Aquaculture research, tập 41, số 4, 2010

Aquaculture Research, 2010, 41, 451^467 doi:10.1111/j.1365-2109.2009.02339.x REVIEW ARTICLE Lactic acid bacteria vs pathogens in the gastrointestinal tract of fish: a review Einar Ringệ1,2, Lisbeth Lệvmo1, Mads Kristiansen1,Yvonne Bakken1w, Irene Salinas3, Reidar Myklebust4, Rolf Erik Olsen2 & Terry M Mayhew5 Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsệ,Tromsệ, Norway Institute of Marine Research, Bergen, Norway Fish Innate Immune System, Department of Cell Biology, University of Murcia, Murcia, Spain Institute of Anatomy and Cell Biology, University of Bergen, Bergen, Norway School of Biomedical Sciences, Queens Medical Centre, University of Nottingham, Nottingham, UK Correspondence: E Ringệ, Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsệ, N-9037 Tromsệ, Norway E-mail: einar.ringo@nfh.uit.no Present address: Lisbeth Lệvmo, Granễsveien 34, 7048 Trondheim, Norway w Present address: Yvonne Bakken, Skretting, 8450 Storkmarknes, Norway Abstract Intensive Âsh production worldwide has increased the risk of infectious diseases However, before any infection can be established, pathogens must penetrate the primary barrier In Âsh, the three major routes of infection are the skin, gills and gastrointestinal (GI) tract The GI tract is essentially a muscular tube lined by a mucous membrane of columnar epithelial cells that exhibit a regional variation in structure and function In the last two decades, our understanding of the endocytosis and translocation of bacteria across this mucosa, and the sorts of cell damage caused by pathogenic bacteria, has increased Electron microscopy has made a valuable contribution to this knowledge In the Âsh-farming industry, severe economic losses are caused by furunculosis (agent, Aeromonas salmonicida spp salmonicida) and vibriosis [agent, Vibrio (Listonella) anguillarum] This article provides an overview of the GI tract of Âsh from an electron microscopical perspective focusing on cellular damage (speciÂc attack on tight junctions and desmosomes) caused by pathogenic bacteria, and interactions between the good intestinal bacteria [e.g lactic acid bacteria (LAB)] and pathogens Using diĂerent in vitro methods, several studies have demonstrated that co-incubation of Atlantic salmon (Salmo salar L.) foregut (proximal intestine) with LAB and pathogens can r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd have beneÂcial eĂects, the cell damage caused by the pathogens being prevented, to some extent, by the LAB However, there is uncertainty over whether or not similar eĂects are observed in other species such as Atlantic cod (Gadus morhua L.) When discussing cellular damage in the GI tract of Âsh caused by pathogenic bacteria, several important questions arise including: (1) Do diĂerent pathogenic bacteria use diĂerent mechanisms to infect the gut? (2) Does the gradual development of the GI tract from larva to adult aĂect infection? (3) Are there diĂerent infection patterns between diĂerent Âsh species? The present article addresses these and other questions Keywords: probiotics, pathogenic bacteria, gut integrity, Âsh Introduction With the development of commercial aquaculture, it has become apparent that diseases can be a signiÂcant limiting factor Major bacterial pathogens of Âsh include the Gram-negative species, Aeromonas salmonicida,Vibrio (Listonella) anguillarum,Vibrio (Aliivibrio) salmonicida and Yersinia ruckeri, the aetiological agents of furunculosis, vibriosis, cold-water vibriosis and red mouth disease respectively In addition, Aeromonas hydrophila may cause infections in Âsh and is 451 Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Aquaculture Research, 2010, 41, 451^467 generally associated with small surface lesions, sloughing of scales, local haemorrhage and septicaemia All these diseases are common worldwide and produce considerable economic losses during intensive aquaculture of trout and salmon (Austin & Austin 1999) The best way to avoid disease problems in a system seems to be through eĂective management practices, i.e management of stock, soil, water, nutrition and environment A number of other approaches have been applied in an attempt to address the disease problem including sanitary prophylaxis, disinfection and chemotherapy, with particular emphasis on the use of antibiotics The application of antibiotics and other chemicals to pond culture is also quite expensive and undesirable and might lead to antibiotic resistance In Norway, the use of antimicrobial drugs has decreased from approximately 50 metric tonnes in 1987 to 746.5 kg in 1997, measured as active components (Verschuere, Rombaut, Sorgeloos & Verstraete 2000) In 2007, use was still at the same low level as in 1997 (Norwegian ScientiÂc Committee for Food Safety 2009) and, recently, vaccinations against speciÂc diseases have been used Intensive Âsh production has increased the risk of infectious diseases worldwide (Press & Lillehaug 1995; Karunasagar & Karunasagar 1999) but, to prevent microbial entry, Âsh deploy protective mechanisms to hinder translocation of pathogens across the primary barriers These include production of mucus by goblet cells, the apical acidic microenvironment of the intestinal epithelium, cell turnover, peristalsis, gastric acidity, lysozyme and antibacterial activity of epidermal mucus (Birkbeck & Ringệ 2005) At the same time, pathogenic microorganisms have evolved mechanisms to penetrate these barriers Probiotics may reduce the incidence of disease or lessen the severity of outbreaks One of the proposed deÂnitions of probiotics used in aquaculture is live microbial cultures added to feed or environment (water) to increase viability (survival) of the host (Gram & Ringệ 2005) Probiotic mechanisms include the production of inhibitory substances against pathogens, competition for essential nutrients and enzymes resulting in enhanced nutrition in the host and the modulation of interactions with the environment and development of beneÂcial immune responses (Ringệ & Gatesoupe 1998; Verschuere et al 2000; Balcazar, de Blas, Ruiz-Zarzuela, Cunningham, Vendrell & Muzquiz 2006; Gomez & Balcazar 2008) Today, it is generally accepted that lactic acid bacteria (LAB) form part of the normal intestinal micro- 452 biota of Âsh from the Ârst few days of life (Ringệ & Gatesoupe 1998; Ringệ 2004; Ringệ, Schillinger & Holzapfel 2005) One of the most important goals for microbiologists has been to obtain a stabile indigenous microbiota in Âsh The practical eĂect of this activity is the exclusion of invading populations of non-indigenous microorganisms, including pathogens that attempt to colonize the gastrointestinal (GI) tract (Ringệ et al 2005) The antagonistic eĂects of gut microbiota against pathogens and other organisms are possibly mediated by competition for nutrients and adhesion sites, formation of metabolites such as organic acids and hydrogen peroxide and production of bacterocins (for a recent review, devoted to antimicrobial activity of LAB isolated from aquatic animals, see Ringệ et al 2005) A fundamental question arises when discussing the protective role of the GI tract microbiota: can the GI tract of Âsh serve as a port of entry for pathogens? During the last 25 years, numerous papers have suggested that the alimentary tract is involved in Aeromonas and Vibrio infections (GroĂ & LaPatra 2000; Birkbeck & Ringệ 2005; Harikrishnan & Balasundaram 2005; Ringệ, Salinas, Olsen, Nyhaug, Myklebust & Mayhew 2007; Ringệ, Myklebust, Mayhew & Olsen 2007; Salinas, Myklebust, Esteban, Olsen, Meseguer & Ringệ 2008) Therefore, one can hypothesize that LAB and other beneÂcial bacteria colonizing the GI tract by producing, for example, bacterocins may oĂer protection against invading Âsh pathogens The aim of this review is to present information on the interaction between beneÂcial bacteria, in our case LAB, and pathogenic agents in the digestive tract of Âsh using in vitro methods Pathogens and cell damage Historically, Aeromonas salmonicida ssp salmonicida (A salmonicida), the causative agent of furunculosis, has been recognized as one of the most important bacterial salmonid pathogens because of its severe economic impact, especially on the aquaculture industry (Olivier 1997; Bricknell, Bron & Bowden 2006) As early as 1930, the Furunculosis Committee suggested the intestine as a valuable site for isolating the bacteria (Mackie, Arkwright, Pyrce-Tannatt, Mottram, Johnston & Menzies 1930) Since then, controversy has existed as to whether or not the gut can function as an infection route for this and other pathogenic bacteria The presence of A salmonicida in the intestine of some Âsh species (Ringệ, Olsen, ềverli & Lệvik 1997; Lệdemel, Mayhew, Myklebust, r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Olsen, Espelid & Ringệ 2001; Pedersen & Dalsgaard 2003), together with evidence that salmonids fed diets containing probiotic bacteria or soybean meal showed changes in mortality rate after cohabitant challenge with A salmonicida (Krogdahl, BakkeMcKellep, Rệd & Bìverfjord 2000; Robertson, ODowd, Burrels, Williams & Austin 2000), indicates that the intestine can be an important route of infection It is known that pathogenic bacteria produce a wide array of virulent factors (including haemolysins, cytotoxins, enterotoxins, endotoxins and adhesins), which can aĂect intestinal barrier function and facilitate translocation (Chopra, Xu, Ribardo, Gonzalez, Kuhl, Peterson & Houston 2000) Translocation mechanisms include increased receptor-mediated endocytosis (Skirpstunas & Baldwin 2002), increased paracellular permeability mediated by eĂects on junctional complexes and the cytoskeleton, and direct damage to the intestinal cells (Fasano 2002) In Âsh, bacterial pathogens can enter the host by one or more of three diĂerent routes: (a) skin, (b) gills and (c) GI tract (Birkbeck & Ringệ 2005; Ringệ, Myklebust, et al 2007) If the GI tract is involved as an infection route, mucosal adhesion is considered to be a critical early phase in all infections caused by pathogenic bacteria (Knudsen, Sệrum, McLPress & Olafsen 1999; Namba, Mano & Hirose 2007) When the bacteria are able to colonize the intestinal mucus, they can cross the GI tract lining by transcellular or paracellular routes However, it is suggested that translocation of bacteria across the intestine, an essential and prerequisite step for bacterial invasion, cannot be studied eĂectively using in vivo models Table1presents an overview of in vivo and in vitro studies of cell damage in the GI tract of Âsh Two diĂerent in vitro methods, the Ussing chamber and intestinal sac, have been used to evaluate translocation and cell damage caused by pathogenic bacteria (Ringệ, Jutfelt, Kanapathippillai, Bakken, Sundell, Glette, Mayhew, Myklebust & Olsen 2004; Jutfelt, Olsen, Glette, Ringệ & Sundell 2006; Lệvmo 2007a, b; Ringệ, Salinas, et al 2007; Salinas et al 2008) The translocation of viable bacteria from the digestive tract into enterocytes has been reported in several investigations (for a review, see Ringệ, Olsen, Mayhew & Myklebust 2003; Ringệ, Myklebust, et al 2007) The phenomenon has mainly been observed for non-pathogenic indigenous gut bacteria, but not for pathogenic bacteria, and does not normally compromise cellular integrity The situation for pathogenic bacteria is completely diĂerent, as severe damage with loss of cellular integrity has been noted in an in vitro study where the foregut of Atlantic salmon was exposed toA salmonicida (Ringệ et al 2004) as well as in vivo infection of Atlantic salmon (Bakken 2002) and spotted wolsh fry (Anarhichas minor Olafsen) by V anguillarum (Ringệ, Mikkelsen, Kaino, Olsen, Mayhew & Myklebust 2006) As no cell damage was observed in control Âsh (not exposed to pathogenic bacteria), we concluded that the indigenous bacteria not aĂect cellular integrity Ringệ et al (2004) used the Ussing chamber technique and observed detached, but almost intact, enterocytes in the foregut lumen after exposure to A salmonicida in vitro A similar result has been reported in the pyloric caeca of Atlantic salmon in an in vivo challenge experiment (Fig 1) and in an in vitro intestinal sac preparation (Ringệ, Salinas, et al 2007; Salinas et al 2008) However, in the in vitro experiment of Ringệ et al (2004), a quite diĂerent situation seems to occur in the hindgut region (distal intestine) as no intact enterocytes were found in the lumen, the microvilli were disintegrating and some damage to intercellular tight junctions and desmosomes was observed The diĂerences between foregut and hindgut have not