Research News

Estimates of seaweed nutrient sequestration ability in open-water, Integrated Multi-Trophic Aquaculture (IMTA) required to ‘balance’ nutrients from fish cages have typically assigned a specific nutrient load for a specific fish biomass. The resultant culture area and densities of seaweeds required for full equivalent nutrient sequestration may have meaning only to experienced aquaculturists. Consequently, a novel ratio model is proposed which determines the weight ratio of harvested seaweeds required to sequester an equivalent weight of soluble inorganic nutrients loaded per unit growth of fish. Soluble inorganic nutrient excretion from Atlantic salmon, Salmo salar, was estimated using a semi-stochastic nutritional mass balance approach. Oxygen demand was estimated using respiratory quotients. Nutrient contents of the IMTA kelps, Alaria esculenta and Saccharina latissima, were measured at harvest times, and net oxygen production was estimated using the photosynthetic equation. To quantify uncertainty, input parameters were assigned theoretical distributions (based on empirical or literature data) and the model was run using a stratified sampling approach (Latin Hypercube) over multiple iterations, to generate distributions of weight ratios for the various nutrients. A mathematical simulation of nutrient loading from a salmon farm over a full production cycle was estimated using mean loading values per unit growth, with monthly growth estimated by a thermal growth coefficient (TGC). Results showed that one kilogramme of Atlantic salmon growth (large fish fed a typical commercial feed, ±standard deviation) resulted in the excretion of 29.49±4.20g nitrogen, 2.26±2.25g phosphorus and the respiration of 243.38±48.28g carbon. Dissolved oxygen requirements for 1kg of growth were 455.29±86.24g. Salmon smolts placed in cages in spring and harvested 21months later, load approximately 4 times more nutrients in the second grow-out season. The mean ratios of A. esculenta weight required to sequester nutrients excreted per unit weight of S. salar production were 6.7(±1.5):1 for nitrogen, 4.8(±3.0):1 for phosphorus, and 5.8(±1.4):1 for carbon. Oxygen could be supplied at a weight ratio of 4.1(±1.0):1. The mean ratios of S. latissima were 12.9(±2.7):1 for nitrogen, 10.5(±6.2):1 for phosphorus, and 10.2(±2.2):1 for carbon. Oxygen could be supplied at a weight ratio of 7.2(±1.5):1. A. esculenta appears to have almost twice the nutrient sequestration capacity per wet weight than S. latissima. However, culture densities of S. latissima are 1.5 times greater than those for A. esculenta and when spatially weighted this difference is reduced to 1–1.5 times. Numbers of rafts for both kelp species required for full nutrient sequestration from a commercial scale salmon farm exceed the number of rafts which can be practically deployed within a typical site lease area. However, not all inorganic nutrients from cultured fish will be available to IMTA seaweeds, nor should 100% nutrient sequestration need be the only successful endpoint in such systems. These aspects should be considered when assessing the net value of kelps in open-water IMTA systems.

Author(s): G.K. Reid , T. Chopin , S.M.C. Robinson , P. Azevedo , M. Quinton , E. Belyea

Wound healing is a complex and well-organized process in which physiological factors and immune mechanisms are involved. A number of different immune modulators have been found to enhance the non-specific defence system in vertebrates, among which β-glucans are the most powerful and extensively investigated. The aim of the present study was to investigate the biological impact of two different commercially available β glucan containing products on the wound healing process in carp. Throughout a two week experiment fish were kept either untreated (control), or in water supplemented with the two different types of β-glucans. The wound healing process was monitored using a multispectral visualisation system. The correlation between wound closure and immune response was investigated by measuring the gene expression patterns of IL-1β, IL-6 family member M17, IL-8 and Muc5b, and measurement of production of radical oxygen species. PAMPs/DAMPs stimulation caused by the wounding and or β-glucans resulted in an inflammatory response by activating IL-1β, IL-6 family member M17 and IL-8 and differences in the expression pattern were seen depending on stimuli. IL-1β, IL-6 family member M17 and IL-8 were activated in all wounds regardless of treatment. Expression of all three interleukins was highly up regulated in control wounded muscle already at day 1 post-wounding and decreased at subsequent time-points. The reverse was the case with control wounded skin, where expression increased from day 1 through day 14. The results for the β-glucan treated wounds were more complex. The images showed significantly faster wound contraction in both treated groups compared to the control. The obtained results clearly demonstrated that a β glucan enriched bath promotes the closure of wounds in common carp and induce a local change in cytokine expression.

Author(s): D.A. Przybylska , J.G. Schmidt , N.I. Vera-Jimenez , D. Steinhagen , M.E. Nielsen

Research on sea slugs production has steadily increased in the last decades as a result of their use as model organisms for biomedical studies, bioprospecting for new marine drugs and their growing demand for academic research and the marine aquarium trade. However, standardized methods for culturing sea slugs are still limited to a reduced number of species. The main bottlenecks impairing sea slugs aquaculture are the lack of knowledge on suitable larval diets and settlement cues that can induce metamorphosis in competent larvae. Additionally, the stenophagous feeding regime displayed by several species requires the collection and/or culture of their prey, which commonly impairs large-scale production. Nevertheless, significant breakthroughs have been achieved in recent years through the development of innovative culture techniques. The present review summarizes the major issues impairing the culture of sea slugs and presents relevant biological and ecological data that can assist on the development of suitable culture protocols. Information on the most suitable husbandry, larviculture and grow-out techniques are critically discussed, with emphasis to their application on some of the most relevant groups of sea slugs from an academic and commercial point of view: sea hares (Aplysia spp.), nudibranchs (e.g., the marine ornamental species Aeolidiella stephanieae) and the “solar powered” sacoglossan (e.g., Elys ia spp.).

