results demonstrate that ecological factors other than a change in diet can influence the differences in δ¹³C and δ¹⁵N signatures between muscle and liver tissues. However, the impact of ecological factors on studies of diet change depends on how the magnitude of the factor effects compared with a biologically meaningful change in diet.     

The use of stable isotope ratios δ13C and δ15N to track the incorporation of Ulva and other important dietary ingredients into the gonads of the sea urchin Tripneustes gratilla

In this study, we demonstrate how stable carbon (C) and nitrogen (N) isotope analysis can be used to estimate the allocation of specific dietary ingredients, incorporated into four artificially formulated feeds supplemented with varying amounts of dried seaweed Ulva armoricana (0, 50, 150 and 200 g/kg), to the production of gonads by Tripneustes gratilla over a 20‐week growth trial. Results indicate that U. armoricana is an important dietary ingredient for gonad production, accounting for an average of 37.33 ± 1.03% of the isotopic signal recorded for gonads at the end of the growth trial. This is significantly more than fish meal and soya, which only contributed an average of 11.40 ± 1.81 and 3.80 ± 0.09%, respectively, of the isotopic signal of the gonads. Maize and wheat each account for almost 25% of all the dietary ingredients used to formulate the feeds, but their relative contribution to gonad production only averaged 8.55 ± 0.58 and 3.80 ± 0.05%, respectively, among the diets. This indicates that, at least for gonad development, these ingredients may not be required at such high levels. These findings support previous data on the importance of Ulva in abalone and urchin diets, and demonstrate the value of stable isotope analysis for assessing the contribution of specific dietary ingredients in new feed formulations, particularly where growth of specific tissues is being investigated.     

The primary culture and subculture of lymphoid cells from shrimp,Penaeus chinensis on thermo‐sensitive CS/α, β‐GP hydrogel

A thermo‐sensitive hydrogel composed of chitosan and α, β‐glycerophosphate was successfully synthesized and used as basic substrate for primary culture and subculture of lymphoid cells from shrimp, Penaeus chinensis. When the ambient temperature increases from 4°C to 25°C, the chitosan/α, β‐glycerophosphate solution transforms into a semi‐solid three‐dimensional hydrogel with a rough surface. Primary lymphoid cells formed were round and grew homogenously over the surface of the hydrogel. The peroxidase activity indicated that cells seeded on the hydrogel suffered less stress than cells suspended in control culture flasks. The higher relative viability and alkaline phosphatase activity of primary cultured lymphoid cells indicated that the chitosan/α, β‐glycerophosphate hydrogel enhanced the growth and proliferation of lymphoid cells. Subsequent subculture of lymphoid cells starting from crumbled primary hydrogel culture was also successful.     

A3α‐peptidoglycan extracted from Bifidobacterium sp. could enhance immunological ability of Apostichopus japonicus

To assess the effects of A3α‐peptidoglycan (A3α‐PG) extracted from Bifidobacterium sp. on the immune response and disease resistance of sea cucumber, different concentrations (0, 0.5, 5 and 50 mg mL^?1) of A3α‐PG suspensions were used to perform hypodermic injection on Apostichopus japonicus, followed by a Vibrio splendidus challenge. Total coelomocyte count (TCC), phagocytosis activity and activities of four immunological enzymes in both cell‐free coelomic fluid (extra‐cellular, EC) and coelomocyte lysate supernatant (intracellular, IC), including acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD) and peroxidase (POD), were measured at 2, 6, 14 and 24 h post injection (hpi). The TCC was not significantly affected (P > 0.05) by A3α‐PG, ranging from 1.84 × 10⁶ to 3.53 × 10⁶ cells mL^?1. The coelomocyte phagocytosis activity was significantly activated (P < 0.05) in all the A3α‐PG treatments, whereas no significant difference was observed between them except 24 hpi (P > 0.05). The EC‐ACP activity in the 5.0 mg mL^?1 treatment increased significantly (P < 0.05) at all sampling times, while the IC‐ACP activity in the 50 mg mL^?1 treatment increased significantly (P < 0.05) at 2 hpi. Also, the 5.0 mg mL^?1 treatment had significant (P < 0.05) increase in the EC‐ALP activity within 14 hpi and the EC‐POD activity at 2 hpi, respectively, while significantly (P < 0.05) enhanced IC‐ALP and IC‐POD activities were observed in the 50 mg mL^?1 treatment within 6 hpi and at 2 hpi, respectively. Only the 5.0 mg mL^?1 treatment showed significant (P < 0.05) increase in the EC‐SOD activity at 2 hpi and IC‐SOD activity within 14 hpi, respectively. The challenge test showed that the animals treated with 50 mg mL^?1 of A3α‐PG had notably lower cumulative mortality after 14 days following V. splendidus exposure. All together, these results suggest that A3α‐PG could positively enhance immune response that effectively promotes the health status of A. japonicus against V. splendidus infection.     