been elucidated, but it is probable that enterocytes in diĂerent regions of the GI tract vary in their susceptibility to pathogen-induced damage This may be linked to diĂerent regional rates of epithelial turnover or diĂerent mechanisms of enterocyte loss by apoptosis or necrosis (Mayhew, Myklebust,Whybrow & Jenkins 1999) Apoptosis-dependent processes tend to preserve junctional integrity while necrosis-like processes tend to be associated with junctional complex disruption and loss of microvillous morphology (T M Mayhew, pers comm.) In vitro exposure of Atlantic salmon foregut to Vibrio anguillarum at two concentrations [6 104 and 106 colony-forming units (CFU) mL 1] resulted in clear changes in the intestinal epithelium compared with samples exposed only to Ringer solution (control) (Ringệ, Salinas, et al 2007) At the highest dose there was an inÊammatory response of gut-associated lymphoid tissue involving leucocytes migrating from the lamina propria towards the lumen The diĂerence in bacterial translocation between indigenous intestinal bacteria and pathogens might be due to the production, by pathogens, of various virulence factors such as extracellular enzymes, outer surface components such as S-layer or secretory proteins and pore-forming toxins (Fivaz & van der Goot 1999) These factors could result in severe cell damage as demonstrated in the foregut of Atlantic salmon (Ringệ et al 2004) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 453 454 FG FG HG and HGC HG and HGC In vitro In vitro In vitro In vitro Carnobacterium divergens Vibrio anguillarum Carnobacterium maltaromaticum V anguillarum In vitro Edwardsiella tarda Intestine In vitro PC, MG, HG, HGC in vivo HG FG In vitro/ In vivo V anguillarum Different bacteria In vivo V anguillarum FG NI NI NI Yes NI NI Yes Yes Yes Yes Yes Yes NI NI NI Yes NI NI NI NI NI NI NI NI NI NI to mucus Adhesion wAs only 13 autochthonous bacterial strains were isolated one can not draw a general conclusion zUssing chamber PC, pyloric cacea; FG, foregut; MG, midgut; HG, hindgut; HGC, hindgut chamber; NI, not investigated Intestinal sac Several fish species Spotted wolffish Vibrio alginolyticus Five probiotics, A hydrophila and In vitroz A salmonicida Spotted grunter HG In vitroz A salmonicida Intestine In vitro Three LAB strains and four pathogens Intestine Intestine Rainbow trout In vitro In vitro Listonella anguillarum and potential probiotics Intestine Lactobacillus L15 In virto Seven LAB of human and animal orgin Intestine Intestine Intestine Flounder Gilthead seabream In vitro Aeromonas hydrophila In vivo Common carp FG In vitro A salmonicida Three different LAB species FG In vitro Lactobacillus delbruăeckii Brown trout FG In vitro V anguillarum MG FG In vitro A salmonicida In vivo FG In vitro C divergens A salmonicida HG FG In vitroz In vitroz A salmonicida A salmonicida In vivo Aeromonas salmonicida Arctic charr Atlantic salmon PC segment Atlantic cod GIT used species in vivo Bacteria Fish In vitro/ Yes Yes Yes NI Yes No NI NI NI NI NI NI NI Yes Yes No Yes Yes No Less Yes Yes No No No No damage Cell Yes Yes No NI No No NI NI NI NI NI NI No Yes No No No No No No No No No No No No location Trans- NI NI NI NI NI NI NI NI NI NI NI NI microbiota Allochthonous NI NI NI NI NI NI NI NI NI Yes Yes microbiotaw Autochthonous microbiotaw Autochthonous microbiota Effect on gut NI NI NI NI NI NI NI NI NI NI NI NI Yes NI NI NI NI NI NI NI NI NI NI NI NI NI response Immune Ringứ et al (2007b) Ringứ et al (2006) Ringứ et al (2006) Vine et al (2004b) E Ringứ et al (unpubl obs.) (unpubl obs.) E Ringứ, R E.Olsen, K Sundell & R Myklebust Balcazar et al (2007) Ying, Lei, Jiazhong, Zhantao & Liguo (2007) Chabrillon, Arijo, Diaz-Rosales and Balebona (2006) Chabrillon et al (2006) Van der Marel et al (2008) Van der Marel, Schroers, Neuhaus & Steinhagen (2008) Balcazar et al (2007) Lứdemel et al (2001) Salinas et al (2008) Salinas et al (2008) Ringứ et al (2007) Ringứ et al (2007) Ringứ et al (2007) Ringứ et al (2004) Ringứ et al (2004) Bakken (2002) Lứvmo (2007b) Lứvmo (2007b) Lứvmo (2007a) Lứvmo (2007a) References Table In vitro and in vivo investigations demonstrating adhesion to mucus, cell damage, translocation and eĂect of gut microbiota caused by lactic acid bacteria and pathogenic bacteria in the gastrointestinal tract (GIT) of Âsh Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Aquaculture Research, 2010, 41, 451^467 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Figure Transmission electron microscopy micrograph of the pyloric caeca from Atlantic salmon (Salmo salar L.) challenge with Aeromonas salmonicida ssp salmonicida The enterocytes are damaged and their organelles are exposed to the gut lumen Several detached enterocytes are seen in the gut lumen (arrows) After Bakken (2002) MV, microvilli Human studies have shown that diĂerent bacteria species colonize diĂerent parts of the GI tract (Madigan, Martinko & Parker 2000), and that diĂerent pathogenic bacteria adhere to, and infect, diĂerent parts of the GI tract Ringệ et al (2004) suggested that the foregut of Atlantic salmon is an infection site for A salmonicida However, as loosening of cell junctions was observed in the hindgut, this region is also probably involved in A salmonicida infection but to a lesser extent than the foregut When discussing intestinal cellular damages, the results presented for the foregut by Ringệ et al (2004) are quite similar to the severe epithelial damage associated with intracellular fat accumulation from dietary linseed oil (Olsen, Myklebust, Ringệ & Mayhew 2000) In these studies, cell debris was also observed in the lumen, providing free access to the epithelial basal membrane As no information per se is available about cell damage and dietary components and pathogens, this topic should be given high priority in future studies, especially as there is increasing interest in substituting Âsh oils and Âsh meal with vegetable products GI microbiota and probiotics in fish Generally, probiotic strains have been isolated from indigenous and exogenous microbiota of aquatic animals Earlier publications stated that bacteria belonging to genus Photobacterium, Pseudomonas andVibrio were retrieved among the dominant genera in the in- testine of marine Âsh (Cahill 1990; Sakata 1990; Ringệ Strệm & Tabachek 1995) while the indigenous microbiota of freshwater Âsh species tend to be dominated by members of the genera Aeromonas, Plesiomonas, representatives of the family Enterobacteriaceae, and obligate anaerobic bacteria of the genera Bacteroides, Fusobacterium and Eubacterium (Cahill 1990; Sakata 1990; Ringệ et al 1995) Recent publications allow one to question whether this is true, particularly the knowledge and experience gained from bacteriological studies using, for example, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) (Grith, Melville, Cook, Vincent, St Pierre & Lanteigne 2001; Jensen, ềvreễs, Bergh & Torsvik 2004; Pond, Stone & Alderman 2006; Hovda, Lunestad, Fontanillas & Rosnes 2007; Kim, Brunt & Austin 2007; Liu, Zhou,Yao, Shi, He, Benjamisen Hệlvold & Ringệ 2008; Zhou, Liu, Shi, He,Yao & Ringệ 2009) It is important to note that the population of endogenous microbiota may depend on genetic, nutritional and environment factors However, microorganisms present in the immediate environment of aquatic species have a much larger inÊuence on health status than is the case with terrestrial animals or humans The gut microbiota of aquatic animals probably comprise indigenous microbiota together with artiÂcially high levels of allochthonous bacteria maintained by their constant ingestion from the surrounding water (Ringệ & Birkbeck 1999) While several studies on probiotics have been published during the last decade, the methodological and ethical limitations of animal studies make it dicult to understand the mechanisms of action of probiotics, and only partial explanations are available Nevertheless, possible beneÂts linked to administering probiotics have been suggested They include: (i) competitive exclusion of pathogenic bacteria (Moriarty 1997; Gomez-Gil, Roque & Turnbull 2000; Balcazar, de Blas, Ruiz-Zarzuela, Vendrell & Muzquiz 2004; Vine, Leukes & Kaiser 2004; Ringệ et al 2005; Baccazar, Vendrell, de Blas, Ruiz-Zarzuela, Girones & Muzquiz 2007); (ii) source of nutrients and enzymatic contribution to digestion (Prieur, Nicolas, Plusquellec & Vigneulle 1990; Sakata 1990; Ringệ & Birkbeck 1999); (iii) direct uptake of dissolved organic material mediated by the bacteria (Moriarty 1997); (iv) enhancement of the immune response against pathogenic microorganisms (Andlid, Vazquez-Juarez & Gustafsson 1995; Scholz, Garcia-Diaz, Ricque, CruzSuarez, Vargas-Albores & Latchford 1999; Rengpipat, Rukpratanporn, Piyatiratitivorakul & Menasaveta r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 455 Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Aquaculture Research, 2010, 41, 451^467 2000; Gullian & Rodr|Ô guez 2002; Irianto & Austin 2002a, b; Balcazar 2003; Balcazar et al 2004); and (v) antiviral eĂects (Kamei,Yoshimizu, Ezura & Kimura 1988; Girones, Jofre & Bosch 1989; Direkbusarakom, Yoshimizu, Ezura, Ruangpan & Danayadol 1998) Most probiotics proposed as biological control agents in aquaculture belong to the LAB group (Lactobacillus, Lactococcus, Carnobacterium, Pediococcus, Enterococcus and Streptococcus), genus Vibrio (Vibrio alginolyticus), genus Bacillus, genus Pseudomonas, genus Roseobacter although other genera or species have also been mentioned (Aeromonas, Alteromonas and Flavobacterium) Readers with special interest in the diĂerent aspects of the use of probiotics in aquaculture are referred to the comprehensive reviews of Ringệ and Gatesoupe (1998), Gatesoupe (1999), Ringệ and Birkbeck (1999), Skjermo and Vadstein (1999), Gomez-Gil et al (2000), Irianto, Roberwen, Austin and Pandalai (2000), Olafsen (2001), Verschuere et al (2000), Irianto and Austin (2002a), Ringệ (2002, 2004), Burr, Gatlin and Ricke (2005), Gram and Ringệ (2005), Hong, Duc and Cutting (2005), Ringệ et al (2005), Balcazar, de Blas, et al (2006), Balcazar, Decamp,Vendrell, de Blas and RuizZarzuela (2006), Farzanfar (2006), Vine, Leukes and Kaiser (2006), Gatesoupe (2007, 2008), KesarcodiWatson, Kaspar, Lategan and Gibson (2008) and Tinh, Dierckens, Sorgeloos and Bossier (2008) LAB It is well documented that LAB constitute a part of the indigenous gut microbiota of several Âsh species (Ringệ & Gatesoupe 1998; Ringệ 2004; Ringệ et al., 2005; Balcazar, de Blas, Ruiz-Zarzuela,Vendrell, Calvo, Marquez, Girones & Muzquiz 2007; Michel, Pelletier, Boussaha, Douet, Lautraite & Tailliez 2007) In most of these studies, diĂerent species of Carnobacterium have been isolated, but Lactobacillus species have also been isolated from the digestive tract of Âsh (for a review, see Ringệ & Gatesoupe 1998; Hagi,Tanaka, Iwamura & Hoshino 2004; Ringệ 2004; Ringệ et al 2005; Balcazar et al 2007; Michel et al 2007; Liu et al 2008) Carnobacterium divergens vs A salmonicida and V anguillarum in Atlantic salmon foregut In two reviews devoted to LAB in Âsh and Âsh farming, Ringệ (2004) and Ringệ et al (2005) suggested that LAB and other beneÂcial intestinal bacteria might be involved in the primary defence system 456 Table Experimental treatments applied to Atlantic salmon (Salmo salar L.) intestine during in vitro exposure to various bacterial strains (colony-forming units, CFU) Treatment number 10 11 Bacterial strain and dose (CFU mL 1) Salmon Ringer solution A salmonicida 106 V anguillarum 104 V anguillarum 106 C divergens 104 C divergens 106 A salmonicida 106 and C divergens 106 V anguillarum 104 and C divergens 104 V anguillarum 104 and C divergens 106 V anguillarum 106 and C divergens 104 V anguillarum 106 and C divergens 106 After Ringệ et al (2007) The estimate bacterial exposure of the foregut was measured by plate counts Stock solution was diluted in sterile 0.9% saline, and 0.1mL volumes of appropriate dilutions were spread on the surface of brainheart infusion agar (Merck, Darmstadt, Germany) (BHI) (Aeromonas salmonicida) and tryptic soy agar (TSA; Oxoid, London, UK) added glucose (5 g L 1) and NaCl (15 g L 1) TSA plates (V ibrio anguillarum and Carnobacterium divergens) against pathogenic colonization and adherence of pathogenic bacteria in the GI tract Several LAB isolated from Âsh and aquatic animals display antagonistic activity against Âsh pathogenic agents (Ringệ et al 2005; Ringệ 2008) To test the hypothesis that LAB can prevent pathogen-induced damage, Ringệ, Salinas, et al (2007) used C divergens strain 6251, originally isolated from the