Author(s): Gisela Dionísio , Rui Rosa , Miguel Costa Leal , Sónia Cruz , Cláudio Brandão , Gonçalo Calado , João Serôdio , Ricardo Calado

Mussel raft culture as traditionally practiced in Galicia (Spain) represents an extreme case of aggregation, where culture density along growth ropes is maximized to achieve greater commercial yields. However, to maintain high growth rates during the entire cultivation period the initial density must be reduced through a process called “thinning-out” when rope weight increases by a factor of 10. In raft culture, the annual commercial cycle is superimposed on a production cycle that can last up to 18months. For this reason, the thinned-out mussel ropes can only occupy 75% of the usable surface area of the raft. Additionally, the rope handling process has a pronounced impact on production costs, affecting both labor and material costs. With the objective of optimizing economic yields in mussel raft culture, we conducted a study to compare a new technique without thinning-out with the traditional method, and we evaluated the effect of density on mussel growth for both techniques. For this purpose, three different densities were prepared for the no thinning-out ropes (800n, 1000n and 1200n mussels/m), and the other three for the thinning-out ropes (400y, 500y and 600y mussels/m). At the end of the experiment, average length and average live weight for mussels cultured without thinning-out were significantly lower than values observed for mussels cultured with thinning-out. However, the no thinning-out technique did not result in a lower total biomass production. The technique without thinning-out has a production cycle of 12months, which coincides with annual production. On the other hand, the culture with thinning-out technique has a 15-month cycle (seeding and thinning-out processes); thus annual production was 20% lower than total production. Under these conditions, production at the two highest densities (1000n and 1200n) was significantly higher than production at all densities with thinning-out. If we consider economic yield per raft per year, which also takes into account the higher price of mussels with increasing size, the difference between the two culture methods is smaller. Only the 1200n ropes still produced a higher economic yield than the 600y ropes. On the other hand, the shortening of the cultivation time and the elimination of the thinning-out reduce the production costs. In conclusion, the results obtained with the new culture technique without thinning-out at high seed densities enable substantial improvements in terms of biomass, economic yields, and operating costs for mussel production.

Author(s): Alejandro Pérez-Camacho , Uxío Labarta , Vanessa Vinseiro , Ma José Fernández-Reiriz

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N Suanyuk, M Rogge, R Thune, M Watthanaphiromsakul, N Champhat, W Wiangkum

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N Tanaka, T Izawa, M Kuwamura, N Higashiguchi, C Kezuka, O Kurata, S Wada, J Yamate

With the aim of evaluating the effect of a Mongolian dairy product derived Lactobacillus paracasei spp. paracasei (strain 06TCa22) (Lpp) on the cytokine-mediated immune responses to Vibrio harveyi infection, we examined 16 cytokine expressions in the Japanese pufferfish, Takifugu rubripes. Fish were orally treated with the heat-killed Lpp at 1 mg g^-1 body weight d^-1 for 3 days. At 24 h post treatment, fish were infected by an intramuscular injection of 0.1 mL V. harveyi bacterial suspension (10⁸ cfu mL^-1). Additionally, superoxide anion production (SAP) and phagocytic activity (PA) of head kidney cells were assessed during 120 h post infection period. Significant up-regulation of pro-inflammatory (IL-1β, IL-6, IL-17A/F-3, TNF-α and TNF-N), cell-mediated immune inducing (IL-12p35, IL-12p40 and IL-18), antiviral/ intra-cellular pathogen killing (I-IFN-1 and IFN-γ), anti-inflammatory (IL-10) and lymphocyte agonistic (IL-2, IL-7, IL-15, IL-21 and TGF-β1) cytokines was observed in the treated fish compared to control ones during the pathogen infection. Furthermore, significantly increased SAP and PA (P<0.01; 0.05) were recorded in the treated fish compared to untreated fish. These results suggest the beneficial role of Lpp in enhancement of cytokine-mediated immunity in the Japanese pufferfish against V. harveyi infection and application of this product as a potential fish immunostimulant.

Author(s): G. Biswas , H. Korenaga , R. Nagamine , S. Kawahara , S. Takeda , Y. Kikuchi , B. Dashnyam , T. Yoshida , T. Kono , M. Sakai

Full-sibling, size-matched ‘all-fish’ growth hormone transgenic (TG; gene construct EO-1α) and non-transgenic (NTG) Atlantic salmon (Salmo salar L.) comprised of conventional diploid (DIP) and reproductively-sterile triploid (TRIP) fish were fed the same experimental grower diet in freshwater until they tripled their weight. The study was conducted to provide baseline data on growth performance, feed efficiency, nutrient digestibility, skeletal disorders, bone ash content and bone mineral composition of NTG/DIP, NTG/TRIP, TG/DIP and TG/TRIP fish and to determine if a practical grower diet for conventional NTG/DIP Atlantic salmon requires modification for TG/TRIP fish. TG fish consumed a significantly higher amount of feed on a daily basis but due to enhanced growth rates, better feed conversion ratios and higher nitrogen retention efficiency they achieved target weight gain in a considerably shorter period (40%) than NTG fish. Total feed required to produce the same fish biomass was reduced by 25%; representative of a significant reduction in overall feed intake. Of TG fish, TRIP had some significant effects on production traits primarily due to lower feed intake relative to DIP fish. Although feed intake was lower in TG/TRIP fish, feed efficiency, digestibility and nutrient retention efficiencies were equal to those of TG/DIP fish and, without exception; TG/TRIP fish out-performed their conventional NTG counterparts, regardless of ploidy. TG/DIP and TG/TRIP fish demonstrated a higher cellular capacity to direct dietary non-protein energy towards satisfying their daily metabolic energy requirements, allowing for a higher proportion of dietary amino acids to be directed towards protein biosynthesis; rather than catabolised as a dietary energy source. Since dietary protein is the largest and most expensive component of salmon feeds and also the major source of nitrogenous pollution from salmon farming, this could represent a highly beneficial alteration of energy metabolism which could result in more economical and ecologically-sustainable Atlantic salmon aquaculture, especially when conducted in closed-containment land-based systems. Although bone ash content of TG fish was slightly lower than NTG fish, no significant differences in key bone mineral composition were observed and the occurrence of skeletal disorders was low (<4%), regardless of transgenics or ploidy.

