Biochemical and Physiological Advances in Finfish … and Physiological Advances in Finfish Aquaculture William Driedzic Scott McKinley Don MacKinlay International Congress on the

Biochemical and

Physiological Advances in Finfish Aquaculture

William Driedzic Scott McKinley Don MacKinlay

International Congress on the Biology of Fish

University of British Columbia, Vancouver, CANADA

Biochemical and

Physiological Advances in

Finfish Aquaculture

SYMPOSIUM PROCEEDINGS

William Driedzic

Scott McKinley

Don MacKinlay

International Congress on the Biology of Fish University of British Columbia, Vancouver, CANADA

Copyright © 2002 Physiology Section,

American Fisheries Society All rights reserved

International Standard Book Number (ISBN) 1-894337-24-7

Notice This publication is made up of a combination of extended abstracts and full papers, submitted by the authors without peer review. The papers in this volume should not be cited as primary literature. The Physiology Section of the American Fisheries Society offers this compilation of papers in the interests of information exchange only, and makes no claim as to the validity of the conclusions or recommendations presented in the papers. For copies of these Symposium Proceedings, or the other 50 Proceedings in the Congress series, contact: Don MacKinlay, SEP DFO, 555 West Hastings St.,

Vancouver BC V6B 5G3 Canada Phone: 604-666-3520 Fax 604-666-6894

E-mail: [email protected]

Website: www.fishbiologycongress.org

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CONGRESS ACKNOWLEDGEMENTS

This Symposium is part of the International Congress on the Biology of Fish, held at the University of British Columbia in Vancouver B.C., Canada on July 22-25, 2002. The sponsors included: • Fisheries and Oceans Canada (DFO) • US Department of Agriculture • US Geological Service • University of British Columbia Fisheries Centre • National Research Council Institute for Marine Biosciences • Vancouver Aquarium Marine Science Centre In addition, this Symposium was assisted by funding from Aquanet. The main organizers of the Congress, on behalf of the Physiology Section of the American Fisheries Society, were Don MacKinlay of DFO (overall chair, local arrangements, program and proceedings) and Rosemary Pura of UBC Conferences and Accommodation (facility arrangements, registration and housing). Thanks to Karin Howard for assistance with Proceedings editing and word-processing; to Anne Martin for assistance with the web pages; and to Cammi MacKinlay for assistance with social events. I would like to extend a sincere ‘thank you’ to the many organizers and contributors who took the time to prepare a written submission for these proceedings. Your efforts are very much appreciated.

Don MacKinlay Congress Chair

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TABLE OF CONTENTS AquaNet - research opportunities in biochemistry, physiology,

and behaviour of fish under aquaculture. Driedzic, W. R........................................................................................ 1 Genomics research on Atlantic salmon: its application to salmonid

aquaculture Davidson, W. et al.................................................................................. 3 Transgenic salmon for culture and consumption Fletcher, Garth ...................................................................................... 5 Metabolic determinants of glucose utilization in rainbow trout Bennett M.T. and K.J. Rodnick ............................................................ 15 Dietary Lipids, Immune Function and Pathogenesis of Disease in Fish Lall, S. et al.......................................................................................... 19 Fish proteomics and nutrition Houlihan, Dominic and S. Martin........................................................ 25 Digestive enzyme expression in the exocrine pancreas during the

ontogeny of the winter flounder Murray, Harry M. et al. ....................................................................... 27 Induction of digestive enzymes in the Brazilian catfish

Pseudoplatystoma Lundstedt, Licia Maria ........................................................................ 33 Aquaculture of tambaqui and its vitamin C requirements Rodrigo Roubach, et al ....................................................................... 45 Effects of photoperiod manipulation on reproductive cyclicity in

haddock Martin-Robichaud, D.J. and D.L. Berlinsky ........................................ 51 The influence of alternate supplemental dietary lipids on the growth

and health of Atlantic salmon in seawater Balfry, S., et al. .................................................................................... 55