foregut of the Artic charr (Salvelinus alpinus L.) (Ringệ & Olsen 1999) This strain has growth-inhibitory eĂects against both A salmonicida and V anguillarum (Ringệ, Sepploa, Berg, Olsen, Schillinger & Holzapfel 2002; Ringệ 2008) The aim was to investigate, by means of light and electron microscopy, the structural changes that Atlantic salmon intestine underwent following in vitro exposure to A salmonicida, V anguillarum and C divergens at two diĂerent doses (Table 2) In the study by Ringệ et al (2004), only foregut samples were examined as this portion of the gut seems to be a more likely infection route for pathogenic bacteria in Atlantic salmon Furthermore, the potentially protective role of C divergens against pathogen-induced damage has been evaluated by simultaneously exposing intestinal mucosa to one of the pathogenic r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Table Morphological description of Atlantic salmon foregut treated with various types of bacteria (1^11; see Table 2) Bacterial treatment type Morphology 10 11 Presence of cell debris in lumen Disorganized microvilli Protruding epithelial cells Oedema Disintegrated tight junctions Dark cellular bodies under LM Loss of epithelial integrity Phagolysosome-like vesicles with bacteria Bacteria-like particles close to tight junctions Column totals 3 0 2 0 0 0 2 2 3 3 3 3 3 1 3 3 3 3 3 0 0 0 0 0 0 0 0 0 0 0 2 2 0 11 20 18 1 9 17 20 After Ringệ et al (2007) Damage and tissue changes were assessed as follows: not observed, 5low frequency, moderate frequency, high frequency Apparent diĂerences between treatments were signiÂcant (Po0.01) strains and to the probiotic strain Both pathogens used in the study of Ringệ, Salinas, et al (2007) have been shown previously to enter Âsh through the GI tract barrier (OToole, Hofsten, Rosqvist, Olsson & Wolf-Watz 2004; Ringệ et al 2004) As shown in Table 3, clear diĂerences were observed between the pathogenic (A salmonicida and V anguillarum) and the non-pathogenic (C divergens) strains Treatment of the foregut with pathogens, at both assayed concentrations, results in various damaging eĂects: epithelial cells with altered microvilli (Fig 2), damaged tight junctions, protruding epithelial cells sloughing into the lumen and the presence of cell debris in the gut lumen Similar Ândings were not observed following exposure to C divergens The diĂerent morphological changes caused byA salmonicida orV anguillarum co-incubated with C divergens also suggest diĂerent invasive and pathogenetic mechanisms between the two pathogenic bacterial strains The foregut exposed to C divergens was histologically similar to control samples showing an intact epithelial barrier This observation is in agreement with the results of Ringệ et al (2004), who suggested that the commensal microbiota not aĂect gut cellular integrity Surprisingly, exposure to V anguillarum at 104 mL results in the presence of phagolysosome-like vesicles containing degraded bacteria in Figure Scanning electron micrograph of the foregut of Atlantic salmon exposed to 106 Vibrio anguillarum Note several detached/detaching enterocytes lacking uniform microvilli at the epithelial surface After I Salinas, E Ringệ and R Myklebust (unpubl obs.) the cytoplasm of enterocytes An immunohistochemical study with anti-V anguillarum antibody might support the nature of these degraded bacteria, together with the observation of bacteria-like structures seen close to the tight junctions between enterocytes treated with V anguillarum and C divergens Whether these particles are the pathogenic or probiotic bacteria remains unclear, and identiÂcation of the unknown bacteria-like particles by immunogold labelling techniques, green Êuorescence protein and in situ Êuorescence hybridization might elucidate some of these questions and will be the subject of further work The Ânding of phagolysosome-like structures suggests that the enterocytes process the bacteria and act as phagocytic cells or as antigenpresenting cells in the same way as M-cells in higher vertebrates The histological changes described above were present in most samples, and so C divergens seems to be unable to completely prevent tissue damage by A salmonicida in Atlantic salmon foregut when the pathogen was present at the highest concentration In the case of V anguillarum at the lowest concentration (3 104 CFU mL 1), reduced tissue damage was observed when Carnobacterium was present at both a high and a low concentration, indicating a possible protective eĂect of C divergens However, this observation might also be attributable to the low concentration of the pathogen in groups in which the intestine was exposed to both the pathogen and the probiont, compared with foregut exposed only to the pathogen To clarify this hypothesis, additional studies are necessary r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 457 Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Aquaculture Research, 2010, 41, 451^467 Table Outcome of comparison of in vitro eĂects of experimental treatments on Atlantic salmon intestine (seeTable 2) ranked by order of severity from 5control (Ringer) to 11 5V ibrio anguillarum at 106 (Pages L test, Po0.01) Ranking Bacterial strain and dose (CFU mL 1) 23 56 Salmon Ringer solution C divergens 104 or 106 V anguillarum 104 and C divergens 106 V anguillarum 104 and C divergens 104; A salmonicida 106 and C divergens 106; A salmonicida 106 V anguillarum 106 and C divergens 104; V anguillarum 106 V anguillarum 106 and C divergens 106; V anguillarum 104 1011 After Ringệ et al (2007) The partially reduced tissue damage observed when the foregut was exposed to C divergens in combination withV anguillarum at the lowest population level may be explained by C divergens inhibiting the growth of the pathogens in vitro (Ringệ et al 2005; Ringệ 2008) and, thereby, colonization However, the probiotic bacteria not seem to reduce the tissue-damaging eĂect when the gut was exposed to a high concentration of V anguillarum The reason for this has not been elucidated, although it may be attributable to the pathogen out-competing the probiotic bacteria When C divergens was present at the highest dose, and the pathogen at the lowest dose, less damage was seen (Table 4) To some extent, this observation might be due to the antagonistic activity of the probiotic bacteria against the pathogens, resulting in fewer live pathogens being available to colonize the foregut Based on this hypothesis, more information on the administration of appropriate levels of probiotic bacteria is needed Lactobacillus delbrueckii ssp lactis vs A salmonicida in Atlantic salmon foregut It is generally accepted that Lactobacillus delbrueckii is a heterogeneous group of bacteria that includes three subspecies: ssp delbrueckii, ssp bulgaricus and ssp lactis (Weiss, Schillinger & Kansdler 1983) Jacobsen, Rosenfelt Nielsen, Hayford, Mệller, Michaelsen, Paerregaard, Sandstrm, Tvede and Jakobsen (1999) suggested that L delbrueckii ssp lactis is a good probiotic candidate according to its bacteriological prop- 458 erties in vitro From an aquaculture point of view, L delbrueckii ssp lactis has been administered in vivo in the diet to gilthead seabream (Sparus aurata L.) and increased the cellular innate immune responses (Salinas, Cuesta, Esteban & Meseguer 2005) The fate of probiotic bacteria in the GI tract of Âsh is less well known, including the histological changes that might take place in the gut following incubation with LAB like L delbrueckii ssp lactis The aims of the work of Salinas et al (2008) were, by means of microscopy techniques, to (i) determine whether an L delbrueckii ssp lactis (CECT 287,Valencia, Spain) of non-aquaculture origin was capable of colonizing the Atlantic salmon GI tract during in vitro incubation, (ii) describe the morphological changes and cellular responses occurring in the intestinal epithelium after in vitro exposure to this probiotic strain and (iii) study the possible protective role of L delbrueckii ssp lactis against tissue-damaging effects caused byA salmonicida in the foregut of Atlantic salmon This information is highly relevant as it is well known that probiotic bacteria increase the host health status and protect mucosal tissue against pathogen-caused damage in mammalian models (Fung, Woo, Wan-Abdullah, Ahmad, Easa & Liong 2009) Using confocal microscopy, their Ândings showed that, following short-term in vitro incubation of Atlantic salmon foregut with tetramethylrhodamine isothiocyanate (TRITC)-labelled L delbrueckii ssp lactis, the probiont was able to colonize the enterocyte surface.When L delbrueckii ssp lactis were observed in the lumen, the bacteria were found in groups or clumps (Fig 3a) Moreover, labelled bacteria were also found at the villus surface, inside the mucosal epithelium or even in the lamina propria (Fig 3b) As the intestines were thoroughly washed three times before samples were taken, only those bacteria able to adhere to the mucus or to the epithelial cells were present Moreover, foregut exposed to the probiotic bacteria only resulted in a healthy intestinal barrier whereas exposure to A salmonicida disrupted its integrity However, pre-treatment of salmon intestine with L delbrueckii ssp lactis prevented Aeromonas damaging eĂects (Table 5) These results are promising in the context of the use of non-autochthonous probiotic bacteria as prophylactic agents against Âsh bacterial infections in the GI tract Although Nikoskelainen, Salminen, Bylund and Ouwehand (2001) studied the adhesion capacity of diĂerent LAB strains to rainbow trout (Oncorhynchus mykiss Walbaum) mucus in vitro, probiotic bacteria had never been shown to cross the Âsh gut before r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs pathogens in the gastrointestinal tract of Âsh E Ringệ et al Whereas some authors (Fuller 1973; Mèyrè-Mèkinen, Manninen & Gyllenberg 1983) have postulated that probiotic bacteria are host speciÂc (and their eĂects are limited to their natural hosts), three studies have reported the absence of speciÂcity in LAB when binding host intestinal mucus (Gildberg & Mikkelsen 1998; Rinkinen, Westermarck, Salminen & Ouwehand 2003; Salinas et al 2008) It was also evident that the LAB species used by Salinas et al (2008) ex(b) LP (a) V L M N L LP L L V L 20à 40à Figure Confocal microscopy of the fate of tetramethylrhodamine isothiocyanate-labelled Lactobacillus delbrueckii ssp lactis in the foregut of Atlantic salmon (a) Fluorescent image of Atlantic salmon foregut cryosection after incubation for 30 with L debreckii subsp lactis [107 colony-forming units (CFU) mL 1] labelled with TRITC L, lumen; M, mucus Note the red Êuorescence of labelled bacteria associated with mucus in the gut lumen Scale bar 20 mm (b) Fluorescent image of Atlantic salmon foregut cryosection after incubation for 30 with L debreckii subsp lactis (107 CFU mL 1) labelled with TRITC Note the red Êuorescence of labelled bacteria located inside the villi (V) at the level of the lamina propria (LP) Scale bar 40 mm After Salinas et al (2008) L, lumen; N, nuclei of enterocytes erted a pronounced local eĂect on the GI tract lining The authors qualitative Ânding, that the concentration of immune cells in the gut epithelium increased following incubation with the probiotic strain, resembled the response that occurred when the intestine was exposed to A salmonicida (Ringệ, Salinas, et al 2007) This is consistent with a recent study that found increased numbers of acidophilic granulocytes in the gut of seabream (S aurata L.) larvae after a probiotic, a mixture of Lactobacillus fructivorans and Lactobacillus plantarum, was delivered through the diet (Picchietti, Mazzini, Taddei, Renna, Fausto, Mulero, Carnevali, Cresci & Abelli 2007) Some information is available demonstrating that A salmonicida causes cell damage in the foregut of Atlantic salmon (Ringệ et al 2004; Ringệ, Salinas, et al 2007; Salinas et al 2008), but these studies used the pathogen without washing the culture supernatant The eĂects observed in these studies might be due either to toxins, to the bacteria themselves or to both toxins and bacterial cells However, as a blocking eĂect was observed when the intestine was exposed to the lactobacilli (Salinas et al 2008), it can be speculated that the lactobacilli compete with the pathogen in adherence to the mucus layer, by producing bacteriocins, organic acid, H2O2, etc., which would inhibit pathogenic colonization The hypothesis proposed by Salinas et al (2008) is in accord with the results of Chabrillon, Ouwehand, Diaz-Rosales, Arijo, Martinez-Manzanares, Balebona