Author(s): S.M. Tibbetts , C.L. Wall , V. Barbosa-Solomieu , M.D. Bryenton , D.A. Plouffe , J.T. Buchanan , S.P. Lall

The innate immune system is a fundamental defense weapon of fish, especially during early stages of development when acquired immunity is still far from being completely developed. The present study aims at looking into ontogeny of innate immune system in the brown trout, Salmo trutta, using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs and hatchlings at 0, 1 h and 1, 2, 3, 4, 5, 6, 7 weeks post-fertilization was subjected to RT-PCR using self-designed primers to amplify some innate immune relevant genes (TNF-α, IL-1β, TGF-β and lysozyme c-type). The constitutive expression of β-actin was detected in all developmental stages. IL-1β and TNF-α transcripts were detected from 4 week post-fertilization onwards, whereas TGF-β transcript was detected only from 7 week post-fertilization onwards. Lysozyme c-type transcript was detected early from unfertilized egg stage onwards. Similarly, tissues such as muscle, ovary, heart, brain, gill, testis, liver, intestine, spleen, skin, posterior kidney, anterior kidney and blood collected from adult brown trout were subjected to detection of all selected genes by RT-PCR. TNF-α and lysozyme c-type transcripts were expressed in all tissues. IL-1β and TGF-β transcripts were expressed in all tissues except for the brain and liver, respectively. Taken together, our results show a spatial-temporal expression of some key innate immune-related genes, improving the basic knowledge of the function of innate immune system at early stage of brown trout.

Author(s): Stefano Cecchini , Mariateresa Paciolla , Elio Biffali , Marco Borra , Matilde V. Ursini , Maria B. Lioi

Cathepsin L (CTSL) is a lysosomal cysteine protease involved in immune responses in vertebrates. However, few studies exist regarding the role of cathepsin L in bivalves. In this study, we isolated and characterized four cathepsin L genes from the razor clam Sinonovacula constricta, referred to as CTSL1, CTSL2, CTSL3 and CTSL4. These four genes contained typical papain-like cysteine protease structure and enzyme activity sites with ERWNIN-like and GNFD-like motifs in the proregion domain and an oxyanion hole (Gln) and a catalytic triad (Cys, His and Asn) in the mature domain. Expression analysis of the four transcripts revealed a tissue-specific pattern with high expression of CTSL1 and CTSL3 in liver and gonad tissues and high expression of CTSL2 and CTSL4 in liver and gill tissues. During the developmental stages, the four transcripts showed the highest expression in the juvenile stage; however, CTSL3 had a much higher expression level than the other three transcripts during embryogenesis. The four transcripts showed significant changes in expression as early as 4 h or 8 h after infection with Vibrio anguillarum. The fact that bacterial infection can induce expression of the four CTSL transcripts suggests that these transcripts are important components of the innate immunity system of the clam.

Author(s): Donghong Niu , Kai Jin , Lie Wang , Bingbing Feng , Jiale Li

Abstract

To determine the potential for improving the conversion and deposition of the omega-3 fatty acids docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) in fish, 44 families of rainbow trout were fed a diet low in these components and then evaluated for their ability to convert and store plant oils that did not contain DHA and EPA in their muscle tissue. The range for EPA and DHA detected in the tissues of different families varied between 1.2 and 2.9%, and 3.8 and 7.1% total fatty acid, respectively. After adjusting for covariates, an averaged heritability of 0.78 ± 0.11 for DHA and 0.61 ± 0.17 for EPA was calculated. Expression analysis of genes related to the elongation and conversion of lipids were also analyzed, and significant differences were found in the expression of some genes between groups of families that were delineated as having relative high, medium, and low capabilities of depositing EPA and DHA in their muscle tissue after being reared on the mainly soy/flax oil containing diet. However, none of these genes showed a positive correlation with the high conversion/deposition group.

Ken Overturf, Tom Welker, Frederick Barrows, Richard Towner, Rich Schneider, Scott LaPatra

Abstract

This study was conducted to determine the ontogenetic development of the digestive tract and its accessory structures (liver, pancreas, and gall bladder) in agastric larval Chinese sucker Myxocyprinus asiaticus with the histological and ultrastructural approaches from hatching to 56 days after hatching (DAH). On the basis of its feeding mode, and analyzing the main histological features of the digestive system, larval development in Chinese sucker was divided into three stages from hatching: stage 1 (endotrophic period): 1–6 DAH; stage 2 (endoexotrophic period): 7–14 DAH; stage 3 (exclusively exotrophic period): from 15 DAH onward. At hatching, the digestive tract of the larvae consisted of an undifferentiated straight tube. At 4 DAH, the mouth opened, and the digestive tract was differentiated into buccopharyngeal cavity, esophagus and intestine. At 7 DAH, fish started to feed exogenously. Yolk sac was completely exhausted at 15 DAH. Until 56 DAH, the digestive tract of the larvae displayed regularly arranged microvilli, abundant vacuoles, and protein inclusion bodies. The pancreas, liver, and gall bladder were functional from 6 DAH, which enabled larvae to ingest, digest, and assimilate the first exogenous food. In comparison with teleosts that have a stomach, the development of the digestive tract of the agastric Chinese sucker seemed relatively slow.

Cai-Xia Liu, Zhi Luo, Xiao-Ying Tan, Shi-Yuan Gong

Abstract

Aquaponics is an integrated fish and plant recirculating production system. The University of the Virgin Islands' raft aquaponic system uses a cylindro-conical clarifier as a primary solids removal device; however, a swirl separator may offer advantages. The objectives of the 8-wk experiment were to compare water quality parameters, Nile tilapia, Oreochromis niloticus, production and water spinach, Ipomoea aquatica, production in a raft aquaponic system using a clarifier or swirl separator for primary treatment of solids in the waste stream.