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Effects of ß-agonist feeding on rainbow trout muscle growth and

myosatellite cells Levesque Haude, and T.W. Moon ........................................................ 61 Diet influences proteolitic enzyme profile of the South American

catfish Rhamdia quelen Lundstedt, Licia Maria ........................................................................ 65 Cloning and characterization of glucose transporters from cod (Gadhus

morhua) heart Long, Jennifer R. and William R. Driedzic .......................................... 73 RNA-DNA ratio in extracts of fish scales can indicate feeding

condition Smith, Todd and L.J. Buckley .............................................................. 77 Ontogeny of digestion in larval of Atlantic cod and haddock Perez-Casanova, Juan Carlos ............................................................. 83 Cloning of rainbow trout (Oncorhynchus mykiss) α-actin and myosin

regulatory light chain 2 genes and α-tropomyosin 5’-flanker. Functional assessment of promoters Aleksei Krasnov, Heli Teerijoki and Hannu Mölsä ............................. 89

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AQUANET -

RESEARCH OPPORTUNITIES IN BIOCHEMISTRY,

PHYSIOLOGY, AND BEHAVIOUR OF FISH UNDER AQUACULTURE

William R. Driedzic, Memorial University of Newfoundland,

Ocean Sciences Centre, St. John’s, NF, A1C 5S7, (709) 737-3282, Fax: (709) 737-3220, [email protected]

EXTENDED ABSTRACT ONLY- DO NOT CITE

Insights into the fundamental aspects of biochemistry and physiology are now about to contribute to a step jump in the aquaculture industry. Recent findings in what features to select for, how to select and indeed how to design fish will result in substantial advancements in farmed production. This symposium brings together the worlds leading experts of applied biochemistry, physiology and molecular biology as relates to finfish aquaculture. This symposium and the one entitled " Behavioural and physiological comparisons of cultured and wild fish" also serve as a forum to present research supported by AquaNet. AquaNet is a funding program supported under the umbrella of the Networks of Centres of Excellence (NCE) in Canada. There are direct funding opportunities for members of the Canadian university professoriate and opportunities of partnership collaboration with non-Canadian scholars under this program. Details of AquaNet will be presented and may be found at:

www.aquanet.mun.ca

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GENOMICS RESEARCH ON ATLANTIC SALMON:

ITS APPLICATION TO SALMONID AQUACULTURE

William Davidson Department of Molecular Biology and Biochemistry

Simon Fraser University Burnaby, BC, V5A 1S6, Canada

(Tel) 604-291-3771:(Fax) 604-291-3424: [email protected]

Ben Koop Department of Biology University of Victoria Victoria, BC, Canada

Bjorn Hoyheim

Norwegian School of Veterinary Medicine Oslo, Norway

Jim Thorsen Children’s Hospital Oakland Research Institute

Oakland, Ca, USA

EXTENDED ABSTRACT ONLY - DO NOT CITE

Greater than 80% of the salmon farmed in British Columbia are Atlantic salmon and worldwide it has become the industry standard. It is expected that production of Atlantic salmon will continue to grow and this expansion is anticipated to occur primarily through gains in productivity. Selective breeding programs and genetics will play an increasingly important role in achieving improvements in the performance of salmon broodstock. Traits that may be amenable to genetic improvement include growth, delayed maturation, flesh quality, pigment uptake, temperature tolerance, and disease resistance. Disease is one of the predominant obstacles slowing the growth of aquaculture and it remains the largest cause of economic losses. The use of marker assisted selection and the development of vaccines have been helped in agriculture species through genomics. It is now quite feasible to construct both a linkage map and a physical map for an economically

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important organism and indeed it is really essential for any future research initiatives. The Genomics Research on Atlantic Salmon Project (GRASP), funded by Genome BC, and a corresponding initiative in Norway have been designed to provide the foundation for understanding the genome of Atlantic salmon. It should be noted, however, that genomic information gained from Atlantic salmon will be applicable to other salmonid species. Moreover, this information will be useful for more than just the development of aquaculture. It will benefit conservation and enhancement of wild stocks, commercial harvesting through the identification of specific stocks, the maintenance of lucrative sports fisheries, and will enable fundamental scientific questions concerning the evolution of salmonid genomes to be answered. The specific aims of the Atlantic salmon genome projects are: (1) to tie together the linkage map based primarily on microsatellite markers with the physical map based on BAC contigs and position these on the chromosomes; (2) to locate genes of known function on the physical map, to gain a better appreciation of the structure and function of constituents of the immune system, and to compare specific regions of the Atlantic salmon genome in order to understand how a duplicated genome reorganizes itself, controls sex-determination, and is related to the genomes of other vertebrates; and (3) to examine gene expression at the transcriptional level and the translational level in several tissues under different conditions, and to identify molecules induced by physiological responses to stress, acclimatization, and exposure to pathogens. More than a score of cDNA libraries have been constructed from a wide variety of tissues and different developmental stages. At the end of May 2002, more than 30,000 reads of the 3' ends of these expressed sequence tags (ESTs) have been completed and these constitute approximately 13,000 contigs or independent gene products. Preliminary analysis of these data reveals the presence of many duplicated gene products. A BAC library has been constructed and 313,000 clones with an average insert size of 170,000 to 190,000 base pairs have been selected for DNA fingerprinting and contig construction at the BC Cancer Agency’s Genome Sciences Centre. This represents a 15 fold coverage of the genome. A microarray with 1,000 genes represented is being prepared and it is anticipated that this will provide salmonid researchers with the opportunity to initiate expression studies. This presentation will give an update on the status of the genomics projects and what the next steps will be.