and Morinigo (2006), who demonstrated that attachment of the pathogenic bacteria (V anguillarum, Photobacterium damselae ssp piscicida,V alginolyticus and Vibrio harveyi) to intestinal mucus of Gilthead seabream was signiÂcantly reduced by Lactobacillus rhamnosus and Table Morphological changes undergone byAtlantic salmon foregut following exposure toAeromonas salmonicida ssp salmonicida, Lactobacillus delbreckii ssp lactis or both bacterial strains Morphological observations Control Aeromonas only Lactobacillus only Lactobacillus1 Aeromonas Presence of cell debris in the lumen Disorganized microvilli Oedema Disintegrated tight junctions Loss of epithelial integrity Leucocyte regrouping Presence of rodlet cells Bacteria in lumen or between microvilli Bacteria paracellularly 0 0 0 0 2 2 0 0 0 3 3 1 0 3 Tissue changes were assessed as follows: (0) not observed; (1) low frequency; (2) moderate frequency; (3) high frequency After Salinas et al (2008) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 459 Aquaculture Research, 2010, 41, 594^597 Rainfall and temperature are WSSV risk factors E A Tendencia et al parameters of the water, plankton proÂle and bacterial level of the rearing water and cultured species and the presence of WSSV in shrimp and crab.White spot syndrome virus detection was carried out on the gills of juvenile or adult shrimp Two methods were used to detect the presence of WSSV: the polymerase chain reaction (PCR) techniques described by de la Penỡa, Lavilla-Pitogo, Villar, Paner, Sombito and Capulos (2007), from January 2000 to April 2006, and the Shrimp Biotechnology Business Unit (SBBU) kit from May 2006 to December 2007 All tests included positive and negative controls The SBBU kit is a test kit for the molecular diagnosis of WSSV developed by the SBBU of Mahidol University, Thailand (Withyachumnarnkul 1999) The kit uses the lateral Êow dipstick (LFD) method BrieÊy, DNAwere extracted from the postlarvae or the gills of juvenile and adult shrimp to be tested and ampliÂed using the kits reagents The ampliÂed DNA was applied onto the LFD and placed in an upright position into the assay buffer to allow the bands to develop The bands in the LFD were then interpreted Results of the WSSVanalysis on shrimp from 2000 to 2007 were evaluated Prevalence was computed by dividing the number of positive samples by the total number of processed samples The mean prevalence and the average rainfall between years and months were compared using the one-way analysis of variance and Duncans multiple range test following the statistical program for the social sciences (SPSS v 12) The relationship between WSSV prevalence and the diĂerent factors was analysed by means of Pearsons Correlation for normally distributed data and Spearman for non-normally distributed data, using the same program The number of shrimp samples analysed Êuctuated and the annual prevalence of WSSV tended to increase with time (Table 1) The annual prevalence of WSSV-positive shrimp was signiÂcantly higher from 2004 to 2006 than in 2000^2001, with immediate values not signiÂcantly diĂerent from those either in 2003 and 2007 The lowest annual prevalence was observed in 2001, when the observed lowest monthly temperature was the highest (21.1 1C), and the number of consecutive months with at least 14 rain days was the lowest (4 months) Higher WSSV prevalence was observed in November, December, January and February, when the observed average minimum atmospheric temperature was generally lower (Table 2) Correlations between the prevalence of WSSV-positive adult shrimp with the monthly rainfall (s 0.25; Po0.05) and the number of rain days (s 0.24; Po0.05) are weakly negative, and somewhat stronger with minimum atmospheric temperature (s 0.41; Po0.01) The average minimum temperature is correlated with the number of rain days (s 50.68; Po0.01) and monthly rainfall (s 0.46; Po0.01) The negative correlation of WSSV prevalence with decreased temperature is in consonance with the results obtained by Liu et al (2006) and Guan et al (2003) Rahman, Escobedo-Bonilla, Corteel, DantasLima, Wille, Alday Sanz, Pensaert, Sorgeloos and Nauwynck (2006) and Reyes, Salazar and Granja (2007) reported that a decrease in temperature weakens the immune response of the shrimp, thereby making them susceptible to WSSV infection Furthermore, the same authors mentioned that a decrease in temperature also increases viral replication and load Table Total number of samples, prevalence (%) of the white spot sydrome virus (WSSV) in juvenile/adult Penaeus monodon, number of months wherein WSSV was observed (NMW), number of rain days, number of consecutive months with ! 14 raindays (CMR), observed lowest monthly atmospheric temperature ( 1C), and the mean of minimum temperature ( 1C) observed in Negros Island, Philippines, from 2000 to 2007 Year # of samples Annual prevalence NMW Total rain days CMR Lowest monthly temperature ( 1C) Mean minimum temperature ( 1C) 2000 2001 2002 2003 2004 2005 2006 2007 77 33 70 107 179 303 963 302 6ab 0a abc 10 20abcd 32cd 34d 36d 28bcd 4 12 234 190 160 175 181 192 198 194 7 8 20.9 21.1 20.1 17 16.2 17 18.9 17.5 22.4 22.5 22.0 22.0 22.0 21.95 22.0 22.27 Superscript value a,b,c,d represent P40.05 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 594^597 595 Rainfall and temperature are WSSV risk factors E A Tendencia et al Aquaculture Research, 2010, 41, 594^597 Table Monthly mean prevalence of white spot syndrome virus (WSSV) shrimp, percentage WSSVoutbreaks, average rainfall, number of rain days, average minimum atmospheric temperature and observed lowest temperature in Negros Island, Philippines, from 2000 to 2007 WSSV Rainfall Temperature ( 1C) Month Monthly prevalence Percentage outbreaksw Number of days Average (mm) Average minimum Observed lowest January February March April May June July August September October November December 39de 34cde 13abcd 17abcd 10abc 1a 2a 6ab 5ab 31bcde 35cde 56e 75abcd 84cd 61abcd 42abcd 48abcd 25abc 16a 21ab 39abcd 89d 79bcd 88d 7a 9a 10a 9a 18bc 22c 22c 22c 21c 23c 16b 15b 33a 64ab 76ab 52ab 262abc 711d 497cd 509cd 377abcd 393bcd 218abc 166abc 20.7a 20.7a 21.5b 22.4cd 23.3g 23.0fg 22.9efg 22.8defg 22.7def 22.5cde 22.1c 21.6b 19.6 19.7 20.1 21.5 22.0 22.2 22.2 22.5 22.1 22.0 21.6 20.7 Computed by dividing the number of WSSV-positive samples by the total number of processed samples wComputed by dividing the number of WSSV-positive samples that resulted in an outbreak by the total number of WSSV-positive samples Values with the same superscripts are not signiÂcantly diĂerent (P40.05) The eĂect of 14 days of continuous rain on WSSV prevalence observed by farmers and the data on the number of consecutive months with 414 rain days in the present study are in accordance with the observation of Karunasagar, Otta and Karunasagar (1997) in the West Coast of India, wherein a WSSVoutbreak occurred when the monsoon was at its peak, salinity near ppt, with heavy surface runoĂ that may carry contaminants and turbidity in natural waters Furthermore, farmers in Kalimantan, Indonesia, associate poor shrimp harvest with high water turbidity due to rain (personal communication) Rainfall aĂects water temperature and salinity through various processes An increase in rainfall could dilute the pond water, which would result in lower salinity and cooler water (Abney & Rakocinski 2004) Water temperature is negatively related to rainfall, but on the other hand, is positively related to air temperature (Nargis & Pramanik 2008) Thermal pollution due to casual runoĂs from upland during rains increases the temperature of surface water (Jones, Hunt & Hunt 2007) These runoĂs not modify the usual thermal stratiÂcation in tropical regions Clouds during the rainy season reduce cooling during the night and the higher average minimum water temperature also prevents ponds from cooling This higher temperature of pond water explains the weak negative correlation between rainfall and WSSV occurrence, which is the dominant 596 trend However, after prolonged rains the thermal pollution of runoĂ water has an inverse eĂect and cools down water and thus disturbs thermal stratiÂcation (Jones et al 2007) Together with the negative relation between rain and pond water, this might explain the eĂect of 14 continuous rain days Most farmers in the Philippines not stock during the cold season because it is during these months that they experience most WSSV-related problems They prefer stocking during the warm months, although they experience problems after 14 days of heavy rains This may be related to the fact that Vibrios are dominant during the warm season (Ortigosa, Esteve & Pujalte 1989), making them more susceptible to secondary infection by other pathogens like the WSSV (Phuoc, Corteel, Nauwynck, Pensaert, Alday-Sanz, Van den Broeck, Sorgeloos & Bossier 2008) This study conÂrmed that low atmospheric temperature is a hazard factor in the development of WSSV in cultured shrimp Penaeus monodon We explained that rainfall on subsequent days is also a hazard factor while the dominant trend is a negative correlation between rainfall and WSSV In general, the average temperatures during the rainy season are higher and thus the water temperature; however, temperatures of runoĂ water and of air are aĂected by several continuous rain days, thus a eĂecting pond water temperature Continuous exposure to a tem- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 594^597 Aquaculture Research, 2010, 41, 594^597 Rainfall and temperature are WSSV risk factors E A Tendencia et al perature lower than the optimum level for shrimp culture acts as a stressor (Baliao & Tookwinas 2002) Although the recent literature and practice demonstrate that pond lining (P Sorgeloos pers comm.) and increased bio-security reducesWSSVoccurrence and outbreaks (Mohan, Corsin & Padiyar 2004), our analysis shows that WSSV will remain a risk in outdoor shrimp culture Cost-eĂective measures to mitigate the eĂect of low atmospheric temperature and other natural phenomena need to be explored Acknowledgment The authors wish to thank the Government of Japan for funding the study through the Trust Fund awarded to the Aquaculture Department Southeast Asian Fisheries Development Center (SEAFDEC AQD) under study code 8001 T FD FH0206 References Abney M.A & Rakocinski C.F (2004) Life-history variation in Caribbean Gambusia, Gambusia puncticulata punticulata (Poeciliidae) from the Cayman Islands, British West Indies Environmental Biology of Fish 70, 67^79 Baliao D.D & Tookwinas S (2002) Best Management Practices for a Mangrove-Friendly Shrimp Farming Aquaculture Extension Manual No 35 ISBN 971-8511- 64- Aquaculture Department Southeast Asian Fisheries Development Center,Tigbauan, Iloilo, Philippines Corsin F., Turnbull J.F., Mohan C.V., Hao N.V & Morgan K.L (2005) Pond-level risk factors for white spot disease outbreaks In: Diseases in Asian AquacultureV (ed by P.Walker, R Lester & M.G Bondad-Reantaso), pp 75^92 Fish Health Section, Asian Fisheries Society, Manila, Philippines de la Penỡa L.D., Lavilla-Pitogo C.R.,Villar C.B.R., Paner M.G., Sombito C.D & Capulos G.C (2007) Prevalence of white spot syndrome virus (WSSV) in wild shrimp Penaeus monodon in the Philippines Diseases of Aquatic Organisms 77,175^179 Guan Y., Yu Z & Li C (2003) The eĂect of temperature on white spot syndrome infections in Marsupenaeus japonicus Journal of Invertebrate Pathology 83, 257^260 Jones M., Hunt E & Hunt B (2007) The eĂect of urban stormwater BMPs on runoĂ temperature in trout sensitive water NWQEP Notes No 124, 11pp ISSN 1062-9149 Karunasagar I., Otta S.K & Karunasagar I (1997) Histopathological and bacteriological study of white spot syn- drome of Penaeus monodon along the west coast of India Aquaculture 153, 9^13 Liu B.,Yu Z., Song X., Guan Y., Jian X & He J (2006) The effect of acute salinity change on white spot syndrome (WSS) outbreaks in Fenneropenaeus chinensis Aquaculture 253,163^170 Mohan C.V., Corsin F & Padiyar P.A (2004) Farm-level biosecurity and white spot disease (WSD) of shrimp Aquaculture Health International 3, 16^20 Nargis A & Pramanik S.H (2008) Physico-chemical parameters in relation to meterological and climatic conditions in a Âsh pond Bangladesh Journal of ScientiÂc and Industrial Research 43, 405^410 Ortigosa M., Esteve C & Pujalte M.J (1989) Vibrio species in seawater and mussels: abundance and numerical taxonomy Systematic and Applied Microbiology 12, 316^325 Peinado-Guevara L.I & Lopez-Meyer M (2006) Detailed monitoring of white spot syndrome virus (WSSV) in shrimp commercial ponds in Sinaloa, Mexico by nested PCR