No significant differences (P ≥ 0.05) existed between treatments for temperature, oxygen, pH, total suspended solids, alkalinity, electrical conductivity, total ammonia-nitrogen, nitrite-nitrogen, nitrate-nitrogen, macronutrients, and micronutrients concentrations in the culture water. There were no significant differences (P ≥ 0.05) between treatments for Nile tilapia production, average weight, survival, or feed conversion ratio. There were no significant differences (P ≥ 0.05) between treatments for water spinach production or plant tissue analysis. In conclusion, the swirl separator used in this experiment performed similar to the clarifier and water spinach grew vigorously in the raft aquaponic system.

Jason J. Danaher, R. Charlie Shultz, James E. Rakocy, Donald S. Bailey

Abstract

Three sources of soybean meal (SBM) were biochemically characterized and evaluated in diets for juvenile Litopenaeus vannamei. Three biological techniques were utilized to evaluate the nutritional quality of the meals including an outdoor growth trial as well as determination of in vivo apparent digestibility and in vitro digestibility including pepsin digestibility (0.0002%), pH Stat degree of hydrolysis, and immobilized digestive enzyme assay (IDEA). The growth trial was conducted over a 10-wk period using soybean meal as the primary protein source with each meal being included at 54–58% of the diet based on an equal protein inclusion. At the conclusion of the growth trial there were no significant differences between the test diets indicating equivalent biological availability under practical conditions. The in vivo digestibility trial utilized chromic oxide as the inert marker and 70:30 replacement techniques resulted in typical results. Apparent protein digestibility (APD) and apparent energy digestibility (AED) of the diets ranged from 66.8 to 80.2% and 65.4 to 74.7%, respectively. Ingredient digestibility values for protein and energy ranged from 78.8 to 93.5% and 60.5 to 85.0%, respectively. One of the three SBM evaluated had numerically lower protein digestibility and significantly lower energy digestibility than the other two meals.

Xuezhi Zhu, D. Allen Davis, Luke A. Roy, T. M. Samocha, J. P. Lazo

Abstract

To determine the genotype with better traits for aquaculture practices of the loach, factorial crosses were made among diploid (D), tetraploid (T) dojo loach, Misgurnus anguillicaudatus, and large-scale loach, Paramisgurnus dabryanus (P), producing DD, DT, DP, TD, TT, TP, PD, PT, and PP genotypes (female listed first). The growth performance and survival of different genotypes in culture were evaluated through a 48-wk rearing trail. The average fertilization rate in genotypes with P. dabryanus as female parental was significantly higher than those of M. anguillicaudatus as female parental. The average fertilization level of sperm from P. dabryanus was about the same as diploid M. anguillicaudatus but significantly higher than tetraploid M. anguillicaudatus. The highest survival rate was found in DD group (52.77%) and PP group (50.31%) (P > 0.05). The intergenetic crosses between P. dabryanus and M. anguillicaudatus, especially for PD (19.05%), DP (17.89%), and PT (15.44%) groups, exhibited poor survival rates. Under the similar rearing conditions, PP group showed significant growth advantage. Growth rates of the interspecific hybrid progenies were found to be intermediate in comparison with the parental species. According to these results, the PP genotype is more suitable for aquaculture practice.

Lei Cui, Li-Bao Fang, Yong-Yao Yu, Wei-Min Wang, Xiao-Yun Zhou, Khalid Abbas

Abstract

Culturing the longsnout seahorse, Hippocampus reidi, remains challenging because unlike most cultured seahorse species, Artemia is not an optimal first feed. Ideally, more nutritious live feeds such as wild plankton or copepods should be used, but are not always readily available. Therefore, facilities commonly utilize enriched rotifers. Numerous feed enrichment products exist, including Dan's Feed Artemia Enrichment^®, (Dan's Feed) which is a blend of commercially available enrichment products. The aim of this study was to assess the survivorship, growth, and condition factor (CF) of 0–28  DPR (days post release) H. reidi fed with rotifers and Artemia enriched with Dan's Feed. This enrichment was evaluated against (1) rotifers and Artemia enriched with live alga (Isochrysis galbana), and (2) wild plankton and Artemia enriched with Dan's Feed. At 28 DPR, in terms of survivorship and growth, seahorses fed with rotifers and Artemia enriched with Dan's Feed had a lower CF and outperformed those fed rotifers and Artemia enriched with I. galbana. Seahorses fed with rotifers and Artemia enriched with Dan's Feed performed on par with those fed with wild plankton and Artemia with no significant difference in CF. These findings demonstrated that the culture of H. reidi may be feasible with rotifers and Artemia enriched with Dan's Feed.

Nancy Kim Pham, Junda Lin

Abstract

High-resolution melting (HRM) has been considered as a fast and simple single-nucleotide polymorphism (SNP) scanning and genotyping method for identifying differences in the shapes of melting curves between different genotypes. Fifty-six SNPs were developed in the Pacific oyster, Crassostrea gigas by mining expressed sequence tags database, using the HRM method. The frequency of the SNPs was estimated at 1 per 113 bp of contig sequences. Analysis of segregation in a full-sib family showed that 28 SNPs were polymorphic, with 15 in accordance to expected Mendelian ratios. Linkage grouping of the 28 markers resulted in six linkage groups. The combined power of exclusion of the 42 SNPs, which conformed to Hardy–Weinberg equilibrium, was greater than 99.98%, while the average polymorphic information content was 0.2223. The simulation results showed that the success rate of parentage analysis could be 97% with the 42 SNPs. These SNPs will be useful for pedigree analysis, association studies, and marker-associated selection of this species.

