Volume 5 Special Issue 1 2011
Aquaculture II
~Sustainable Aquaculture~

ISBN 978-4-903313-9-5

Guest Editor

Riccardo Russo

Public Health Research Institute - University of Medicine and Dentistry of New Jersey, USA

www.umdnj.edu

CONTENTS AND ABSTRACTS

Esther Gómez, Jessica Méndez, Roberto Navais, Pilar Reimundo, David Pérez-Pascual, José A. Guijarro (Spain) Genetic Approaches for the Identification of Genes Involved in the Pathogenesis of Bacterial Infections in Fish (pp 1-11)

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Invited Mini-Review: Over the last 15 years new approaches have been developed for the identification and study of the functions of genes and proteins involved in the virulence mechanisms of bacteria. Some of these approaches are based on the identification of genes required by the bacteria to survive and grow inside the host. Most of these techniques have recently been applied to bacterial fish pathogens successfully. Some of these techniques involve the “In vivo expression technology (IVET)”, which identifies specific promoters expressed in bacteria grown inside the fish body but not in conventional culture media; “Signature-tagged mutagenesis (STM)”, a useful tool for the screening of null mutants which show a decreased virulence capability in the host; and “Suppression Subtractive Hybridisation (SSH)”, a genome-wide technology, which enables the identification of genes present only in virulent strains of bacteria or the identification of genes expressed only inside the fish host. This paper reviews the application of these techniques to important pathogenic bacteria for the aquaculture field.

Sofia Carbajosa, Paloma Encinas, Amparo Estepa, Julio Coll (Spain) Possible Applications to Aquaculture of Salmonid Sleeping Beauty Transposon Vectors (pp 12-20)

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Invited Review: The sleeping beauty (SB) vectors have been designed from transposons “sleeping” in thousands of incomplete copies in the salmon/trout genomes. The SB system consists of one vector containing the transposase enzyme and of second vector carrying the inverted terminal repeats flanking an exogenous gene. The SB dualvectors system originated from fish, but it has been mostly used in mammals that lack these transposon systems. Several human clinical trials for gene therapy are using this SB system. Some of the advantages of SB vectors include the introduction of a defined, non-viral sequence as a single copy with each transposition (instead of a random recombination), thus avoiding the carrier over of prokaryotic sequences. This manuscript reviews the development of SB transposase versions and their derived vectors for possible use in fish cultured at a commercial level. There is a potential use for the SB vectors for the creation of transgenic fish, for the improvement of fish DNA vaccines and for the identification of genes in fish that might be important to improve the culture of these animals. However, the presence of thousands of incomplete SB transposon-related copies in many fish genomes and the possible activity of their endogenous SB transposases might interfere with the use of SB vectors in fish. Those possible limitations will depend on the fish specie and will have to be investigated in each case.

Fatimah Md. Yusoff, Sanjoy Banerjee, Helena Khatoon, Mohamed Shariff (Malaysia) Biological Approaches in Management of Nitrogenous Compounds in Aquaculture Systems (pp 21-31)

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Invited Review: Aquaculture is the fastest growing food-producing sector accounting for almost 43% of the world’s food fish. There is however a need to increase aquaculture production in the next two decades in order to satisfy the minimum protein requirement for human nutrition. There are many constraints that limit the maximum production in aquaculture systems such as water quality and adequate live feeds. With the development of modern aquaculture farming, extensive culture has given way to intensive culture systems. In intensive systems, cultured organisms are fed protein-rich formulated feeds. Uneaten feed along with metabolic wastes and other organic matters decompose resulting in an increase of toxic nitrogenous compounds causing deterioration of water quality which is toxic to cultured organisms. The discharge of a large amount of nutrient-rich wastes from these aquaculture systems, the majority of which are nitrogenous compounds, promotes eutrophication in water bodies. In general, an increase of nitrogenous compounds has adverse effects on the environment and on aquaculture production. The aim of this paper is to highlight some of the trends in biological management of nitrogenous substances in aquaculture systems.