Aquaculture 251, 33^45 Phuoc L.H., Corteel M., Nauwynck H.J., Pensaert M.B., Alday-Sanz V., Van den Broeck W., Sorgeloos P & Bossier P (2008) Increased susceptibility of White Spot Syndrome Virus infected Litopenaeus vannamei to Vibrio campbellii Environmental Microbiology 10, 2718^2727 Rahman M.M., Escobedo-Bonilla C.M., Corteel M., DantasLima J.J.,Wille M., Alday Sanz V., Pensaert M.B., Sorgeloos P & Nauwynck H.J (2006) EĂect of high water temperature (33 1C) on the clinical and virological outcome of experimental infections with white spot syndrome virus (WSSV) in speciÂc pathogen-free (SPF) Litopenaeus vannamei Aquaculture 261, 842^849 Reyes A., Salazar M & Granja C (2007) Temperature modiÂes gene expression in subcuticular epithelial cells of white spot syndrome virus infected Litopenaeus vannamei Developmental and Comparative Immunology 31, 23^29 Rodriguez J., Bayot B., AmanoY., Panchana F., de Blas I., Alday V & Calderon J (2003) White spot syndrome virus infection in cultured Penaeus vanamei (Boone) in Ecuador with emphasis on histopathology and ultrastructure Journal of Fish Diseases 26, 439^450 Withyachumnarnkul B (1999) Results from black tiger shrimp Penaeus monodon culture ponds stocked with postlarvae PCR-positive or -negative for white-spot syndrome virus (WSSV) Diseases of Aquatic Organisms 39, 21^27 Yu Z., Li C & GuanY (2003) EĂect of salinity on the immune responses and outbreak of white spot syndrome in the shrimp Marsupenaeus japonicus Ophelia 57, 99^106 Keywords: WSSV, P monodon, risk factors, prevalence, rainfall, atmospheric temperature r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 594^597 597 Aquaculture Research, 2010, 41, 598^601 doi:10.1111/j.1365-2109.2009.02342.x SHORT COMMUNICATION Algicidal and cyanocidal effects of selected isoquinoline alkaloids Daniel Jancula1,2, Jana Gregorova3 & Blahoslav Marsalek1,2 Centre for Cyanobacteria and Their Toxins, Institute of Botany, Academy of Sciences of the Czech Republic, Brno, Czech Republic RECETOX, Masaryk University, Brno, Czech Republic Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno, Czech Republic Correspondence: Daniel Jancula, Centre for Cyanobacteria and their Toxins, Institute of Botany, Czech Academy of Sciences; Kvetna 8, 603 65 Brno, Czech Republic E-mail: jancula@sinice.cz The formation of noxious phytoplankton blooms causes deterioration in water quality in aquaculture ponds and reservoirs, but also in swimming pools and aquaria (Joska & Bolton 1996; Burford 1997) Among the most eĂective and commonly used algicides/cyanocides for water management are chemicals including copper sulphate, aluminium salts (polyaluminium chloride), simazine and many others Nevertheless, due to their negative eĂects on aquatic ecosystems, scientists all over the world seek to discover or develop alternatives to this chemical treatment One way is by searching for algicidal/cyanobacterial compounds in natural resources like plants (both terrestrial and aquatic), bacteria, fungi, protozoa, etc (Schrader 2003; Volk & Furkert 2006) Probably, the most widely known example is the use of decomposing barley straw (Welch, Barrett, Gibson & Ridge 1990) Many studies have demonstrated that isoquinoline alkaloids (naturally found in the families Papaveraceae, Fumariaceae, Meliaceae, CaprioÂlaceae, Ranunculaceae and Rutaceae) display a wide spectrum of biological activities They show anti-inÊammatory, anti-tumour and antimicrobial activities against a variety of organisms including bacteria, viruses, fungi and protozoa (Lenfeld, Kroutil, Marsalek, Slavik, Preininger & Simanek 1981; Colombo & Bosisio1996) Our study examines the algicidal and cyanobacterial properties of eight isoquinoline alkaloids and evaluates their potential use in water management of noxious phytoplankton development 598 In our study, we focused on four groups of isoquinoline alkaloids, namely: protoberberine alkaloids ^ berberine, coptisine; protopine alkaloids ^ protopine; and aporphine alkaloids ^ magnoÊorine and benzophenanthridine alkaloids ^ chelidonine, sanguinarine, chelerythrine and chelirubine (Fig 1) These alkaloids were obtained from the Department of Biochemistry, Faculty of Medicine, Masaryk University, Czech Republic A common process for isolation of isoquinoline alkaloids from Chelidonium majus was described by Slav|Ô k and Slav|Ô kova (1954, 1977) Each drug was extracted (family Papaveraceae plants were used as a source of alkaloids) by methanol in a Soxhlet apparatus; the extract was evaporated to dryness and subsequently dissolved in 1% sulphuric acid Insoluble remnants were Âltered out and the Âltrate was made alkaline with a bicarbonate solution The alkaline solution was extracted by diethylether, the organic layer was evaporated to dryness and then fractionated by column chromatography on an acid oxide of aluminium During isolation of alkaloids, the column was washed with a mixture of benzene and1% acetic acid and various amounts of ethyl alcohol (Slav|Ô k & Slav|Ô kova 1954) The identity of the tested alkaloids was veriÂed by EI-MS, 1H NMR and 13C NMR and their purity was not o98% (tested by HPLC) (Suchomelova, Bochor akova, Paulova, Musil & Taborska 2007) Selected alkaloids were tested on laboratory strains of three cyanobacteria (Microcystis aerugino- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd Aquaculture Research, 2010, 41, 598^601 Algicidal and cyanocidal eĂects D Janc ula et al Figure Structures of examined compounds Table EĂects of selected alkaloids on phytoplankton species growth Chelidonine Chelirubine Chelerythrine Sanguinarine Berberine Coptisine Magnoflorine Protopine Pseudokirchneriella subcapitata Chlorella vulgaris 41000 (ND) 420.0 (ặ 71.2) 433.3 (ặ 59.1) 73.3 (ặ 6.0) 41000 (ND) 301.6 (ặ 21.3) 41000(ND) 41000(ND) 41000 41000 41000 41000 41000 41000 41000 41000 (ND) (ND) (ND) (ND) (ND) (ND) (ND) (ND) Scenedesmus quadricauda Microcystis aeruginosa Synechococcus nidulans Aphanothece clathrata 41000 (ND) 242.0 (ặ 78.6) 256.7 (ặ 99.8) 42.6 (ặ 9.1) 756.7 (ặ 75.9) 576.3 (ặ 108.6) 41000 (ND) 41000 (ND) 41000 (ND) 41000 (ND) 570 (ặ 77.9) 291.3 (ặ 58.7) 276.7 (ặ 98.4) 543.6 (ặ 57.9) 41000 (ND) 41000 (ND) 41000 (ND) 41000 (ND) 790.0 (ặ 86.4) 573.3 (ặ 85.7) 575.0 (ặ 73.8) 756.6 (ặ 89.9) 41000 (ND) 41000 (ND) 41000 (ND) 41000 (ND) 816.6 (ặ 97.4) 290.0 (ặ 70.7) 641.6 (ặ 98.1) 663.3 (ặ 92.8) 41000 (ND) 41000 (ND) EC50 values in mg L and its standard deviations (ặ SD) ND, not determined sa, Synechococcus nidulans and Aphanothece clathrata) and three green algae (Pseudokirchneriella subcapitata, Scenedesmus quadricauda and Chlorella vulgaris) obtained from the Algal Culture Collection CCALA, Tr ebooỉ, Czech Republic Phytoplankton cultures were cultivated at 25 1C under white light of 80 mmol m s in a mixture of Z-medium (Staub 1961) and BB-medium (Bristol modiÂed by Bold 1949) (1:1) The tests were performed in transparent, 96-well microplates, each well having a volume of 250 mL, three replicates for each concentration and a control Organisms in the tests were cultivated for 96 h At the end of the test, the density of the algal cells was assessed using an absorbance measurement (microplate reader, Sunrise,Tecan, Austria) at a wavelength of 680 nm The values of EC50 (the con- centration required to cause 50% inhibition in growth) were determined using linear regression analysis of inhibition percentage versus the extract concentration The values of EC50 for the three green algae and three cyanobacteria are listed in Table The highest toxicity against algae was found for sanguinarine (EC50o100 mg L for P subcapitata and S quadricauda) Most of the other alkaloids tested showed an eĂective toxicity of between 100 and 1000 mg L On the other hand, protopine, magnoÊorine and chelidonine were found to be less toxic (EC50 higher than 1000 mg L 1) compared with the other alkaloids An alkaloid selectively toxic (one order more toxic to cyanobacteria than to other non-target species) to cyanobacteria was not found On the other r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 598^601 599 Algicidal and cyanocidal eĂects D Janc ula et al hand, the toxicity of berberine was statistically lower for algae (Po0.05, ANOVA,Tukeys test) than for M aeruginosa, which is one of the most abundant species in central Europe (Komarek & Komarkova 2002) In this regard, berberine seems to be an interesting alkaloid for further research in this scientiÂc area For example, inhibition eĂects on natural colonial strains of M aeruginosa should be experimentally evaluated in the future The toxic eĂects of most eĂective alkaloids tested in this study are comparable with other chemicals that show algicidal properties like copper sulphate, hydrogen peroxide or phthalocyanines For example, the copper EC50 value for green alga Raphidocelis subcapitata was found to be 137 mg L (de Oliveira, Lopes & Paumgartten 2004) and 270 mg L for hydrogen peroxide toxicity against cyanobacterium M aeruginosa (Drabkova, Admiraal & Mars alek 2007) Singlet oxygen generators as phthalocyanines can develop toxicity towards cyanobacteria even at concentrations below 100 mg L (Jancula, Drabkova, Cớerny, Karaskova, Kor |Ô nkova, Rakus an & Mars alek 2008) Toxic eĂects of the alkaloids tested were reported in many studies However, knowledge of isoquinoline alkaloids is rather broad in medical sciences The only information relating to ecotoxicological data is the study of Jancula, Suchomelova, Gregor, Smutna, Mars alek and Taborska (2007), where the eĂects of extracts from the family Papaveraceae plants on aquatic organisms were tested Our Ândings in the present study conÂrm that the biological eĂects of extracts from Papaveraceae plants are apparently related to high amounts of alkaloids in their tissues On the other hand, it has been proven (Jancula et al 2007) that the total amount of alkaloids in plant tissues (crude extracts) does not correspond with their total toxicity However, while EC50s of plant extracts range in orders of milligrams per litre, EC50s of isolated alkaloids range in orders of micrograms per litre as can be seen from our results Because of this, we suppose that the presence of biologically active alkaloids can play an important role in toxicity on phytoplankton species, but probably other active compounds may also be responsible for toxic eĂects Furthermore, there may also occur synergic/antagonistic eĂects between the number of components within one plant species Our study presents the Ârst evidence of toxicity of the mentioned isoquinoline alkaloids to algae and cyanobacteria Inhibition eĂects on phytoplankton species were found to be strong (EC50o100 mg L 1) and so isoquinone alkaloids seem to be a potentially inter- 600 Aquaculture Research, 2010, 41, 598^601 esting group of compounds for further ecotoxicological evaluation and possibly for phytoplankton management Acknowledgments The research was supported by a grant from the Ministry of Education, Youth and Sports of the Czech Republic, no 1M0571 Research Centre for Bioindication and Revitalization, grant no AVOZ60050516 (Institute of Botany ASCR) and grant no 525/08/0819 from the Grant Agency of the Czech Republic References Bold H.C (1949) The morphology of Chlamydomonas chlamydogama, sp nov Bulletin of the Torrey Botanical Club 76,101^108 Burford M (1997) Phytoplankton dynamics in shrimp ponds Aquaculture Research 28, 351^360 Colombo M.L & Bosisio E (1996) Pharmacological activities of Chelidonium majus L (Papaveraceae) Pharmacological Research 28, 351^360 de Oliveira E.C., Lopes R.M & Paumgartten F.J.R (2004) Comparative study on the susceptibility of freshwater species to copper-based pesticides Chemosphere 56,369^374 Drabkova M., Admiraal W & Mars alek B (2007) Combined exposure to hydrogen peroxide and light ^ selective eĂects on cyanobacteria, green algae, and diatoms Environmental Science and Technology 41, 309^314 Jancula D., Drabkova M., Cớerny J., Karaskova M., Kor |Ô nkova R., Rakus an J & Mars alek B (2008) Algicidal activity of phthalocyanines ^ screening of 31 compounds EnvironmentalToxicology 23, 218^223 Jancula D., Suchomelova J., Gregor J., Smutna M., Mars alek B & Taborska E (2007) EĂects of aqueous extracts from Âve species of the family Papaveraceae on selected aquatic organisms EnvironmentalToxicology 22, 480^486 Joska M.A & Bolton J.J (1996) Filamentous freshwater macroalgae in South Africa ^ a literature review and perspective on the development and control of weed problems Hydrobiologia 340, 295^300 Komarek J & Komarkova J (2002) Review of the European Microcystis ^ morphospecies (Cyanoprokaryotes) from nature Czech Phycology 