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TRANSGENIC SALMON FOR CULTURE

AND CONSUMPTION

Garth L. Fletcher, Ocean Sciences Centre, Memorial University, St. John’s,

NF, A1C 5S7, Canada & Aqua Bounty Canada, PO Box 21233, St. John’s,

NF, A1A 5B2, Canada. Phone 709-738-4638, Fax 709-738-4644

[email protected]

Margaret A. Shears,

Ocean Sciences Centre, Memorial University, St. John’s, NF, A1C 5S7, Canada

Madonna J. King, Ocean Sciences Centre, Memorial University, St. John’s,

NF, A1C 5S7, Canada

Sally V. Goddard Aqua Bounty Canada, PO Box 21233, St. John’s,

NF, A1A 5B2, Canada.

Abstract Over the past 20 years we have generated stable lines of transgenic Atlantic salmon possessing either antifreeze protein (AFP) genes or a salmon growth hormone (GH) gene construct. The AFP transgene is expressed and AFP secreted into the blood of all generations to date. However antifreeze protein levels remain low and a means to improve these levels needs to be developed. Our GH transgene enhances growth rates to the point where Atlantic salmon can reach market size (4-6kg) a year earlier than can non-transgenics grown commercially in Atlantic Canada. The characteristics of the transgenic salmon are described, and the hurdles to be overcome before products derived from transgenics can take their place in the world

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market are discussed (Fletcher et al 1999b). Introduction World fisheries are in crisis. Most are exploited to the maximum extent, or over fished, and many are in danger of commercial extinction (Pauly et al. 1998; Watson and Pauly 2001). As the world population continues to grow exponentially, it is clear that if fish are to maintain their current status as an essential food resource, production must be dramatically improved. Aquaculture stands alone as the only major means of meeting demands for fish in the future (New 1997).

A key element to enhanced production of cultured species is the development of genetically superior broodstocks that are tailored to their culture conditions and to the market. Characteristics that are generally desirable include improvements in growth rates, feed conversion efficiencies, disease resistance, cold and freeze resistance, tolerance to low oxygen levels and the ability to utilize low cost, or non-animal protein diets (Hew and Fletcher 1997).

Aquaculture is still in its infancy relative to the farming of terrestrial livestock. Despite the acknowledged power of traditional selection and breeding methods, the development of superior broodstock using this process is still relatively slow, and while such broodstock development programs have been underway for salmon since 1971 (Gjedrem 1997), many aquaculture ventures are still reliant on broodstock fish collected from the wild. If we are to realize the increased production needed to meet the requirements of the 21st century, a quantum leap in broodstock development is needed.

Transgenic technology provides a means by which such a quantum leap in production is possible (Hew and Fletcher 2001; Melamed et al. 2002). The identification, isolation and reconstruction of genes responsible for desirable traits, and their transfer to broodstock, offer powerful methods of genetic/phenotypic improvement that would be difficult, if not impossible to achieve using traditional selection and breeding techniques (Devlin, 1997).