Constantina Nasopoulou, Haralabos C. Karantonis, Ioannis Zabetakis (Greece) Nutritional Value of Gilthead Sea Bream and Sea Bass (pp 32-40)

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Invited Mini-Review: Fish and fish oil have a high nutritional value and protect against cardiovascular diseases. Given the intrinsic difficulties in sustaining the availability of wild fish, the industry has moved, over the past three decades, towards establishing aquaculture units where fish are produced under controlled conditions. Sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) are the two most commercial aqua cultured species in Europe. In this mini review, the studies of the nutritional value of these two species are evaluated in terms of contents of ω-3 fatty acids and micro constituents of fish lipids with a particular focus on their ability to inhibit or not the initiation of atherogenesis. Platelet Activating Factor (PAF), a key trigger molecule in atherogenesis, and PAF antagonists, that are present in fish, play a crucial role in inhibiting the formation of atheromatic lesions. The diet of aqua cultured fish is also evaluated for its contents of ω-3 fatty acids and its capacity to inhibit PAF activity. New trends are presented towards modifying this diet to produce fish with a higher nutritional value.

Francesco Buonocore, Elisa Randelli, Daniela Casani, Catia Marozzi, Giuseppe Scapigliati (Italy) Cloning of Interferon in Sea Bream (Sparus aurata): An Important Marker for Immune Response after Viral Infection (pp 41-48)

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Original Research Paper: The interferons (IFNs) are soluble cytokines involved in the immune response against viral pathogens. In mammals, three families of IFNs have been identified (types I, II and III) and, recently, homologues of type I have been found in different fish species. In this paper we report the cloning of IFN genes from sea bream (Sparus aurata), expression analysis and gene structure. The sea bream IFN cDNA consists of 914 bp that translate in one reading frame to give the entire molecule containing 186 amino acids. The analysis of the sequences revealed the presence of a putative 20 amino acid signal peptide, two cysteine residues and two potential N-glycosylation sites and, finally, a high percentage of identity with type I IFN from mammals compared to type III IFN. Real time PCR was performed after poly I:C stimulation of head kidney leukocytes from sea bream to investigate the expression of sea bream IFN and Mx and an induction was observed for both genes at different time points and with different intensities. The sea bream IFN genes contains four introns, as type III IFN from mammals. The presented results will give the opportunity to investigate more in detail antiviral immune responses, vaccination and immunostimulation effects in sea bream, an economical important species for the South Mediterranean aquaculture.

Jenny Rodríguez, Ricardo Cedeño, Bonny Bayot, Fabrizio Echeverría (Ecuador), Jaime A. Teixeira da Silva (Japan), Alejandra Valladares, Daniel Aguayo, Stanislaus Sonnenholzner (Ecuador) Effects of the Vibrio alginolyticus Probiotic, b-1,3/1,6-Glucans and Temperature on Shrimp Production (pp 49-55)

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Original Research Paper: The main task of this research was to test certain health management strategies for cultured shrimp in order to improve overall production variables (yield, survival rate, weight and feed conversion ratio - FCR). To do so, four thorough studies were conducted with an emphasis on the use of three factors: the probiotic Vibrio alginolyticus (Ili strain), b-1,3/1,6-glucans and controlled water temperature. In the first experiment 45-day old postlarvae (PL45) were fed (or not) b-1,3/1,6-glucans, and the water temperature was kept at 24 or 31°C. As a consequence, less shrimp with white spot disease (WSD) injuries were detected when they were fed b-1,3/1,6-glucans and kept at 31°C. In the second experiment, the effect of administrating b-1,3/1,6-glucans from a nursery phase was tested. Larvae (Nauplius 5 - PL4) were reared with the probiotic. At the nursery and grow-out phases shrimp were separated into two groups: the first received b-1,3/1,6-glucans while the other did not. In the ponds where animals received immunostimulants, yield increased while the feed conversion ratio became lower. A third experiment was designed to determine the effect of applying the probiotic at an early stage of larviculture. Shrimp received (or not) the probiotic at early stages and were fed (or not) b-1,3/1,6-glucans in the grow-out phase. Shrimp yield and FCR increased and decreased significantly, respectively, if the larvae received only the probiotic. A fourth trial was designed based on the results of the first three experiments. Two management protocols were compared. In the first, shrimp received the probiotic in the early larviculture phase. In the second protocol, shrimp received the probiotic at the early larviculture stage and then were fed b-1,3/1,6-glucans during the nursery and grow-out stages. The resulting output was significantly improved using the second protocol.