Xiaoxiao Zhong, Qi Li, Hong Yu, Lingfeng Kong

Vibriosis outbreaks caused by Vibrio harveyi and related species are amongst the major obstacles for the further expansion of giant freshwater prawn (Macrobrachium rosenbergii) larviculture. Vibrio harveyi regulates virulence gene expression through quorum sensing, bacterial cell-to-cell communication, and consequently, quorum sensing disruption has been suggested as an alternative strategy to control infections caused by these bacteria. Previous studies have shown that quorum sensing-disrupting compounds are able to disrupt quorum sensing in Vibrio harveyi. In this study, we demonstrated that the quorum sensing-disrupting compounds cinnamaldehyde, (Z-)-4-bromo-5-(bromomethylene)-2(5H)-furanone and (Z)-4-((5-(bromomethylene)-2-oxo-2,5-dihydrothiophen-3-yl)metoxy)-4-oxobutanoic acid increased the survival of giant freshwater prawn larvae when challenged to pathogenic Vibrio harveyi. Our in vivo challenge test showed that cinnamaldehyde and the thiophenone can protect the larvae from Vibrio harveyi infection when dosed to the culture water at 1μM and 10μM, whereas the brominated furanone offered protection at 1μM but resulted in complete mortality at 10μM. Although there were significant differences in survival between challenged larvae with and without addition of quorum sensing-disrupting compounds, there were no differences in growth (as determined by the larval stage index).

Author(s): Gde Sasmita Julyantoro Pande , Anne Aamdal Scheie , Tore Benneche , Mathieu Wille , Patrick Sorgeloos , Peter Bossier , Tom Defoirdt

The isolation of a betanodavirus strain from juvenile turbot showing unspecific symptoms and very low mortality is reported. The presence of an IPNV-type virus was also revealed in the fish tissues, but only after nested-PCR, suggesting a very low viral load. In addition, different Vibrio species were isolated from some of the individuals. Both viral RNAs were sequenced and phylogenetic analysis indicated that the strain showed 99% identity with the RGNNV genotype. Experimental infections (via immersion and intraperitoneal route) were conducted in order to determine the susceptibility of turbot juveniles (2 and 5g) to the NNV isolate at 15 and 18°C (range of turbot rearing temperatures in our area). The results obtained indicated that although the viral isolate was able to replicate in the turbot tissues, it did not induce clinical disease in this fish species. These findings suggest that the existence of a reservoir of NNV-RGNNV type in wild fish in the area represents a low risk for the turbot farming industry.

Author(s): J.G. Olveira , S. Souto , C.P. Dopazo , I. Bandín

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are the numbers of the lipid transfer protein/lipopolysaccharide-binding protein family and play crucial roles in the innate immune response to Gram-negative bacteria. A novel Sb-BPI/LBP1 from ark shell Scapharca broughtonii was isolated by expressed sequence tag (EST) and RACE techniques. The Sb-BPI/LBP1 cDNA encoded a polypeptide of 484 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 463 amino acids. The deduced amino acid sequence of Sb-BPI/LBP1 contained an N-terminal BPI/LBP/CETP domain BPI1 with three functional regions that display LPS-binding activity, and a C-terminal BPI/LBP/CETP domain BPI2. In structure and sequence, Sb-BPI/LBP1 showed highly similar to those of the BPI/LBPs from invertebrate and non-mammalian vertebrate, the LBPs and BPIs from mammal. By quantitative real-time RT-PCR, Sb-BPI/LBP1 transcripts could be detected in all normal tested tissues, including hepatopancreas, adductor muscle, mantle margin, heart, gonad, gill and hemocytes, and was universally up-regulatable at 24 h post LPS challenge. The mRNA expression of Sb-BPI/LBP1 in hemocytes was the most sensitive to LPS challenge, significantly up-regulated at 12 h post LPS challenge and peaked at 24 h (16.76-fold, P < 0.05). These results suggested that Sb-BPI/LBP1 was a constitutive and inducible acute-phase protein contributing to the host immue defense against Gram-negative bacterial infection in ark shell S. broughtonii.

Author(s): Yuze Mao , Chunya Zhou , Ling Zhu , Yao Huang , Tingru Yan , Jianguang Fang , Wei Zhu

This study was conducted to evaluate the effects of dietary isoleucine (Ile) on the immune response, antioxidant status and gene expression in the head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Six semi-purified isonitrogenous diets (4.2, 7.0, 9.5, 11.9, 13.9 and 16.9 g Ile kg^-1 diet) were fed to Jian carp (6.9 ± 0.03 g) for 60 days. The results showed that Ile supplementation improved the head kidney index, red and white blood cell counts, anti-hydroxyl radical capacity and the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase (P < 0.05), and decreased the malondialdehyde, protein carbonyl and glutathione contents in the head kidney (P < 0.05). After a 60 day feeding trial, an Aeromonas hydrophila challenge study was conducted for 17 days. Differences in survival rate, leukocyte phagocytic activity, serum lysozyme activity, acid phosphatase activity, haemagglutination titre, complement components 3 and 4, immunoglobulin M level and A. hydrophila agglutination antibody titre followed the same trend as that of the head kidney index (P < 0.05). Furthermore, real time polymerase chain reaction revealed that relative mRNA expression of transforming growth factor β2 and target of rapamycin (TOR) in the head kidney significantly increased with increasing Ile levels (P < 0.05). Conversely, the relative mRNA expression of tumour necrosis factor α, interleukin 10 and eIF4E-binding protein (4E-BP) in the head kidney showed a downward trend (P < 0.05). Collectively, this study indicates that dietary Ile improves the fish immune response, regulates the antioxidant status and cytokine, TOR and 4E-BP gene expression in the head kidney.