This brief communication highlights our progress towards generating genetically engineered Atlantic salmon broodstocks for commercial aquaculture. Our discussion centres around personal experiences with salmon. The issues involved in the production of transgenic fish and their successful integration into the aquaculture industry involve not only science but also food safety, environmental risk assessment, animal welfare, consumer acceptance, and intellectual property

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protection (Fletcher et al. 1999a). Transgenic Salmon

We came into the field of transgenics some eighteen years ago in response to problems faced by the aquaculture industry along the east coast of Canada. Most of these coastal waters are characterized by ice and sub-zero temperatures that are lethal to salmonids. Therefore, sea cage aquaculture of salmon is almost entirely restricted to a relatively small area in the most southerly part of the region (Hew et al. 1995). The challenge for us as scientists was to find a means of producing salmon that would avoid freezing, and thus facilitate the expansion of aquaculture and economic development throughout the entire Atlantic coastal region. The solution became evident when Palmiter et al. (1982) demonstrated the power of transgenic technology as a means of genetically improving commercially important animals.

Two potential ways in which transgenic technologies can be used to solve the problem of overwintering salmon in sea cages in Atlantic Canada are: 1) produce freeze-resistant salmon by giving them a set of antifreeze protein genes, and 2) enhance growth rates by growth hormone gene transfer so that overwintering may not be necessary. Antifreeze Protein Genes Antifreeze proteins (AFP) are produced by a number of marine teleosts that inhabit waters at sub-zero (zero to -1.8°C ) temperatures. These proteins are produced in two locations: 1. Liver, from where they are secreted into the blood, resulting in plasma concentrations as high as 20 mg/ml, serving to reduce the freezing point of the fish extracellular fluids to safe levels, and 2. Epithelial tissues, where AFPs are produced to protect the tissues from damage due to direct ice contact at sub-zero temperatures (Fletcher et al. 2001). Many commercially important fish (salmon, halibut, etc.) lack these proteins and their genes and, as a consequence, they will not survive if cultured in icy sea water (Hew et al. 1995; Fletcher et al. 1998).

In 1982, our transgenic studies were initiated by injecting winter flounder antifreeze genes into the fertilized eggs of Atlantic salmon. A full length gene encoding the major liver secretory AFP was used and the AFP transgene was successfully integrated into the salmon chromosomes, expressed, and found to exhibit Mendelian inheritance over 5 generations to date (Shears et al. 1991). Expressed levels of AFP in the blood of these fish is quite low (100 - 400 µg/ml) and is insufficient to confer

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any significant increase in freeze resistance to the salmon. However, the “proof of the concept” that salmon and other fish species can be rendered more freeze resistant by gene transfer has been established. The challenge now is to design an antifreeze gene construct(s) that will result in enhanced expression in appropriate tissues; epithelia and liver. This is the essence of our current research within AquaNet, a Canadian National Centre of Excellence.

Growth Hormone Genes All aquaculture ventures could benefit commercially from the development of culture species with enhanced growth rates that would reduce the time required to raise fish (or shellfish) to market size. At present, it takes approximately 16-18 months of sea pen culture to produce marketable Atlantic salmon in Atlantic Canada. If growth rates during this phase could be doubled, it may be possible to market the salmon following a single growing season and obviate the need for overwintering in sea-pens. Growth hormone genes are normally expressed in the pituitary gland under the control of the central nervous system (CNS). In order to by-pass the CNS control, it is necessary to modify the tissue specific elements of the gene so that expression can take place elsewhere. Since the AFP genes are expressed predominantly in the liver, we designed our gene construct using the ocean pout AFP promoter (opAFP) linked to the chinook salmon GH cDNA (opAFP-GHc) (Hew et al. 1995). Our GH gene transfer studies were initiated in 1989 with the injection of these constructs into fertilized salmon eggs.

The GH transgene genomic integration frequency was similar to that observed for the AFP genes (2-3 %). All of the GH-transgenic founder fish were germ cell mosaics, and half of them failed to pass on the GH transgene to their offspring. Approximately 40% of the founder transgenic fish exhibited growth rates that were, on average, 3-6 times that of standard (control) salmon over a 30 month period. Mendelian inheritance of the GH transgene and its rapid growth phenotype was established at the F1 generation and has now been demonstrated through the second, third, fourth, and fifth generations.