Alfredo Mena-Herrera, Marco Agustín Liñán-Cabello, Fernando Díaz-Herrera (Mexico) Effects of Salinity on Electrolyte Levels (Na⁺, K⁺, and Cl^-) in the Hemolymph of the Pacific White Shrimp Litopenaeus vannamei (pp 56-59)

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Original Research Paper: Pacific white shrimp (Litopenaeus vannamei) were consecutively exposed for 3 h each to salinity of 40, 30, 20, 10 and 0‰, and the concentrations of Na⁺, K⁺, and Cl^־ in their hemolymph were measured. In females, the Na⁺ concentrations in hemolymph were highest (304.1 mmol/l) after exposure to 20‰ salinity and the K⁺ concentrations (5.32 mmol/l) were highest at 10‰ salinity, meanwhile in males, the Na⁺ concentrations in hemolymph were highest (316.0 mmol/l) after exposure to 40‰ salinity and the K⁺ concentrations (5.62 mmol/l) were highest at 30‰ salinity. The Cl^־ concentrations in males decreased with salinity, with concentrations of 261.5, 254.6, 237.1, 224.5, and 191.0 mmol/l at 40, 30, 20, 10 and 0‰ salinity, respectively. Similar decreases were seen in females, except that the Cl^־ concentration at 10‰ salinity was 247.6 mmol/l. The overall interaction effect of Na⁺, K⁺, and Cl^־ differed significantly in males and females (p < 0.05). These results indicate that hemolymph electrolyte concentrations generally decrease with salinity, indicating that shrimp osmoregulatory and ionoregulatory activities adjust to ionic gradients in their environment.

Terri F. Sutherland, Phillip A. Yeats (Canada) A Comparison of Analytical Digestion Techniques for the Detection of Elemental Tracers Associated with Marine Finfish Aquaculture (pp 60-65)

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Original Research Paper: Two different digestion techniques for trace-element analysis were carried out on marine sediment samples collected in the Broughton Archipelago, British Columbia, Canada, to compare their utility in terms of identifying excess element concentrations using a geochemical normalization technique. The first technique consisted of a partial digestion of sediment samples using the Strong Acid Leachable Metals (SALM) method, while the second technique carried out a total digestion of sample material using hydrofluoric (HF) and nitric (HNO₃) acids. Relationships observed between HF- and SALM-derived trace-element concentrations for Cd, Ca, Cr, Cu, Fe, Pb, Li, Mg, Mo, Ni, P, Na, S, V, and Zn were stronger than those for trace-elements, Al, As, Ba, Be, Co, Mn, K, Sr, Sn, Ti, and U. Regression analysis of the combined data set of the former group showed a strong correlation and an offset from unity between the congruent trace-element concentrations. The weak relationships observed for the latter group were attributed to resolution associated with detection-limit issues (As, Be, Sn, and U) and/or sample heterogeneity (Al, Ba, Co, Mn, K, and Ti). Geochemical normalization against lithium (Li) allowed for the quantification of “excess” levels of trace-element tracers and redox indicators. Both digestion methods clearly identified samples containing excess levels of both direct (Cu, Zn, P) and indirect (Mo) aquaculture tracers through the Li-normalization technique. Stronger element:Li correlations were observed for SALM methods in comparison to HF. Although the SALM method involves a partial digestion of trace-elements, it is useful in identifying both direct and indirect tracers of aquaculture activities once combined with Li-normalization, which can be used to complement benthic organic enrichment indicators used in environmental monitoring programs.