Effects of dietary supplementation with β‐glucan and synbiotics on growth,haemolymph chemistry,and intestinal microbiota and morphology in the Pacific white shrimp

The goal of this study was to investigate the effects of dietary supplementation with β‐glucan and microencapsulated probiotics (Bacillus subtilis or Pediococcus acidilactici) on growth performance, body composition, haemolymph constituents, and intestinal morphology and microbiota of the Pacific white shrimp Litopenaeus vannamei. Four treatment diets [basal diet (C), β‐glucan‐containing diet (β‐glu), β‐glucan plus B. subtilis‐containing diet (β‐glu+Bs), and β‐glucan plus P. acidilactici‐containing diet (β‐glu+Pa)] were fed to L. vannamei for 90 days. Shrimp fed the β‐glu and β‐glu+Pa diets exhibited similar growth performance and body protein content, which were significantly higher than those of shrimp fed the control diet (P < 0.05). No significant differences in haemolymph triglyceride, cholesterol, protein, haemolymph urea nitrogen or chloride were detected among the experimental diets. However, dietary β‐glucan alone increased the haemolymph glucose level and osmolarity (P < 0.05). Synbiotic supplementation had greater effects on intestinal microbiota and morphology than dietary β‐glucan alone. For example, β‐glu+Bs increased the number of intestinal lactic acid bacteria and decreased the number of Vibrio spp. (P < 0.05), and β‐glu+Pa increased the height of intestinal villi.     

Effects of γ‐aminobutyric acid supplementation on the growth performance,serum biochemical indices and antioxidant status of pharaoh cuttlefish,Sepia pharaonis

This study evaluated the effects of dietary γ‐aminobutyric acid (GABA) on the growth performance, serum biochemical indices and antioxidant status of pharaoh cuttlefish, Sepia pharaonis. Cuttlefish were cultured in open‐culturing cement pool systems for 8 weeks. Six practical diets supplemented with graded levels of GABA (0, 20, 40, 60, 80 and 100 mg/kg) were formulated. Each diet was randomly assigned to triplicate groups of 60 cuttlefish (mean weight: 10.33 g), the cuttlefish were fed two times per day to apparent satiation. The results showed that the specific growth rate (SGR), weight gain (WG) and feed efficiency (FE) significantly increased with dietary GABA supplementation (p < .05). The survival rate (SR) and protein content in muscle significantly increased when 58.9 mg/kg GABA supplied. Moreover, the nitric oxide (NO) content and acid phosphatase (ACP) activity in serum were significantly increased with dietary GABA supplementation (p < .05), while the activity of aspartate aminotransferase (AST) in serum decreased significantly when supplied with GABA at 58.9 mg/kg (p < .05). In addition, dietary GABA improved antioxidation activity by significantly increasing the activities of superoxide dismutase (SOD) and catalase (CAT) but decreasing malondialdehyde (MDA) levels in the liver and gill (p < .05). On the basis of the quadratic regression analysis of FE, the optimum content of dietary GABA in S. pharaonis was estimated to be 55.3 mg/kg. The findings of this study demonstrated that dietary GABA had a positive effect on the growth performance, serum biochemical indices and antioxidant status of S. pharaonis.     