In general, the transgenics grow most rapidly during their first year, slow to that of standard salmon at approximately one kilogram, and reach market size (3-4kg) a year earlier than do non-transgenics grown commercially in Atlantic Canada. Prospects and Expectations for the Future of Transgenics

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There is no doubt that transgenic techniques can be used to produce superior fish for domestic consumption. Such improvements could impart significant benefits to the growing world population and, at the same time, have a positive impact on the stability and preservation of the marine and terrestrial environment. However, there are a number of factors to consider and weigh before the final product can enter the marketplace, and these can be grouped under the following headings: -Science

- BioSafety (food safety; environmental protection; animal welfare) - Consumer acceptance

Science Our lessons from salmon have taught us that: 1. Integration frequencies of injected transgenes into the Atlantic salmon genome will be low (2-3%). This will probably be the case when using other gene constructs with other species 2. The transgene can integrate into more than one chromosome, and more than one copy of the gene can integrate into a single chromosome. 3. The transgenes can be rearranged prior to integration, resulting in weak to no expression. 4. All of the founder generation fish will be somatic as well as germ cell mosaics, indicating that the transgene does not integrate into the chromosomes until as late as organogenesis. 5. A Mendelian inheritance pattern cannot be established until the third generation (F2). 6. Transgenic fish homozygous for the transgene cannot be produced with certainty until the fourth generation (F3).

Two general conclusions can be drawn from the above observations. The production of stable lines of desirable and commercially valuable broodstock is not a short term endeavour, and the success of the final product is difficult to predict with certainty from the first two generations. Biosafety

There are three areas to consider under this heading: a) assessment of the safety of the transgenic fish as food for human (or animal) consumption, b) assessment of the possible environmental impact of the living transgenic fish should they be introduced or escape into the wild, and c) health of the transgenic fish produced as a

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result of biotechnology.

Food safety issues will be dealt with by the relevant regulatory body (country specific and also determined by the nature of the genetic modification). In order to bring transgenic Atlantic salmon to market in Canada or the U.S.A., the regulatory bodies involved (Health Canada and the FDA respectively), will require data to demonstrate that the edible tissue is equivalent in composition to that of the product already on the market and that there is no change in allergenicity of the product. Fish do not possess genes that code for toxins. Thus, there can be no rational concern that insertion of the transgene into the host DNA could result in a toxic food product (Berkowitz & Kryspin-Sorensen 1994).

Environmental protection considerations are hard to deal with in general terms, since potential risks will depend on the species being cultivated, the area in which it is being cultivated and the nature of the ecosystem into which transgenic individuals might possibly escape.

At present, salmon are cultured in cages that are located in coastal waters near to the shore. This brings with it a number of problems, one of which is the possibility that fish will escape and interact with the wild resource. When considering a transgenic salmon, it is essential that transgenic broodstock be maintained in secure, contained land-based facilities. Table fish, if they are to be cultured in cages, must be rendered sterile. To date, the only effective and publicly acceptable method of ensuring 100% sterility is the production of triploids (Johnstone 1996; Devlin & Donaldson 1992)

Intensive cage culture of salmon in coastal waters can have a negative impact on the environment and on the natural wild stocks (Stewart 1997). The long-term effect of near-shore aquaculture on the sustainability of the coastal ecosystem is impossible to predict, particularly when growth in production is factored into the equation. Therefore, under certain circumstances, it may be preferable for aquaculture development to take place on land in high quality recirculating water systems, making aquaculture less dependent on good coastal water sites. The challenge of such land-based systems is their commercially viability; their advantage is that they offer growers greater control over disease, parasitic infections, feeding regimens, temperature and photoperiod, making it possible to provide high-value fish in a sustainable, environmentally, and ecologically sound manner. Culture on land would also allow broodstock developers to fully domesticate farmed fish, and free them from concerns over changing the genetic make-up of domesticated fish from that of their wild relatives (Alestrøm 1995).

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Animal welfare is an issue of concern to fish farmers, as well as animal rights groups and, indeed, to all right-thinking individuals. Thus, the transgenic’s overall general welfare and health must be of paramount importance throughout the life cycle of the fish. Whatever the transgenic modification, fish must be healthy and exhibit normal feeding and other behaviour patterns typical of domesticated species. In Canada the appropriate regulatory agencies for food safety and animal health are Health Canada, and the Canadian Food Inspection Agency. Environmental safety is regulated by Environment Canada and the Department of Fisheries and Oceans. In United States the appropriate agency is the Center for Veterinary Medicine within the Food and Drug Administration. In the case of transgenic salmon the transgenes and their products are considered as new animal drugs. Consumer acceptance It is important to think globally when considering consumer acceptance of transgenic technology. What may seem outlandish, unnatural, and unnecessary to inhabitants of one part of the planet, may hold the key to increased prosperity, environmental remediation, and even survival elsewhere. It is also important to learn from past experience - no new technology is risk-free but the benefits may vastly outweigh the risks (for example, the Green Revolution, and the development of prescription medicines).