Author(s): Juan Zhao , Yang Liu , Jun Jiang , Pei Wu , Weidan Jiang , Shuhong Li , Ling Tang , Shengyao Kuang , Lin Feng , Xiaoqiu Zhou

A 120-day feeding trial was conducted to investigate the effects of dietary increasing levels of grape seed oil (GO) and linseed oil (LO), rich in linoleic acid (LA) and α-linolenic acid (ALA) respectively, on growth, fatty acid composition and expression levels of putative Δ5 fatty acyl desaturases (Fads) in muscle of juvenile abalone (Haliotis discus hannai Ino). Seven experimental diets were formulated to contain increasing amounts (0%, 0.875%, 1.75% and 3.5%) of GO or LO as dietary lipids. Tripalmitin (TP), rich in 16:0, was supplemented to reach 3.5% (dry weight) total lipid. These diets were named as 0%GO/LO (TP), 25%GO, 50%GO, 100%GO, 25%LO, 50%LO and 100%LO. With the increase of dietary GO or LO inclusion, growth parameters (such as specific growth rate of weight) first increased and then decreased (R ² >0.72, P =0.000). The survival rate of abalone was not significantly correlated with dietary GO (R ² =0.04, P =0.815) or LO inclusion (R ² =0.50, P =0.046). The contents of muscle n-6 polyunsaturated fatty acids (PUFA), including LA, 20:2n-6, 20:4n-6, 22:2n-6, were significantly positive correlated with dietary GO inclusion (R ² >0.895, P =0.000). A positive relationship was significantly noted between the values of muscle n-3 PUFA (ALA, 20:3n-3, 20:5n-3 and 22:5n-3) and the dietary LO inclusion (R ² >0.905, P =0.000). DHA was significantly increased and then decreased with dietary LO increase (R ² =0.937, P =0.000). The expression levels of putative Δ5 Fads in 50%GO group were significantly higher than those of other GO groups (P <0.05). With the increase of dietary LO, expressions of putative Δ5 Fads first increased and thereafter reached a plateau (R ² >0.929, P =0.000). These results indicated that biosynthesis of long chain polyunsaturated fatty acid in muscle of abalone could be increased in response to increasing levels of dietary LA or ALA through increased expressions of putative Δ5 Fads. High intakes of dietary LA and/or ALA inhibited the biosynthesis of DHA and compromised the growth performance of abalone.

Author(s): Mingzhu Li , Kangsen Mai , Qinghui Ai , Gen He , Wei Xu , Wenbing Zhang , Yanjiao Zhang , Huihui Zhou

Gynura bicolor (Roxb. & Willd.) DC is widely distributed in certain areas of Asia and is very popular in vegetarian cuisine in Taiwan. To investigate the regulatory roles of G. bicolor in various functions in crustaceans, we examined innate non-specific immune responses (including total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RBs), and superoxide dismutase (SOD) activity), physiological responses (including haemolymph glucose, lactate, and lipids), and gene expressions (including prophenoloxidase (proPO), lipopolysaccharide- and b-1,3-glucan-binding protein (LGBP), and peroxinectin (PE) mRNA transcripts) to the pathogen Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) that were individually injected with the water extract from G. bicolor at 2, 4, and 8 μg g⁻¹. Results indicated that PO, RBs, SOD activity, proPO, LGBP, and PE mRNA transcripts of shrimps receiving the water extract of G. bicolor at 2, 4, and 8 μg g⁻¹ significantly increased after challenge with V. alginolyticus for 96 h. However, no significant difference in the THC was seen at any dose. L. vannamei injected with the water extract of G. bicolor at all doses respectively maintained lower glucose, lactate, and lipid levels in response to V. alginolyticus challenge at 12–36, 24–36, and 24–48 h. Survival rates at 24–72 h of L. vannamei that received G. bicolor at any dose was significantly higher than those of shrimp that received saline. It was concluded that the water extract of G. bicolor can maintain physiological homeostasis and enhance immunity against V. alginolyticus infection in L. vannamei.

Author(s): Shu-Ling Hsieh , Chih-Chung Wu , Chun-Hung Liu , Juang-Lin Lian

To determine the effects of dietary organic selenium (OS) supplementation on the growth performance and immune competence of marron, Cherax cainii (Austin, 2002), a group of marron were fed 0.2 g kg⁻¹ of Sel-Plex^® supplemented basal diet and then compared with another group (control) of marron fed basal diet without any supplementation. After 90 days of feeding, final weight, average weekly gains (AWG), relative gain rate (RGR), specific growth rate (SGR), survival, total and differential haemocyte counts (THC and DHC), were compared between the two groups. Surviving marron from each group were then divided into three sub-groups (three tanks per sub-group with seven marron per tank); (1) first sub-group was injected with 20 μL of 3.24 × 10⁶ cfu Vibrio mimicus; (2) the second sub-group was injected with 20 μL normal saline and (3) the third sub-group was not subjected to injection and became the control group. THC, DHC, neutral red retention time (NRRT) and Vibrio ranks of post-injected marron were evaluated for 96 h, at every 24-h interval. The results showed that after 90 days of feeding, final weight, AWG, RGR, SGR, survival, THC, proportion of hyaline cells of OS-fed marron were significantly higher (P < 0.05) than the control group, whereas proportion of granular and semigranular cells were not affected by dietary OS. After challenging with V. mimicus, survival rate of marron without dietary OS significantly decreased (P < 0.05) as compared to the control group of marron. THC of marron in all sub-groups were significantly reduced (P < 0.05) after the challenge. However, THC and granular cells of sub-groups fed OS were higher than other sub-groups. Vibrio ranks and NRRT of marron fed OS were significantly lower and slower, respectively, than marron fed without OS. These findings demonstrated the benefits of OS inclusion in the marron diet in terms of growth, health and disease resistance.