Terri F. Sutherland, Phillip A. Yeats (Canada) Elemental Indicators of Benthic Organic Enrichment Associated with Marine Finfish Aquaculture (pp 66-75)

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Original Research Paper: Sediment samples were collected from the west and east coasts of Canada and analyzed for trace-element content to identify inorganic tracers and indicators of aquaculture waste material and benthic organic enrichment events. Samples were collected at varying distances from fish-farm sites in the Broughton Archipelago (BA), British Columbia and in Letang Inlet, southwest New Brunswick (SWNB). The results from a principal component analysis revealed that copper (Cu), molybdenum (Mo), phosphorus (P), and zinc (Zn) were consistently associated with established aquaculture indicators of benthic organic enrichment (porewater sulfide, redox potential, and/or organic matter) across different operational (active vs fallow) and oceanographic/bathymetric settings (archipelago vs estuarine). Although cadmium (Cd) and uranium (U) were linked to this aquaculture tracer/indicator grouping in SWNB, they did not show similar trends in BA, likely due to higher background concentrations (Cd) and analytical detection issues (U). In general, these findings suggest that certain trace-elements (Cu, P, Zn) serve as direct tracers of aquaculture waste material (feed, faeces and anti-fouling agents), while other trace-elements (Cd, Mo, U) serve as indirect “redox” indicators of benthic organic enrichment and associated sediment anoxia. Geochemical normalization allowed for the quantification of “excess” levels of tracers and redox indicators and, in turn, permitted correlation of these estimates with other aquaculture indicators (organic matter), spatial gradients, and meiofaunal diversity. In the BA, excess concentrations of Cu, Mo, P and Zn showed strong inverse relationships with distance from net pen systems as well as with meiofaunal abundance (Kinorhyncha). Principal component analysis and Li-normalization facilitates identification of both direct and indirect tracers of aquaculture activities that can be used to complement benthic organic enrichment indicators in environmental monitoring programs.

Riccardo Russo (USA) An Attenuated Strain of Edwardsiella ictaluri is Killed by Channel Catfish (Ictalurus punctatus) Macrophages and Confers Protection in Few Days (pp 76-82)

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Original Research Paper: In channel catfish protective immunity against Edwardsiella ictaluri is mediated by cellular immune response, but E. ictaluri is able to survive inside macrophages. The aim of this study was to determine if catfish macrophages were able to kill an avirulent strain of E. ictaluri and to observe if there was a difference in the dispersion in the catfish body among virulent and avirulent E. ictaluri. Channel catfish macrophages were exposed to a virulent or to an attenuated strain of E. ictaluri transformed with plasmids coding a green or a red fluorescent protein. Macrophage killing efficiency, reactive oxygen species and nitric oxide production were determined. Catfish were intraperitoneally (IP) injected with fluorescent virulent or attenuated E. ictaluri to determine the location of the bacteria within internal organs of the fish. Catfish were also IP injected or bath exposed to the attenuated strain of E. ictaluri 3, 2, or 1 day before, or at the same time of the challenge with the virulent strain of E. ictaluri. The virulent strain of E. ictaluri survived and replicated inside macrophages, while the attenuated strain was killed. Fluorescent virulent bacteria were observed in all organs of the fish and attenuated bacteria were observed only in lymphoid organs. Injection of the attenuated strain of E. ictaluri at least one day prior to challenge conferred protection (survival from 83 to 100%) in fish and upregulated the expression of the toll-like receptor 5. The results of this study suggest that the channel catfish immune system can kill and control the dispersion of the attenuated E. ictaluri strain within the fish. The protection conferred by the E. ictaluri attenuated strain highlights the possibility to use this strain as a “therapeutic” during disease outbreak.