In Europe and, to a lesser extent North America, fear of the unknown, distrust of Government and Big Business, and a desire (in the absence of hunger) to return to nature has resulted in something approaching biotechnophobia. It will take time, and considerable dialogue between all those involved for this situation to change.

From the perspective of the general public, information concerning genetically modified food must come from an objective, unbiased source that the consumer has confidence in, and must provide the consumer with the ability to assess the product and then make an informed, rational choice as to whether to buy it or not. The general public should be kept informed about upcoming products in advance of their appearance in the marketplace. They must be certain that new products of biotechnology are safe, useful and beneficial to their well being and to the environment. Producers must also be kept informed about new products, and given the confidence that they will not lose their markets because they choose to grow fish using the most advanced methods available to them.

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Acknowledgements The authors gratefully acknowledge NSERC, MRC, IRAP-NRC, ACOA, DFO, Aqua Bounty Canada, and AquaNet. for providing funding for this research. References Alestrøm, P. 1995. Genetic engineering in aquaculture. In Helge Reinertsen and

Herborg Haaland (eds), Sustainable Fish Farming. A.A.Balkema/ Rotterdam/ Brookfield

Berkowitz, D.B. and Kryspin-Sorensen, I. 1994. Transgenic fish: Safe to eat?

Bio/Technology 12: 247-252. Devlin, R.H. and Donaldson, E.M. 1992. Containment of Genetically Altered Fish

with emphasis on Salmonids. In Hew, C.L. and Fletcher, G. L. (eds) Transgenic Fish: 229-265. World Scientific.

Devlin, R.H. 1997. Transgenic Salmonids. In Louis Marie Houdebine (ed) Transgenic Animals, Generation and Use. Harwood Academic Publishers. Pp. 105-117.

Fletcher, G.L., Hew, C.L., and Davies, P.L. 2001. Antifreeze Proteins of Teleost

Fishes. Annu Rev.Physiol. 63:359-390. Fletcher, G.L, Goddard., S.V. Davies., P.L. Gong., Z. Ewart, S.V., and Hew, C.L.

1998. New insights into antifreeze proteins in fish: physiological significance and molecular regulation. In: H.O. Portner and R. Playle (eds.), Cold Ocean Physiology, Cambridge University Press. Pages 239-265.

Fletcher, G.L., Alderson, R., Chin-Dixon, E.A., Shears, M.A., Goddard, S.V., and

Hew, C.L. 1999a Transgenic fish for sustainable aquaculture. Proceedings of the 2nd International Symposium on Sustainable Aquaculture, N. Svennevig, H. Reinertsen, & M. New (eds.). A.A. Balkema, Rotterdam. Pages 193-201.

Fletcher, G.L., Goddard, S.V., and Wu, Y. 1999b. Antifreeze proteins and their

genes: from basic research tobusiness opportunity. Chemtech 29:17- 28. http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html

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Gjedrem, T. 1997. Selective breeding to improve aquaculture production. World Aqua. 28: 33-45.

Hew, C. L., and Fletcher, G. L. 1997 Transgenic fish for aquaculture. Chemistry

and Industry. April 21, 311-314. Hew, C. L., and Fletcher, G. L. 2001. The role of aquatic biotechnology in

aquaculture. Aquaculture 197: 191-204. Hew, C.L., Fletcher, G. L. and Davies P. L. 1995 Transgenic salmon: tailoring the

genome for food production. Journal of Fish Biology 47 (Supplement A): 1-19.

Johnstone, R. 1996 Experience with salmonid sex reversal and triploidisation

technologies in the United Kingdom. Bull. Aquacul. Assoc. Canada 96 (2): 9-13.

Melamed, P., Gong, Z., Fletcher, G., and Hew, C.L. 2002. The potential impact of

modern biotechnology on fish aquaculture. Aquaculture 204: 255-269. New, M. B. 1997 Aquaculture and the capture fisheries - balancing the scales.

World Aquaculture 28 (2): 11-30. Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer, M.E., Rosenfeld, M.G.,

Brinberg, N.C., and Evans, R.M. 1982 Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 30: 611-615.

Pauly, D., Christensen, V., Dalsgaard, J., Froese, R., and Torres, F. Jr. 1998.