Purification and characterization of a β–glucan binding protein from the haemolymph of freshwater prawn Macrobrachium rosenbergii

β‐glucan binding protein (βGBP), a pattern recognition protein was purified from the haemolymph of freshwater prawn Macrobrachium rosenbergii by heparin affinity chromatography that showed a single band in native gradient PAGE. The β‐glucan binding property of the purified protein was confirmed in a phenoloxidase (PO) assay, where addition of βGBP along with β‐glucan increased the specific PO activity compared with that of β‐glucan alone. The molecular weight of the βGBP was found to be ~316 kDa on gel filtration chromatography. In SDS‐PAGE, βGBP molecule was reduced to one polypeptide chain of molecular weight ~113 kDa. Thus the βGBP in M. rosenbergii is possibly a homotrimeric molecule. The purified sample run on unreduced condition in SDS‐PAGE also revealed a similar size band (~113 kDa) and hence, the polypeptide chains of βGBP are held by non‐covalent interactions. The purified βGBP samples run in native PAGE was stained positively with alcian blue for carbohydrates and Sudan black for lipids indicating the βGBP to be a glycolipoprotein. With rabbit polyclonal anti‐βGBP serum developed, an indirect ELISA was standardized and the normal βGBP concentration in adult M. rosenbergii serum was quantified to be ~2 mg mL^?1. Furthermore, the applicability of the developed ELISA is discussed.     

Effect of γ‐aminobutyric acid supplementation on growth performance,endocrine hormone and stress tolerance of juvenile Pacific white shrimp,Litopenaeus vannamei,fed low fishmeal diet

An 8‐week feeding trial was conducted to evaluate the optimum dietary γ‐aminobutyric acid (GABA) level in low fishmeal diet for juvenile Pacific white shrimp, Litopenaeus vannamei. Six practical diets (449 g kg^?1 protein, 87 g kg^?1 lipid) supplemented with graded levels of GABA (0, 50, 100, 150, 200, 250 mg kg^?1) were formulated. Each diet was randomly assigned to triplicate groups of 30 shrimps (approximately 0.44 g), and the shrimps were fed four times a day to apparent satiation. Weight gain and gain rate were significantly increased with the supplementation of GABA (P < 0.05). Hepatosomatic index and survival were also significantly influenced by the dietary GABA levels (P < 0.05) and show a similar trend to those of growth performance. Insulin and neuropeptide Y concentrations in blood were increased with the supplementation of GABA. In total, 150 mg GABA kg^?1‐supplemented diets significantly increased the survival after 12, 24 and 36 h of NH₃ stress, also increased the total antioxidant capacity, total superoxide dismutase, catalase, antihydroxyl radical and phenoloxidase activities before and after the 36 h NH₃ stress compared to basic group. These results clearly indicated that GABA could improve growth performance, antioxidative capacity and resistance against NH₃ stress in L. vannamei, and 150 mg kg^?1 GABA supplementation was suitable for L. vannamei fed with low fishmeal diet.     

Effects of β‐conglycinin on growth performance,antioxidant capacity and intestinal health in juvenile golden crucian carp,Carassius auratus

The objective of this study was conducted to research the effects of β‐conglycinin in the diets on the growth performance, immunity function, antioxidant capacity and intestinal health of juvenile golden crucian carp (Carassius auratus). Five diets contained respectively (0, 20, 40, 60 and 80 g/kg) β‐conglycinin, and were used to feed juvenile golden crucian carp for 56 days. Final weight, weight gain and specific growth rate were significantly reduced by dietary β‐conglycinin (20–80 g/kg). Feed efficiency and protein efficiency were significantly reduced by dietary β‐conglycinin (40–80 g/kg). In hepatopancreas, the activities of T‐SOD, ACP, ALT and T‐AOC were significantly suppressed by dietary β‐conglycinin (20–80 g/kg). The activities of LZM, AKP, CAT and GPx were significantly reduced by dietary β‐conglycinin (40–80 g/kg). The activities of protease were significantly reduced and the content of MDA was significantly increased by dietary β‐conglycinin (60–80 g/kg). In proximal intestines, the activities of protease and CAT were significantly decreased by dietary β‐conglycinin (40–80 g/kg). In mid and distal intestines, the activities of protease and CAT were significantly inhibited by dietary β‐conglycinin (20–80 g/kg). In intestines, T‐AOC and GPx were significantly declined by dietary β‐conglycinin (20–80 g/kg). In proximal and mid intestines, the content of MDA were significantly increased by dietary β‐conglycinin (40–80 g/kg). In distal intestines, the content of MDA was significantly increased by dietary β‐conglycinin (20–80 g/kg). The expression of IGF‐I was significantly decreased and the expression of IL‐1β and TNF‐α was significantly increased by dietary β‐conglycinin (20–80 g/kg). The structural integrity of intestinal tissues were damaged by dietary β‐conglycinin (20–80 g/kg), the part of intestinal villus were shed, the part of epithelial cells were separated from lamina propria. Ultimately, these results suggested dietary β‐conglycinin should be <20 g/kg in formula feed of golden crucian carp.     