2, 1^24 Lenfeld J., Kroutil M., Marsalek E., Slavik J., Preininger V & SimanekV (1981) Isolation, chemistry and biology of alkaloids from plants of the Papaveraceae.84 Anti-inÊammatory activity of quaternary benzophenanthridine alkaloids from chelidonium-majus Planta Medica 43, 161^165 Schrader K.K (2003) Natural algicides for the control of cyanobacterial-related oĂ-Êavor in catÂsh aquaculture In: OĂ-Flavors in Aquaculture (ed by A.M Rimando & K.K r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 598^601 Aquaculture Research, 2010, 41, 598^601 Schrader), pp 195^208 American-Chemical-Society, Orlando,Florida Slav|Ô k J & Slav|Ô kova L (1954) Alkaloidy rostlin Makovitych (Papaveraceae) II Delen|Ô chelerythrinu a sanguinarinu a nalez dvou novych alkaloidu' ve Vlas tovicn|Ô ku (Chelidonium majus L) ChemickeÔ Listy 48,1382^1386 Slav|Ô k J & Slav|Ô kova J (1977) Alkaloids of Papaveraceae Minor alkaloids from Chelidonium majus L Collection of Czechoslovak Chemical Communications 42, 2686^ 2693 Staub R (1961) Research on physiology of nutrients of the planktonic cyanobacterium Oscillatoria rubescens Schweizerische Zeitschrift fur Hydrologie 23, 82^198 Suchomelova J., Bochor akova H., Paulova H., Musil P & Taborska E (2007) HPLC quantiÂcation of seven quaternary Algicidal and cyanocidal eĂects D Janc ula et al benzo[c]phenanthridine alkaloids in six species of the family Papaveraceae Journal of Pharmaceutical and Biomedical Analysis 44, 283^287 Volk R.B & Furkert F.H (2006) Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth Microbiological Research 161,180^186 Welch I.M., Barrett P.R.F., Gibson M.T & Ridge I (1990) Barley straw as an inhibitor of algal growth I: studies in the ChesterÂeld Canal Journal of Applied Phycology 2, 231^239 Keywords: cyanobacteria, algae, toxicity, management, alkaloids r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 598^601 601 Aquaculture Research, 2010, 41, 602^606 doi:10.1111/j.1365-2109.2009.02343.x SHORT COMMUNICATION Effects of moving acclimation cages before release of cultured fish: alternate release strategies for a juvenile winter flounder Pseudopleuronectes americanus stock enhancement effort Elizabeth A Fairchild1, James A Sulikowski2, Nathan Rennels1,W Huntting Howell1 & Paul C W Tsang3 Department of Biological Sciences, University of New Hampshire, Durham, NH, USA Marine Science Center, University of New England, Biddeford, ME, USA Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, NH, USA Correspondence: E A Fairchild, Department of Biological Sciences, University of New Hampshire, Spaulding Hall, 38 Academic Way, Durham, NH 03824, USA E-mail: elizabeth.fairchild@unh.edu Biomass of winter Êounder Pseudopleuronectes americanus, a target species of both recreational and commercial Âsheries, is at an all-time low (Northeast Fisheries Science Center 2008), and due to new unprecedented regulations, the largest of the three stocks is closed to all Âshing activities in federal waters (National Oceanic and Atmospheric Activities 2009).Winter Êounder population recovery could be expedited by stock enhancement (Waters 1996; Le Francois, Lemieux & Blier 2002), and experimental stocking studies have been conducted since 1996 in New Hampshire The goal of past projects has not been to initiate large-scale releases Instead,responsible approach (Blankenship & Leber 1995) has been applied to develop the processes needed to successfully enhance winter Êounder by answering key questions in the event that large-scale stocking efforts occur Much of the research has focused on optimal release strategies for juvenile winter Êounder In many stock enhancement programmes, initial survival rates of newly released cultured Âsh are low (Pitman & Gutreuter 1993; Stottrup & Sparrevohn 2007; Sudo, Kajihara & Fujii 2008) These may be due, in part, to stress responses in hatchery-reared Âsh causing departures from behavioural norms which result in a higher probability of mortality (Schreck, Olla & Davis 1997) Developing release stra- 602 tegies that minimize stress responses and reduce post-release mortality is essential to any enhancement eĂort and can be done with a combination of hatchery and Âeld techniques One such technique is using acclimation cages in situ For many Âsh species, acclimation cages improve post-release survival, growth, and site Âdelity (Koshiishi, Itano & Hirota 1991; Jonssonn, Brannas & Lundqvist 1999; Kuwada, Masuda, Shiozawa, Kogane, Imaizumi & Tsukamoto 2000; Brennan, Darcy & Leber 2006; Sparrevohn & Stottrup 2007) For juvenile winter Êounder, predation by green crabs Carcinus maenas is of special concern (Fairchild & Howell 2000;Taylor 2005) Acclimation cages have been used at the release site under the assumption that these cages reduce immediate post-release mortality Cultured Êounder, stocked into these cages for 48 h, adjust to the release site, hone their burial skills, begin pigment change, recover from the stress of transport to the release site and maintain high site Âdelity post release (Fairchild & Howell 2004; Sulikowski, Fairchild, Rennels, Howell & Tsang 2005, 2006; Fairchild, Rennels & Howell 2009) However, the cages also can be detrimental by attracting predators to the release site In a recent study, Fairchild, Rennels and Howell (2008) compared green crab density when cages were absent, present but empty r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd Aquaculture Research, 2010, 41, 602^606 Moving in situ cages stresses cultured Âsh E A Fairchild et al and present and containing Êounder They found that crab density increased dramatically from 1.8 to 4.1 crabs 50 m when empty cages were deployed They hypothesized that the cages themselves provided desirable crab habitat on the relatively featureless, sandy, estuary bottom.When cages stocked with Âsh were deployed, crab density increased even higher to 6.35 crabs 50 m 2, indicating that the dense assemblages of Êounder further attracted this crustacean predator Based on these Ândings, it is apparent that modiÂcation of the release strategy is necessary in order to oĂset this predator problem, and alternate release strategies are being investigated One suggested alternate release strategy is to stock and set acclimation cages at the release area for the required 48 h Instead of releasing the Âsh in the same spot, the cages would be moved gently to a nearby secondary release site to oĂset any predatory crab aggregations, and the Âsh would be released there However, this site transfer could negatively affect the Âsh by inducing more stress The objective of this study was to determine if this alternate release strategy would be eĂective by evaluating the stress levels of Êounder, as measured by cortisol concentrations, when stocked acclimation cages were moved between two sites The study was conducted in August 2006 at two sites (A and B) in the Hampton-Seabrook Estuary, NH (Fig 1) Site A is a well-studied location in the Hampton River that has been used as a release site for previous winter Êounder stock enhancement studies Site B is approximately 250 m downriver from Site A and has been examined as an alternate release site (Fairchild, Rennels et al 2008) Both sites are sandy areas devoid of any macroalgae or structure, about m deep at mean high water, have high salinity and dissolved oxygen, and naturally occurring wild juvenile winter Êounder populations (Fairchild, Sulikowski, Rennels, Howell & Gurshin 2008) August bottom water temperature in these areas typically ranges from 12 to 21 1C (Fairchild, Sulikowski et al 2008) Two acclimation cages (32 32 10 cm; bottom surface area 0.1m2) constructed of cm plasticcoated wire and lined with mm nylon were lowered from the boat to the bottom at release site A Each cage contained 50 cultured juvenile winter Êounder (118 days post hatch; mean TL 40 ặ mm; 102% stocking density) reared at the University of New Hampshires Coastal Marine Laboratory (per the methodology of Fairchild, Rennels, Howell & Wells 2007) Fish were allowed to acclimate over days in the cages to simulate the normal Êounder release strategy After 48 h, one cage was hauled to the surface and the Âsh were immediately snap frozen on dry ice The other cage was raised and tethered to the Figure The Hampton-Seabrook Estuary in New Hampshire Winter Êounder release studies have been conducted in the Hampton River at site (a) An alternate release site (b) was tested approximately 250 m downriver r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 602^606 603 Moving in situ cages stresses cultured Âsh E A Fairchild et al Aquaculture Research, 2010, 41, 602^606 boat so that it was still submerged, and towed at 1kt to release site B There the cage was lowered to the bottom for 10 to simulate a release, then raised, and all Âsh were snap frozen on dry ice Samples for whole body cortisol analyses were prepared according to Sulikowski et al (2005, 2006) Individual juvenile Êounder from each acclimation cage were thawed, weighed to the nearest gram, and dissected into smaller segments to facilitate the homogenization process To ensure cortisol levels fell within detectable levels, Âve juvenile winter Êounder were combined in a 50 mL test tube to yield approximately g of tissue which was equivalent to an individual sample In this scheme, each treatment (cage) consisted of 10 samples of pooled Âsh The control values were obtained from previous studies by Sulikowski et al (2005, 2006) BrieÊy, hatchery-reared Âsh (those that were not moved into or part of the acclimation cage experiment) were hand netted, immediately frozen on dry ice ( 70 1C), and stored at 20 1C for later use This procedure took about 10 s and yielded ng g wet weight samples that contained consistent and statistically similar (analysis of variance; P40.05) cortisol levels Individual samples (5 g of pooled tissue) were homogenized in ice-cold phosphate-buĂered saline (PBS), centrifuged and the supernatant removed Each sample was extracted three times with a threefold volume of ether (anaesthesia grade) and the aqueous phase was frozen in an acetone/dry ice bath to facilitate removal of the organic phase Following evaporation of the ether under a stream of nitrogen, the dried extracts were reconstituted in PBS with 0.1% gelatin Approximately 1000 counts of tritiated cortisol were added to account and correct for procedural losses The overall mean recoveries were 76% Duplicate samples of cortisol were analysed by the Atlantic Veterinary College (Prince Edward Island, Canada) using standard radioimmunoassay techniques (e.g Tsang & Callard 1987; Sulikowski, Tsang & Howell 2004) The intra- and inter-assay coecients of variance were 4.4% and 5.1% respectively DiĂerences in cortisol concentrations between Âsh that remained in site A and those that were moved to site B were compared using a t-test A probability (P) value of o0.05 was considered statistically signiÂcant Despite very gently moving the caged Âsh to a nearby alternate release site, the Âsh experienced a signiÂcantly higher level of stress (t(9) 4.83, Po0.001), as measured by cortisol concentration, than the caged Âsh which were not moved Caged Âsh that remained in site A had a mean (ặ 1SEM) cortisol concentration of 10.7 ặ 1.9 ng g wet weight, whereas caged Âsh that were moved to site B had a mean cortisol concentration of 45.6 ặ 7.1ng g wet weight Although there is potential for wholebody preparations to contain other extractable, free steroid metabolites that could cross-react with the cortisol antibody and contribute to the total measured hormone concentrations (i.e King & Berlinsky 2006), this was unlikely in this study Given the speciÂcity of the antibody, the consistently low intraand inter-assay coecients of variance (4.4% and 5.1% respectively), and the results from two previous studies on juvenile winter Êounder (Sulikowski et al 2005, 2006), these results clearly denote an observed stress response produced by cage movement In other studies using cultured juvenile winter Êounder, cortisol concentrations under non-stressed conditions were similar (0.8^9.6 ng g 1), but stressed conditions yielded much lower values (7.2^15.5 ng g 1) compared with this study (Breves & Specker 2005; Sulikowski et al 2005, 2006) Winter Êounder which experience a sharp cortisol increase, such as the moved caged Âsh did, likely will have lower survival post release Generally when Âshes are exposed to a stressor, primary stress responses are induced (Mazeaud & Mazeaud 1981) These include an increase in the release of the catecholamines adrenaline and noradrenaline, and the subsequent release of corticosteroid