Fishing Down Marine Food Webs. Science 279: 860-863. Shears, M.A., Fletcher, G. L., Hew, C.L., Gauthier, S., and Davies, P. L. 1991

Transfer, expression, and stable inheritance of antifreeze protein genes in Atlantic salmon (Salmo salar ). Molecular Marine Biology and Biotechnology 1: 58-63.

Stewart, J. E. 1997. Environmental impacts of aquaculture. World Aquaculture 28

(1): 47-52. Watson, R., and Pauly, D. 2001. Systematic distortions in world fisheries catch

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trends. Nature 414: 534-536.

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METABOLIC DETERMINANTS OF GLUCOSE

UTILIZATION IN RAINBOW TROUT

Max T. Bennett Department of Biological Sciences

Idaho State University Pocatello, ID 83209 USA

phone: (208) 282-3790/fax: (208) 282-4570/e-mail: [email protected]

Kenneth J. Rodnick Department of Biological Sciences

Idaho State University Pocatello, ID 83209 USA

[email protected]

EXTENDED ABSTRACT ONLY- DO NOT CITE Introduction It has been well documented that trout, along with other carnivorous fishes, have a limited ability to metabolize glucose (Moon 2001). One possible explanation for glucose intolerance in fishes is a competitive interaction between circulating glucose and other energy substrates. In mammals, elevated plasma free fatty acids (FFA) reduce the organism’s ability to transport and utilize glucose (Randle 1963). The relationship of plasma FFA and in vivo glucose metabolism has not been investigated in fish. Similar to mammals, we hypothesized that an elevation in plasma FFA would decrease the rate of glucose uptake in rainbow trout (Oncorhynchus mykiss). Methods Immature male and female rainbow trout (n = 14, fork length = 30.5 ± 0.4 cm were fed the up to and including the day of experimentation. Trout were anesthetized with a 0.007% MS222 solution containing 5.0 mM NaHCO3 and 1% NaCl. Animals were cannulated via the dorsal aorta using no heparin. Cannulae were filled with 1% NaCl. Trout were then allowed to recover in black Perspex® boxes 48 h prior to performing intravenous glucose tolerance tests with normal and elevated FFA. FFA were elevated by injecting an emulsion of fish oil (250 mg/kg body wt.) and heparin (50 U/kg body wt.) 60 min prior to

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receiving a glucose load (250 mg/kg body wt). Control fish were given an equivalent volume of saline the same dose of glucose. Blood samples (200 µl) were taken at –60, 0, 30, 60, 120, 180, and 420 min. Whole blood was mixed with 0.6 mg EGTA, centrifuged at 7200g for 2 min, and plasma was isolated and frozen at the temperature of liquid N2. Blood glucose was measured using a One Touch® Ultra glucometer at the times above and at 5, 10, and 20 min. FFA and triglycerides were measured with NEFA C (Wako) test kit and Triglyceride Infiniti assay (Sigma) respectively. Glucose elimination rates (KG) were calculated as percent fall of glucose after log transformation between 5 and 20 min. Additionally, non-log transformed values were used to calculate KG from 30-420 min. Calculated KG values were compared using t-tests. FFA values were compared using repeated measures ANOVA (p<0.05) Results and Discussion Intravenous injection of fish oil and heparin increased plasma FFA levels 60%, but not triglycerides or glucose after 60 min. This elevation did not appear to affect glucose utilization.

2

7

12

17

22

-60 0 60 120 180 240 300 360 420

Time (min)

Glu

cose

(mm

ol) Fish oil and heparin

Saline

Fig. 1 Blood glucose levels for rainbow trout with elevated FFA (increased by bolus IV injection of fish oil (250 mg/kg body wt.) and heparin (50 U/kg body wt.) at -60 min and saline controls during intravenous glucose tolerance tests.

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All fish received an injection of glucose (250 mg/kg body wt.) at the start of the test (0 min). We noted similar KG values between 5 and 20 min for fish with elevated FFA (1.21 ± 0.13%/min) and control trout (1.19 ± 0.11%/min, p=0.89). KG between 30 and 420 min were also similar between experimental groups (0.14 ± 0.01%/min for fish with elevated FFA and 0.11 ± 0.01%/min for controls (p=0.13)). These data do not support our initial hypothesis and suggests that, in contrast to mammals, the acute elevation of circulating FFA does not affect intravenous glucose tolerance of rainbow trout. We therefore question whether the glucose intolerance exhibited by trout is due to a reciprocal metabolic interaction between glucose and fatty acids in peripheral tissues. References Moon, T. W. (2001) Glucose intolerance in teleost fish: fact or fiction?