Ratanaporn Leesing (France/Thailand), Daniel Dijoux, Doan Duy Le Nguyen, Gérard Loiseau (France), Ramesh Chandra Ray (France/India), Didier Montet (France) Improvement of DNA Extraction and Electrophoresis Conditions for the PCR-DGGE Analysis of Bacterial Communities Associated to Two Aquaculture Fish Species (pp 83-87)

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Original Research Paper: Improvement of a denaturing gradient gel electrophoresis (DGGE) method for analyzing bacterial microbiota 16S rDNA in fish is presented. Tilapia and sea bass were collected from different locations in Thailand and France, respectively. The DGGE electrophoresis conditions were optimized which indicated that a decrease in electrophoresis applied voltage and an increase in the running time (80 V for 12 h) provided optimal band resolution. DNA extraction procedure was also optimized. PCR-DGGE analysis patterns of bacterial DNA from tilapia and sea bass showed that there was a unique band pattern for each type of fish and dissimilarity was observed even within the same species. The differences in bacterial community in fish were probably due to different locations of collections.

Thaís Billalba Carvalho, Eliane Gonçalves-de-Freitas (Brazil) Social Stability Related to Sex Group Composition in the Cichlid Nile Tilapia (pp 88-91)

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Research Note: Aquaculture practices usually put the Nile tilapia in an artificial social environment, which males predominate due to their faster growth desirable for aquaculture purposes. Such a situation can increase male-male fighting because males are generally more aggressive than females, and also because fighting ability is similar within the same sex, leading to longer contests. As behavior has been used to infer welfare in several fish species, the aim of this study was to investigate whether sex composition affects agonistic interactions, social hierarchy and energetic demand in groups of Nile tilapia (Oreochromis niloticus; L.). Size-matched adult fish were divided in two treatments: MM = four males and MF = two males and two females (10 repetitions for each treatment). The experiment lasted for 11 days and social interactions (aggressiveness and rank order) were recorded at the 2^nd, 6^th and 10^th days (15 min per day). Fish were food deprived and body weight loss was used to infer energetic cost. A higher frequency of lateral threat (Student’s t independent test; t = 2.55; p = 0.02) and total interactions (Student’s t independent test; t = -2.81; p = 0.01) was observed in the MF treatment. MM group showed unstable hierarchy (Binomial test, p = 0.04), which is considered a social stressor. However, mean weight loss was not affected by treatments (Student’s t independent test; t = -0.74; p = 0.47). These results support the idea that sexual composition affects aggressive interactions and destabilizes social hierarchy, but not energy cost.

Emmanuel M. Vera Cruz (USA/The Philippines), Christopher L. Brown (USA) Dynamics of Increase in Insulin-like Growth Factor-I mRNA Expression in Nile Tilapia, Oreochromis niloticus,in Response toElevated Temperature (pp 92-97)

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Original Research Paper: Insulin-like Growth Factor-I (IGF-I) is a physiological mediator and a potentially important growth indicator candidate in teleost fishes. In this study, the effects of increased temperature on the growth and hepatic IGF-I gene expression in Oreochromis niloticus were evaluated. Twenty all-male fish were reared separately at temperatures below 24°C for 12 days and then water temperature in 15 aquaria was gradually raised to 30°C within a day. Growth and hepatic IGF-I gene expression in five fish were obtained before the temperature change and after 2, 5 and 7 days of increasing the water temperature. The growth rate of the fish reared in the warmer temperature for 2, 5 and 7 days was significantly increased in a time dependent manner (r = 0.93). Mean hepatic IGF-I mRNA levels in fish reared at warm temperature for 2, 5 and 7 days were elevated 1.6-, 2.5-, and 3.6-fold, respectively compared to that of fish reared at cold temperature (<24°C). The IGF-I levels were significantly elevated after at least 5 days of exposure to warm temperature, which is consistent with the idea that hepatic IGF-I gene expression can be used as a short-term growth rate indicator for O. niloticus. A significant positive correlation was observed between days of rearing at warm temperature and hepatic IGF-I levels (r = 0.92); between specific growth rate (length) and IGF-I levels (r =0.92); and between condition factor and IGF-I levels (r = 0.55). The high positive association between IGF-I mRNA and growth rate validated the assertion that hepatic IGF-I levels are sufficiently sensitive to be used as instantaneous growth rate indicator in this species of fish.