hormones, principally cortisol, from the interrenal tissue The associated distress of this primary stress response can induce a cascade of secondary (physiological) stress responses that subsequently cause tertiary (whole animal) responses For example, elevated cortisol levels can aĂect physiological parameters such as growth (Barton, Schreck & Barton1987; Pickering, Pottinger, Sumpter, Carragher & Le Bail 1991), reproductive status (Pankhurst & Van Der Kraak 1997), as well as disturbances to the immune system (Pickering & Pottinger 1989) In addition, elevated cortisol levels can aĂect Âsh behaviour, which in turn can modify food acquisition, predator avoidance, aggression, learning, habitat selection (Olla, Davis & Schreck 1995) and social interactions with conspeciÂcs (Gregory & Wood 1999) Despite many of the positive attributes benthic acclimation cages aĂord cultured winter Êounder, they are not eĂective since predatory green crabs are attracted to them (Fairchild, Rennels et al 2008), and moving the cages adversely aĂects the Âsh Because there are signiÂcant beneÂts to acclimating winter 604 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 602^606 Aquaculture Research, 2010, 41, 602^606 Moving in situ cages stresses cultured Âsh E A Fairchild et al Êounder in situ before release, it is worthwhile to try other cage designs One such design is a Êoating cage system so that non-swimming green crabs are unable to reach the Êounder These Êoating cages would allow the Âsh to acclimate to the abiotic conditions of the environment and recover from any stress incurred during transport Unlike benthic acclimation cages, the Âsh would have no contact with the substrate; however, pre-release substrate conditioning could occur in the hatchery Because cultured winter Êounder incur a high stress response when acclimation cages are moved to avoid predators pre-release, this release strategy does not seem viable However, the upper stress limit for juvenile winter Êounder is unknown, and further research in this area would be useful for quantifying post-release eĂects of diĂerent release strategies Determining the formula for maximizing post-release survival using combinations of release strategies is essential for a successful enhancement eĂort Evaluating the physiological eĂects of varying stress levels in cultured Âsh, and providing guidelines of cortisol levels that should not be exceeded in released cultured Âsh would be helpful for maximizing the cost eĂectiveness in all enhancement programmes Walbaum) to predators Journal of Experimental Marine Biology and Ecology 325,1^7 Fairchild E.A & Howell W.H (2000) Predator-prey size relationship between Pseudopleuronectes americanus and Carcinus maenas Journal of Sea Research 44, 81^90 Fairchild E.A & Howell W.H (2004) Factors aĂecting the post-release survival of cultured juvenile Pseudopleuronectes americanus Journal of Fish Biology 65, 69^87 Fairchild E.A., Rennels N., Howell W.H & Wells R.E (2007) Gonadal development and diĂerentiation in cultured juvenile winter Êounder Pseudopleuronectes americanus Journal of the World Aquaculture Society 38, 114^121 Fairchild E.A., Rennels N & Howell W.H (2008) Predators are attracted to acclimation cages used for winter Êounder stock enhancement Reviews in Fisheries Science 16, 262^268 Fairchild E.A., Rennels N & Howell W.H (2009) Using telemetry to monitor movements and habitat use of cultured and wild juvenile winter Êounder in a shallow estuary Reviews: Methods andTechnologies in Fish Biology and Fisheries 9, 5^22 Fairchild E.A., Sulikowski J.A., Rennels N., Howell W.H & Gurshin C.W.D (2008) Distribution of winter Êounder, Pseudopleuronectes americanus, in the Hampton-Seabrook Estuary, New Hampshire: observations from a Âeld study Estuaries and Coasts 31, 1158^1173 Gregory T.R & Wood C.M (1999) The eĂects of chronic plasma cortisol elevation on the feeding behaviour, growth, competitive ability and swimming performance of juvenile rainbow trout Physiological and Biochemical Zoology 72, 286^295 Jonssonn S., Brannas E & Lundqvist H (1999) Stocking of brown trout, Salmo trutta L.: eĂects of acclimatization Fisheries Management and Ecology 6, 459^473 KingW.V & Berlinsky D.L (2006) Whole-body corticosteroid and plasma cortisol concentrations in larval and juvenile Atlantic cod Gadus morhua L following acute stress Aquaculture Research 37, 1282^1289 Koshiishi Y., Itano H & Hirota Y (1991) ArtiÂcial stock-size improvement of the Êounder Paralichthys olivaceus: present status of technological achievement In: NOAA Technical Report NMFS 102 (ed by R.S Svrjcek), pp 33^43 Department of Commerce,Washington, DC, USA Kuwada H., Masuda R., Shiozawa S., KoganeT., Imaizumi K & Tsukamoto K (2000) EĂect of Âsh size, handling stresses and training procedure on the swimming behavior of hatchery-reared striped jack: implications for stock enhancement Aquaculture 185, 245^256 Le Francois N.R., Lemieux H & Blier P.U (2002) Biological and technical evaluation of the potential of marine and anadromous Âsh species for cold-water mariculture Aquaculture Research 33, 95^108 Mazeaud M.M & Mazeaud F (1981) Adrenergic responses to stress in Âsh In: Stress and Fish (ed byA.D Pickering), pp 50^75 Academic Press, London, UK Acknowledgments The authors wish to thank N Carlson, L Kintzing, and G Rice for their help in the Âeld This project was funded by NOAA through a grant to the Science Consortium for Ocean Replenishment This is Jackson Estuarine Laboratory Contribution Series number 486 References Barton B.A., Schreck C.B & Barton L.D (1987) EĂects of chronic cortisol administration and daily acute stress on growth, physiological conditions, and stress responses in juvenile rainbow trout Diseases of Aquatic Organisms 2, 173^185 Blankenship H.L & Leber K.M (1995) A responsible approach to marine stock enhancement American Âsheries society symposium 15,167^175 Brennan N.P., Darcy M.C & Leber K.M (2006) Predator-free enclosures improve post-release survival of stocked common snook Journal of Experimental Marine Biology and Ecology 335, 302^311 Breves J.P & Specker J.L (2005) Cortisol stress response of juvenile winter Êounder (Pseudopleuronectes americanus, r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 602^606 605 Moving in situ cages stresses cultured Âsh E A Fairchild et al Aquaculture Research, 2010, 41, 602^606 National Oceanic and Atmospheric Administration (2009) NOAA announces interim rules to reduce overÂshing and rebuild northeast groundÂsh stocks while balancing economic and conservation concerns Press release, April 2009 Available at http://www.noaanews.noaa.gov/stories 2009/20090406_groundÂsh.html (accessed 27 July 2009) Northeast Fisheries Science Center (2008) Assessment of 19 Northeast GroundÂsh Stocks through 2007 Report of the 3rd GroundÂsh Assessment Review Meeting (GARM III), Northeast Fisheries Science Center,Woods Hole, MA, 4^8 August 2008 US Department of Commerce, NOAA FIsheries, Northeast Fish Science Center Ref doi 08-15; 884 p 1xvii Olla B.L., Davis M.W & Schreck C.B (1995) Stress-induced impairment of predator evasion and non-predator mortality in PaciÂc salmon Aquaculture Research 26, 393^398 Pankhurst N.W & Van Der Kraak G (1997) EĂects of stress on reproduction and growth of Âsh In: Fish Stress and Health in Aquaculture (ed by G.K Iwama, J Sumpter, A.D Pickering & C.B Schreck), pp 73^95 Cambridge University Press, Cambridge, UK Pickering A.D & Pottinger T.G (1989) Stress responses and disease resistance in salmonid Âsh: eĂect of chronic elevation of plasma cortisol Fish Physiology and Biochemistry 7, 253^258 Pickering A.D, Pottinger T.G., Sumpter J.P., Carragher J.F & Le Bail P.Y (1991) EĂects of acute and chronic stress on the levels of circulating growth hormone in rainbow trout, Oncorhynchus mykiss General Comparative Endocrinology 83, 86^93 PitmanV.M & Gutreuter S (1993) Initial poststocking survival of hatchery-reared Âshes North American Journal of Fisheries Management 13,151^159 Schreck C.B., Olla B.L & Davis M.W (1997) Behavioral responses to stress In: Fish Stress and Health in Aquaculture (ed by G.K Iwama, J Sumpter, A.D Pickering & C.B Schreck), pp 145^169 Cambridge University Press, Cambridge, UK Sparrevohn C.R & Stottrup J.G (2007) Post-release survival and feeding in reared turbot Journal of Sea Research 57, 151^161 Stottrup J.G & Sparrevohn C.R (2007) Can stock enhancement enhance stocks? Journal of Sea Research 57,104^113 Sudo H., Kajihara N & Fujii T (2008) Predation by the swimming crab Charybdis japonica and piscivorous Âshes: a major mortality factor in hatchery-reared juvenile Japanese Êounder Paralichthys olivaceus released in Mano Bay, Sado Island, Japan Fisheries Research 89, 49^56 Sulikowski J.A., Tsang P.C.W & Howell W.H (2004) Annual changes in steroid hormone concentrations and gonad development in the winter skate, Leucoraja ocellata Marine Biology 144, 845^853 Sulikowski J.A., Fairchild E.A., Rennels N., Howell W.H & Tsang P.C.W (2005) The eĂects of tagging and transport on stress in juvenile winter Êounder, Pseudopleuronectes americanus: implications for successful stock enhancement Journal of theWorld Aquaculture Society 36, 148^156 Sulikowski J.A., Fairchild E.A., Rennels N., Howell W.H & Tsang P.C.W (2006) The eĂects of transport density on cortisol levels in juvenile winter Êounder, Pseudopleuronectes americanus Journal of theWorld Aquaculture Society 37, 107^112 Taylor D.L (2005) Predatory impact of the green crab (Carcinus maenas Linnaeus) on post-settlement winter Êounder (Pseudopleuronectes americanus Walbaum) as revealed by immunological dietary analysis Journal of Experimental Marine Biology and Ecology 324,112^126 Tsang P & Callard I.P (1987) Morphological and endocrine correlates of the reproductive cycle of the aplacental viviparous dogÂsh, Squalus acanthias General Comparative Endocrinology 66, 182^189 Waters E.B (1996) Sustainable Êounder culture and Âsheries University of North Carolina Sea Grant College Program Report UNC-SG-96-14, pp.1^12 606 Keywords: release strategies, cultured Êounder, stress, stock enhancement, post-release mortality, cortisol r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 602^606 Aquaculture Research, 2010, 41, 607^609 doi:10.1111/j.1365-2109.2009.02365.x SHORT COMMUNICATION Capture, transport and acclimatization to captivity of European hake, Merluccius merluccius L: preliminary data on feeding and growth JoseÔ Iglesias, Mar|Ô a JesuÔs Lago, Francisco Javier Sanchez & Rosa Cal Instituto Espanỡol de Oceanograf|Ô a, Cabo Estai ^ Canido,Vigo, Spain Correspondence: J Iglesias, Instituto Espanỡol de Oceanograf|Ô a, PO Box 1.552, Cabo Estai ^ Canido, 36200 Vigo, Spain E-mail: jose.iglesias@vi.ieo.es In recent years, several culture experiments have been carried out up to the juvenile stage using eggs obtained from wild-caught specimens of European hake Merluccius merluccius L (Bjelland 2001; Bjelland & Skiftesvik 2006) and Southern hake Merluccius australis (Hutton) from spawning induced in captivity (Bustos & Landaeta 2005; Bustos, Balbot|Ô n & Landaeta 2007) Other experiments include surveys conducted to capture and tag wild juveniles for studies on stock distribution and growth in the natural environment (Pontual, Bertignac, Battaglia, Bavouzet, Moguedet & Groison 2003; Pinỡeiro, Rey, De Pontual & Gonỡi 2007) Researchers at IFREMER in Brest (France) have succeeded in developing a stock of adults caught with the same type of net used in this work (M Suquet, pers comm.) However, no other experiments dealing with the capture and acclimatization of this species in captivity for extended periods of time have been reported in the literature The purpose of this work was to optimize the capture (by testing the appropriate Âshing gear, transport methods and handling) and acclimatization of live specimens, by providing both an appropriate diet and the most suitable environmental conditions By maintaining a stock of individuals, it would be possible to determine their growth and potential for reproduction in captivity Future research on larval rearing will assess the viability of this species as a candidate for commercial aquaculture Hake were caught during a 3-day survey carried out in July 2007 in the R|Ô a of Vigo (NW Spain), using an experimental Âshing gear (GO-73 modiÂed) with a co- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd dend specially designed to retain water while hauling the net (Pontual et al 2003) Fishing tows were carried out at 25^35 m depth, lasting13^15 at1^1.5 knots Hauling was performed slowly (75 m 1) to minimize the problems associated with gas bladder