Author(s): Rudy Agung Nugroho , Ravi Fotedar

The hot-water extract of Eichhornia crassipes leaves (ECE) was produced and incorporated into the diet of the prawn, Macrobrachium rosenbergii, as an immunostimulant. Survival rates of prawn against Lactococcus garvieae, and its immune parameters including the total haemocyte count (THC), different haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, transglutaminase (TG) activity, haemolymph coagulation time, and phagocytic activity and clearance efficiency against L. garvieae by M. rosenbergii were determined when prawn (23.0 ± 2.8 g) were fed ECE-containing diets at 0, 1.0, 2.0 and 3.0 g kg⁻¹. Prawn fed a diet containing ECE at 2.0 and 3.0 g kg⁻¹ for 12 days showed significantly increased THC, HC, GC, PO activity, RBs, SOD activity, GPx activity, and TG activity, and a significantly decreased coagulation time. The phagocytic activity and clearance efficiency against L. garvieae of prawn fed the ECE-containing diets at 1.0, 2.0, and 3.0 g kg⁻¹ were significantly higher than those of prawn fed the control diet at 3–12 days. Survival rates of M. rosenbergii fed the diet containing ECE at concentrations of 2 and 3 g kg⁻¹ were significantly higher than those fed the control diet after challenge with L. garvieae for 48–144 h. The relative percentage survival of prawn fed the 1.0, 2.0, and 3.0 g kg⁻¹ ECE-containing diets for 12 days were 17.5%, 39.1%, and 52.2%. It was concluded that the ECE can be used as an immunostimulant for prawn through dietary administration to enhance immune responses and resistance of M. rosenbergii against L. garvieae.

Author(s): Chin-Chyuan Chang , Hui-Ching Tan , Winton Cheng

This study attempts to describe the effects of innate immunity responses and field application of mushroom beta-glucan mixture (MBG) in cultured orange-sported grouper, Epinephelus coioides. Chemical analysis for MBG showed that the mixture contains 34.06% of macro-molecular polymers with bio-active linkage such as 3-; 3,4- and 4,6-glucopyranosyl and 6-linked galactopyranosyl residues. Study performed on the innate immunity showed that oral ingestion of MBG at 1.0 g and 2.0 g per kilogram of feed levels may significantly enhance the lysozyme activity, alternative complement activity, phagocytic activity and respiration burst of the experimental groupers. Observation on the experimental challenge of pathogen showed that uses of MBG at 0.1% and 0.2% levels in feed might significantly enhance the protection of grouper against Vibrio alginolyticus. Field trials performed on short and long-term culture showed that feeding of diet containing 0.1% or 0.2% of MBG may significantly enhance the survival of cultured groupers up to 16% when compared with those obtained from controls.

Author(s): Ching-Sheng Chang , Shih-Ling Huang , Sherwin Chen , Shiu-Nan Chen

Lysozyme is an important component of the innate immune system. In this study, four lysozyme genes including one c-type lysozyme and three g-type lysozymes were identified from channel catfish (Ictalurus punctatus). The lysozyme genes are highly conserved in their structural features as compared to those from other species. Phylogenetic analyses were conducted allowing annotation of these genes. Additional analyses using conserved syntenies allowed determination of orthologies for the c-type lysozyme. Phylogenetic analysis indicated that the g-type lysozyme may have gone through species-specific gene duplications leading to multiple copies in some teleost species. Channel catfish possessed three copies of the g-type lysozyme genes. Expression analysis revealed that the catfish lysozyme genes were expressed in a broad range of tissues. The highest levels of expression were found in head kidney, liver, spleen, and trunk kidney, compatible with the immune functions of these tissues/organs. The c-type and g-type lysozymes were drastically induced after bacterial infection, but exhibited large differences in the extent of induction and the tissue with the highest level of induction, with the g-type lysozyme being most highly induced in the head kidney whereas the other three lysozymes being most highly induced in the liver, suggesting their cooperative actions in the immune responses but difference in their detailed functions.

Author(s): Ruijia Wang , Jianbin Feng , Chao Li , Shikai Liu , Yu Zhang , Zhanjiang Liu

Teleosts are the first group of vertebrates possessing an acquired immune system; however, it is less developed than in mammals and is highly influenced by environmental changes. Therefore, innate immunity effectors play a more critical role in survival of pathogen-challenged fish. In a previous study we showed that trout high density lipoprotein (HDL), and its major apolipoprotein (ApoA-I) are widely expressed in primary defense barriers and other immune-relevant tissues, displaying important antibacterial activity in vitro. Here we show that trout HDL inhibits both basal and LPS-induced transcript expression of pro-inflammatory cytokines such as TNF-α and IL-1β, and the acute phase protein serum amyloid A (A-SAA), in head kidney leukocytes (HLK) from rainbow trout. In addition, trout HDL was able to block the respiratory burst of PMA-stimulated HKL, at physiological concentrations and in a dose dependent manner. Moreover, this effect was only partially mimicked by supra-physiologic concentrations of the HDL-transported carotenoid, astaxanthin. These results constitute the first data suggesting that in addition to its antimicrobial activity, HDL would have a relevant immunomodulatory role in salmonid fish.

Author(s): Franz Villarroel , Alin Casado , Rodolfo Amthauer , Margarita I. Concha

Suspended culture of bivalves offers a number of advantages over beach (intertidal) culture, but is often hindered by two issues: biofouling and suboptimal shell shape. This study assessed the efficacy of two novel culture media in Pacific oyster (Crassostrea gigas) grow-out and Manila clam (Venerupis philippinarum) nursery tray culture. We examined the combined effects of culture media type (expanded clay aggregate and lava rock), media volume (0, 15, 30, and 45Lm⁻²), and bivalve stocking density (226, 453, and 679indm⁻² for oysters; 3019, 9057, and 15,094indm⁻² for clams) on biofouling levels, shell characteristics, growth, condition, and survival of bivalves. Adding either media type to culture trays significantly reduced mussel fouling on oysters and effectively eliminated clam entanglement in mussel byssus. Clay aggregate was more effective at controlling tube worms and combined fouling on oysters than lava rock. Oyster shell shape index was not affected by either media type and decreased with increased stocking density. Oyster cup depth index decreased at higher media volumes and stocking densities, likely as a result of space restrictions. Reduced growth and condition were observed in both clams and oysters as media volume and stocking density increased. The presence of culture media slightly decreased oyster survival and improved clam survival. Oysters in all treatments exceeded minimum industry marketability standards. The benefits of culture media for biofouling control were realized at the lowest media volume (15Lm⁻²) suggesting that the efficacy of lower volumes of media should be assessed in the future.