Alleviating effects of β‐glucan in Oreochromis niloticus on growth performance,immune reactions,antioxidant, transcriptomics disorders and resistance to Aeromonas sobria caused by atrazine

Three hundred Oreochromis niloticus were divided into five groups; the control fish (CNT) were fed a basal diet, beta glucan (βG) group was fed 0.5 g/kg^?1 βG, atrazine (ATZ) group was exposed to 1/5 96‐hr LC₅₀ (1.39 mg/L) ATZ, the (βG/ATZ) group was fed βG while being exposed to ATZ, and the (βG then ATZ) group was supplemented with βG for fifteen days before exposed to ATZ. ATZ exposure caused a decline in growth that was ameliorated by βG. ATZ reduced the levels of total and different types of leucocytes. Additionally, ATZ exposure caused reductions in total proteins, globulins, α 1‐globulin, α 2‐globulin, ?‐globulin, immunoglobulin M, lysozymes, superoxide dismutase, nitric oxide and catalase but increases in hepatic transaminases and malondialdehyde without any variations in albumin and β‐globulin. Exposure to ATZ also resulted in a rise in the mRNA level of IL‐8. In contrast, expression of IgM, SOD and CAT were decreased in the tilapias exposed to ATZ. Exposure to ATZ increases the susceptibility response to Aeromonas sobria challenge, as indicated by an increase in cumulative mortality post‐challenge. Supplementation with βG fifteen days before (βG then ATZ group), counteracted the adverse effects of ATZ on the immune, biochemical and antioxidants values, though only slight alleviation was observed with simultaneous treatment (βG/ATZ group). Our results established that ATZ has adverse impacts on immune responses, antioxidant equilibrium and its related genes. While, supplementation with βG before exposure to ATZ may be valuable for counteracting the possible damage caused by ATZ water pollution than its simultaneous treatment with ATZ.     

Characterization of a γ‐aminobutyrate type A receptor‐associated protein gene,which is involved in the response of Portunus trituberculatus to CO2‐induced ocean acidification

The γ‐aminobutyrate type A receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier implicated in membrane trafficking and fusion events involving the γ‐aminobutyrate type A receptor, autophagy and apoptosis. In this study, the gene encoding GABARAP was cloned from swimming crab Portunus trituberculatus (PtGABARAP) based on the expression sequence tag (EST). The full‐length cDNA of 664 bp includes a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 223 bp with a poly(A) tail, and an open reading frame (ORF) of 354 bp encoding a polypeptide of 117 amino acids with a predicted molecular weight of 13.96 kDa. The deduced amino acid sequence shares high similarity (93%–100%) with GABARAPs from other species and includes a conserved Atg8 domain. In a phylogenetic analysis PtGABARAP clustered with GABARAPs from other species, and more widely with other GABARAP family proteins. The impact of elevated ocean acidification (OA) on P. trituberculatus behaviours was investigated, and real‐time RT‐PCR revealed that PtGABARAP expression was up‐regulated after OA exposure. Ocean acidification also caused crabs anxiety‐like behaviours, like the shoal average speed increase, preference for dark environment (scototaxis) and fast exploration. The results indicated that GABARAP might be involved in the interactions of GABA_A receptors and elevated‐CO₂ seawater.     