inÊation After being hauled onboard, the Âsh were placed in a 500 L isothermal tank, equipped with a system pumping water from a depth of 16 m to maintain a temperature close to that of their natural environment (14^16 1C) Dark conditions and oxygen to saturation, measured using an OxyGuard oxygen meter (OxyGuard International A/S, Birkerệd, Denmark), were maintained Fish Êoating on the surface with distended abdomens were punctured with a 0.8 mm 40 mm needle at a 451 angle in an area equidistant from the anus and the dorsal Ân Light pressure was applied to the abdomen to extract the excess of gas accumulated in the gas bladder Punctured and non-punctured Âsh were placed separately in isothermal tanks maintained at 13^14 1C Dead specimens from the catch (150) were measured to the nearest 0.5 cm and weighed to the nearest 0.1g to estimate the mean initial size At day 200 all the survivors were measured to obtain the Ânal size A total of 330 specimens were caught in a shallow zone of the R|Ô a of Vigo (o50 m); 45.5% died owing to capture and the air extraction processes (Table 1) Seventy-eight percent of the remaining Âsh showed abdominal inÊation, Êoating in the tank, and so it was necessary to perforate the gas bladder to extract the excess of gas The other 22% swam to the bottom of the tank and no puncture was needed 607 Capture and acclimatization of European hake J Iglesias et al Table Number of hakes caught during the days Âshing survey Captured Punctured Not punctured Dead Day Day Day Total 138 39 12 87 69 32 10 27 123 69 18 36 330 140 40 150 Fish (n 5180) were transported in the same tank and environmental conditions and was carried out at a slow speed, lasted no longer than 45 and yielded a survival rate of 91.11% The total number of specimens Ânally acclimatized was 164: 136 punctured Âsh and 28 non-punctured Âsh The higher mortality observed in the non-punctured Âsh during transport and acclimatization may be explained by the traumatic eĂect exerted on organs and blood vessels by the excessive dilation of the gas bladder (Keniry, Brofka, Horns & Marsden 1996) Fish were kept in two (4 1) m3 indoor rectangular tanks A light intensity of 80 Lux for 12 L:12 D, a water temperature of 13^14 1C and a water Êow rate of 1500 L h were provided The highest mortality due to catch was registered during the Ârst days After this critical period, 29.26% of Âsh had survived Final survival from day to day 200 was 17 68% Fish were fed to satiation and during the Ârst months, the diet consisted of live sand eels: Ammodytes tobianus L and Hyperoplus lanceolatus (Le Sauvage) The diet was later modiÂed to include fresh and thawed dead Âsh, mainly sardine and sand eel Live sand eels were readily accepted by hakes, which showed a very active feeding behaviour, achieving a consumption level up to kg Âsh day after months of acclimatization This diet was given days a week At day 100 of captivity, they began to accept dead sand eel and sardine, and a few weeks later, they would eat these species thawed The feeding rate decreased substantially until an average of 300^600 g Âsh per ration, supplied every days No diĂerent feeding behaviour was observed in hake fed on fresh or thawed diets Fish spent most of their time resting on the bottom, moving quickly to snatch up the food when administered Growth was calculated on the basis of the initial increase in the mean length (cm) and weight (g) until day 200 of captivity The daily growth rate was also calculated Before sampling, Âsh were anaesthetized in a 100 L bath for 1min, containing a mixture 608 Aquaculture Research, 2010, 41, 607^609 Table Growth in length and weight of European hake maintained in captivity for 200 days Day Size (cm) Weight (g) Day 100 Day 200 30.15 ặ 4.58 37.31 ặ 3.64 38.40 ặ 5.80 194.57 ặ 80.88 410.80 ặ 133.64 434.79 ặ 225.85 of clove essence (0.04 mL L 1) and ethanol (0.04 mL L 1) Table shows the length and weight reached during the feeding stages with the live and dead Âsh During the Ârst 100 days (period of live diet), the growth rate showed values of 0.21cm day and 0.75 g day in length and weight, respectively, significantly higher than those observed in the next 100 days (period of dead Âsh diet), 0.06 cm day and 0.03 g day 1, while during the entire 200-day experiment, the rates were 0.11cm day and 0.40 g day These overall growth rates were higher than those reported by Pontual et al (2003), 0.033 cm day 1, obtained from tagged and released wild Âsh The higher growth rates observed during the live feeding stage may be attributed to the fact that hake consumed larger quantities of live Âsh Hake captured in late July 2007 with a mean starting length of 30.15 cm measured 38.40 cm by day 200 These preliminary growth values of European hake reared in captivity Ât the fast growth model proposed by Pontual, Groison, Pinỡeiro and Bertignac (2006), who reported that European hake reached a total length of 25, 45 and 60 cm during the Ârst, second and third year, respectively, while the values estimated from the classic otolith age determination method, Pinỡeiro and Sa|Ô nza (2003), were 20, 29 and 37 cm in total length Despite the low total survival percentage, our results indicate that it is possible to maintain hake specimens in captivity for extended periods of time There are a number of factors that determine the success of the process: specimens could be captured live due to the high eciency of the Âshing gear used, making it possible to keep the Âsh submerged in water to prevent asphyxia However, Âshing was the factor that most aĂected mortality, which varied considerably during the diĂerent tows, owing to the excoriations caused by spiny species such as horse mackerel Trachurus trachurus L and axillary seabream Pagellus acarne (Risso), which were abundant in some catches Another factor inÊuencing survival was the precision of the abdominal puncture in specimens presenting dilation of the gas bladder It was r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 607^609 Aquaculture Research, 2010, 41, 607^609 essential to act quickly; otherwise, death occurred owing to the eĂect of the pressure on the adjacent organs and blood vessels Other factors inÊuencing the survival were the strict control of environmental parameters (darkness, high levels of oxygen and low temperatures) while the Âsh were being held on board and during their transfer to the laboratory, Âshing in shallow waters, which minimized the effect of depressurisation, and the proximity of the Âshing area, shortening the time spent in transport In short, this paper has contributed to our knowledge of capture methods, transport, acclimatization, feeding and growth in captivity of European hake for the purpose of assessing the viability of this species as a candidate for aquaculture References Bjelland R.M (2001) European hake, Merluccius merluccius (L 1758), a new candidate for aquaculture? Rearing techniques, larval development and startfeeding Hovedfagsoppgave, Institutt for Fiskeri og Marinbiologi, Universitetet i Bergen, p.77s Bjelland R.M & Skiftesvik A.B (2006) Larval development in European hake (Merluccius merluccius L.) reared in a semi-intensive culture system Aquaculture Research 37, 1117^1129 Bustos C.A & Landaeta y M.F (2005) Desarrollo de huevos y larvas tempranas de la merluza del sur, Merluccius Capture and acclimatization of European hake J Iglesias et al australis, cultivados bajo condiciones de laboratorio Gayana 69, 402^408 Bustos C.A., Balbot|Ô n F & Landaeta M.F (2007) Spawning of the southern hake Merluccius australis (Pisces: Merluccidae) in chilean fjords Fisheries Research 83, 23^32 Keniry M.J., Brofka W.H., Horns W.H & Marsden J.E (1996) EĂects of decompression and puncturing the gas bladder on survival of tagged yellow perch North American Journal of Fisheries Management 16, 201^206 Pinỡeiro C & Sa|Ô nza M (2003) Age estimation, growth and maturity of the European hake (Merluccius merluccius L.) from Iberian atlantic waters ICES Journal of Marine Science 60, 1086^1102 Pinỡeiro C., Rey J., De Pontual H & Gonỡi R (2007) Tag and recapture of European hake (Merluccius merluccius L.) oĂ the Northwest Iberian Peninsula: Ârst results support fast growth hypothesis Fisheries Research 88,150^154 Pontual H., Bertignac M., Battaglia A., Bavouzet G., Moguedet P & Groison A.L (2003) A pilot tagging experiment on European hake (Merluccius merluccius): methodology and preliminary results ICES Journal of Marine Science 60, 1318^1327 Pontual H., Groison A.L., Pinỡeiro C & Bertignac M (2006) Evidence of underestimation of European hake growth in the Bay of Biscay, and its relationship with bias in the agreed method of age estimation ICES Journal of Marine Science 63, 1674^1681 Keywords: European hake, Merluccius merluccius, capture, maintenance, feeding, growth r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 607^609 609 [...]... Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 481^495 Aquaculture Research, 2010, 41, 481^495 Aquaculture development and food security K M Jahan et al Table 1 Aquaculture growth rate and percentage contribution to total ¢sh production from 1983/1984 to 2005/2006 Fish production (metric tonnes)Ã Years Inland capture Aquaculture Marine capture Total 1983–1984 1987–1988... Blackwell Publishing Ltd, Aquaculture Research, 41, 481^495 483 Aquaculture development and food security K M Jahan et al Adoption-Income Link Aquaculture Research, 2010, 41, 481^495 AdoptionEmployment Link AdoptionConsumption Link High income effect from aquaculture production and sale-profit Ability to create alternative employment High homeconsumption Higher return to capital from aquaculture Ability... 73, 2947^2955 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 465 Lactic acid bacteria vs pathogens in the gastrointestinal tract of ¢sh E RingÖ et al Aquaculture Research, 2010, 41, 451^467 Moriarty D (1997) The role of microorganisms in aquaculture ponds Aquaculture 151, 333^349 Namba A., Mano N & Hirose H (2007) Phylogenetic analysis of intestinal... autochthonous microbiota in the gastrointestinal tract of yellow grouper (Epinephelus awoara) cultured in cages Aquaculture 286,184^189 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 467 doi:10.1111/j.1365-2109.2009.02334.x Aquaculture Research, 2010, 41, 468^480 Changes to the histological gill structure and haemolymph composition of early blue swimmer... r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 468^480 Aquaculture Research, 2010, 41, 468^480 Changes to the histological gill structure and haemolymph composition N Romano & C Zeng the gill structure post sub-lethal ammonia-N exposure, eight crabs were sampled at 1, 3, 6, 12, 24, 36, 48, 72, 84 and 96 h, respectively, adopting the same procedure mentioned... stages of the lobster Homarus americanus Marine Biology 110, 293^300 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 468^480 Aquaculture Research, 2010, 41, 481^495 doi:10.1111/j.1365-2109.2009.02337.x The impacts of aquaculture development on food security: lessons from Bangladesh Khondker Murshed^e-Jahan1, Mahfuzuddin Ahmed2 & Ben Belton3 1 The WorldFish... Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 463 Lactic acid bacteria vs pathogens in the gastrointestinal tract of ¢sh E RingÖ et al Aquaculture Research, 2010, 41, 451^467 Bowden T.J., Adamson K & Bricknell I.R (2003) Diseases of relevance to haddock In: Early Rearing of Haddock State of the Art (Special Publication), Vol 7 (ed by D.E Aitkin), pp 113^114 Aquaculture Association of... Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467 Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs pathogens in the gastrointestinal tract of ¢sh E RingÖ et al RingÖ E., Myklebust R., Mayhew T.M & Olsen R.E (2007) Bacterial translocation and pathogenesis in the digestive tract of larvae and fry Aquaculture 268, 251^264 Rinkinen M.E.,Westermarck S.,... impact of the project The impact of the aquaculture intervention across project and control farmers was another focus of the research programme Therefore, another set of non-project farmers (control farmers) was monitored from 2003/ r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 481^495 Aquaculture Research, 2010, 41, 481^495 2004 for this purpose One... intralamellar septum and lamellae distortion (Fig 6a^f) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 468^480 473 Changes to the histological gill structure and haemolymph composition N Romano & C Zeng Aquaculture Research, 2010, 41, 468^480 Figure 4 The mean haemolymph ammonia-N levels (mmol L À 1) (Æ SE) of early Portunus pelagicus juveniles exposed