Author(s): R.D. Marshall , A. Dunham

Aerobic exercise training may improve growth and help to protect fish from viral and bacterial diseases. This study was conducted to determine the effect of increased sustained aerobic exercise on the maintenance requirements and the efficiency of utilization of digestible protein (DP), digestible amino acids and metabolizable energy (ME) for growth above maintenance of Atlantic salmon post-smolts. The fish were held in tanks with currents of 7 (low speed) or 23 (high speed) cms⁻¹ (initial speeds 0.32 and 1.06bodylengthss⁻¹) for 62days and fed a fishmeal-based diet in excess or at levels approximating 30, 40, 60 and 80% of the intake of the full-fed groups. In the full-fed groups, there was no significant effect of exercise treatment on feed intake, final weights, growth rates, feed efficiency or whole-body composition. An extrapolation to zero weight gain showed that the increased speed resulted in an increase in the maintenance requirement from 5.3 to 13.4gdrymatterfish⁻¹. The maintenance requirement for ME, which includes the energy used for activity, was 46.5kJkg^−0.8 d⁻¹ in the high exercise groups and 24.7kJkg^−0.8 d⁻¹ in the low exercise groups. Our calculations showed that true maintenance, in which energy used for activity has been removed, was 15.3kJkg^−0.8 d⁻¹ at zero speed. Above maintenance, the efficiency of utilization of ME for energy gain was 0.89 in the high exercise groups and 0.70 in the low exercise groups (P<0.05). The maintenance requirements for DP were three times higher in the high exercise groups than in the low speed groups, but above maintenance, DP was used more efficiently for protein gain in the high exercise groups (0.70) than in the slow groups (0.60) (P<0.05). The efficiency of utilization of digestible arginine, histidine, isoleucine, leucine, lysine and phenylalanine for gain of these amino acids above maintenance tended to be greater in the salmon exercised at the high speed compared with those exercised at the low speed (P≤0.1). The maintenance requirements for most of the digestible amino acids were about twice as high in the high speed groups compared with the low speed groups. These results show that with increased aerobic exercise, the higher activity costs of Atlantic salmon are associated with increased utilization of nutrients and energy for growth.

Author(s): Barbara Grisdale-Helland , Harald Takle , Ståle J. Helland

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Majbritt Bolton-Warberg, Damien O'Keeffe, Richard D. FitzGerald

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Halyna Tkachenko, Natalia Kurhaluk, Joanna Grudniewska

Filter-feeding shellfish could act as biological agents in the control of parasitic sea lice (Caligidae) at salmon farms as a benefit of integrated multi-trophic aquaculture. Determining the extent to which various bivalve species ingest sea lice (Lepeophtheirus salmonis) larvae under controlled laboratory conditions was an important first step towards understanding the potential for shellfish to reduce the number of these copepod larvae in the water column at net pens. A series of laboratory experiments examined the effects of temperature (~5, 10, and 15°C), diet (larvae alone or with phytoplankton), and bivalve size (small, medium, and large) on the amount of L. salmonis larvae ingested by basket cockles (Clinocardium nuttallii), Pacific oysters (Crassostrea gigas), mussels (Mytilus spp.), and Pacific scallops (unconfirmed hybrid: Mizuhopecten yessoensis x Patinopecten caurinus). Feeding rate was assessed by placing bivalves individually in 2-l containers holding approximately 450 larvae in 750ml of filtered seawater. Results of the temperature/diet experiments indicated that all four species of bivalves ingested sea lice larvae, regardless of phytoplankton presence or absence, and that temperature had no significant effect on the proportion of larvae ingested. All three sizes of oysters and scallops ingested sea lice larvae, with large shellfish consuming a significantly greater proportion of the larvae than small individuals. Future research, examining the role of bivalves in the control of sea lice, should be continued at a commercial scale at a salmon farm.

Author(s): Janis L. Webb , Julie Vandenbor , Bradley Pirie , Shawn M.C. Robinson , Stephen F. Cross , Simon R.M. Jones , Christopher M. Pearce

Heteropneustes fossilis is distributed in most of the Southeast Asian countries. It is a promising species for aquaculture due to its high protein and iron content, ability to withstand shifts in salinity and temperature, and survival in oxygen depleted water. In view of the above, this study was undertaken to evaluate the effects of varying levels of dietary l-histidine on growth, protein gain, histidine retention, hematological and body composition of this fish so as to optimize the inclusion of histidine in its commercial feeds. Casein-gelatin based isonitrogenous (380gkg⁻¹ CP) and isocaloric (17.9MJkg⁻¹ GE; 15.3MJkg⁻¹ DE) amino acid diets with six different l-histidine levels (5.0, H1; 6.5, H2; 8.0, H3; 9.5, H4; 11.0, H5; 12.5gkg⁻¹dry diet H6) were fed to quadruplicate groups of fingerling Singhi (6.6±0.2g) twice daily at 07:00 and 17:30h to apparent satiation for 8week. Feeding trial was conducted in a flow-through system (1–1.5Lmin⁻¹) at 28°C water temperature. Growth, feed conversion, protein gain and histidine retention of fingerling Singhi were found to increase significantly with the quantitative increase in dietary histidine from 5.0 (H1) to 9.5gkg⁻¹ diet (H4). Hepatosomatic index, condition factor, hematocrit and hemoglobin were significantly affected in fingerling Singhi fed different concentrations of histidine and were also found to be best in fish fed diet H4. Quadratic regression analysis of the above parameters at 95% maximum and minimum plateau indicated that inclusion of histidine at 9.4gkg⁻¹ of the dry diet, corresponding to 24.8gkg⁻¹ dietary protein is optimum in formulating histidine-balanced, cost-effective, commercial feeds for intensive culture of Singhi.

Author(s): Farhat , Mukhtar A. Khan