The effect of β‐1,3‐glucan derived from Euglena gracilis (Algamune™) on the innate immunological responses of Nile tilapia (Oreochromis niloticus L.)

Algamune^? is a commercial additive produced from Euglena gracilis, providing a rich source of the β‐1,3‐glucan paramylon. Isolated kidney phagocytes of Nile tilapia were incubated with graded doses (0, 8, 16, 32, 64 and 128 μg/ml) of Algamune^? and purified paramylon to gauge their ability to elicit the production of reactive oxygen species. A linear response was observed for extracellular superoxide anion for both sources but only Algamune^? for intracellular superoxide anion. After corroborating the immunostimulant properties ex vivo, a feeding trial was conducted to evaluate the dietary supplementation of Algamune^? (0, 100, 200, 400 and 800 mg/kg of diet) for Nile tilapia. Fish were fed for 3 weeks, after which, fish were sampled for blood and head kidney phagocytes. The remaining fish were challenged with Streptococcus iniae. Macrophage extracellular superoxide anion production was significantly elevated in fish fed diets with 200 mg of Algamune^? kg^?1 when compared to fish fed the basal diet. Even though the disease challenge did not show statistical differences, it is worth mentioning that fish fed intermediate doses of Algamune^? had lowest numerical mortality values. Therefore, Algamune^? was demonstrated to enhance some immunological responses of tilapia both in ex vivo and in vivo.     

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.     

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

Effects of continuous and alternate administration of β‐glucan and mannan‐oligosaccharide on the growth,immunity and resistance against Vibrio splendidus of sea cucumber Apostichopus japonicus (Selenka)

A 4‐week growth trial was conducted to compare the effects of different feeding strategies of dietary immunostimulants on the growth, immunity and resistance against Vibrio splendidus of sea cucumbers Apostichopus japonicus (Selenka). Six feeding strategies were set, including feeding immunostimulants‐free diet continuously (control), feeding dietary β‐glucan (1.25 g kg^?1 diet) continuously, feeding dietary mannan‐oligosaccharides (MOS; 2.00 g kg^?1 diet) continuously, feeding β‐glucan 2 days followed by MOS 5 days alternately, feeding β‐glucan 5 days followed by MOS 2 days alternately and feeding β‐glucan 7 days followed by MOS 7 days alternately. The sea cucumbers fed immunostimulants showed higher specific growth rate (SGR) and lower cumulative mortality than control (P < 0.05). When sea cucumbers were fed with β‐glucan continuously, total coelomocytes counts and superoxide anion were significantly higher than control on the 4th day (P < 0.05). However, these two immune parameters were not significantly higher than those in control after the 18th day (P > 0.05). While sea cucumbers continuously fed MOS, these two immune parameters were not significantly higher than control until the 15th day. All immune parameters of the sea cucumbers fed with β‐glucan and MOS alternately were significantly higher than those in control during the experiment (P < 0.05). The sea cucumbers fed with β‐glucan 7 days followed MOS 7 days alternately showed the highest SGR and second lowest cumulative mortality. It was suggested that this feeding strategy is most suitable for sea cucumbers.     

Effect of treatment of chum salmon Oncorhynchus keta (Walbaum) eggs with 1,3;1,6‐β‐D‐glucans on their development and susceptibility to Saprolegnia infection

The effects of six 1,3;1,6‐β‐D‐glucooligo‐ and polysaccharides with different structures (ranging from 1 to 10 kDa in molecular mass and containing 10–25% of β‐1,6‐linked glucose residues) from brown algae, Saccharina cichorioides, on development of the chum salmon, Oncorhynchus keta (Walbaum), were evaluated. Exposure of chum salmon eggs to 1,3;1,6‐β‐D‐glucans with a molecular mass of more than 2 kDa increased the survival of embryos and juveniles and their resistance to Saprolegnia infection by up to 2.5‐fold, leading to a weight gain in juveniles of 40–55% compared with The control chum salmons. The 1,3;1,6‐β‐D‐glucans with molecular mass of 6–8 kDa and used at a at concentration of 0.5 mg mL^?1 rendered the best stimulative effect.