Comparative Biochemistry and Physiology Part B. 129, 243-249.

Randle, P. J., Garland, P. B., Hales, C. N. and Newsholme, E. A. (1963). The

glucose fatty acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1, 785-789.

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DIETARY LIPIDS, IMMUNE FUNCTION AND PATHOGENESIS

OF DISEASE IN FISH

Santosh P. Lall and Joyce E. Milley National Research Council Canada, Institute for Marine Biosciences,

1411 Oxford Street, Halifax, NS B3H 3Z1 Phone: 902-426-6272/Fax: 902-426-9413/e-mail: [email protected]

David A. Higgs

Department of Fisheries and Oceans, West Vancouver Laboratory, 4160 Marine Drive, West Vancouver, BC, V7V 1N6

Phone: 604-666-7924/Fax: 604-666-3497/e-mail: [email protected]

Shannon K. Balfry Faculty of Agricultural Sciences, University of British Columbia

2357 Main Mall, Vancouver, BC, Canada V6T 1Z4 Phone: 604-666-0034/Fax: 604-666-3497/e-mail: [email protected]


Lipids supply essential fatty acids (EFA) and energy in fish diets. Most fish cannot synthesize (de novo) polyunsaturated fatty acids (PUFA) and therefore they must be supplied in the diet for normal growth, reproduction and health. EFA include PUFA of the n-3 and n-6 series, e.g. α-linolenic acid, 18:3n-3 and linoleic acid, 18:2n-6. Generally, EFA requirements of freshwater fish can be met by the supply of 18:3n-3 and 18:2n-6 fatty acids in their diets. By contrast, the EFA requirement of marine fish can only be met by supplying the correct concentrations and ratios of the long-chain PUFAs, eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) with perhaps some arachidonic acid (20:4n-6; AA), a highly unsaturated member of the n-6 series. (NRC, 1993; Higgs and Dong, 2000). Freshwater fish are able to elongate and desaturate 18:3n-3 to 22:6n-3, whereas marine fish, which lack or have a very low activity of 5-desaturase, require the long-chain PUFAs, mainly from the n-3 series. This presentation will briefly review the status of knowledge on the relationship between EFA and immune functions with emphasis on eicosanoid production. Fish tissues contain relatively higher concentrations of PUFA than are found in those from mammals. PUFAs are important components of all cell membranes,

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mailto:[email protected]



which make fish tissue highly vulnerable to lipid peroxidation. Although the quantitative requirements and deficiency signs of EFA in several freshwater and marine fish have been documented (NRC, 1993; Higgs and Dong, 2000), the functional role of n-3 and n-6 PUFA in nonspecific and specific humoral and cellular immunity has not been studied extensively (Balfry and Higgs, 2001). Twenty-carbon PUFAs derived from EFA are precursors of two groups of eicosanoids that are comprised of prostaglandins and thromboxanes on the one hand, as well as leucotrienes and lipoxins on the other. These may have diverse pathophysiological actions that influence immune response and inflammatory processes especially if there is a preponderance of eicosanoids stemming from AA. Eicosanoids constitute a group of extracellular mediator molecules that are part of an organisms defense system. Further, they are synthesized from di-hommo gamma linolenic acid (20:3, n-6), as well as AA and EPA by the action of two oxygenase enzymes, cyclooxygenase and lipoxygenase. Lipoxygenase yields a range of monohydroxy fatty acids while di- and tri-hydroxy fatty acids, such as leucotrienes (LT) and lipoxins (LX) are also formed via epoxy intermediates (Figure 1).

20:4(n-6) 20:5(n-3) 5-, 12- or 15-

LIPOXYGENASE O2PGH

2O22e-

CYCLOOXYGENASE

Prostanoids PGD2 PGE2 PGF2 PGI2 TXA2 PGD3 PGE3 PGF3 PGI3 TXA3

Hydroperoxyeicosatetraenoic acids (HPETEs) Hydroperoxyeicosapentaenoic acids (HPEPEs)

Hydroxyeicosatetraenoic acids (HETEs) Hydroxyeicosapentaenoic acids (HEPEs)

H2O

2e-

Leucotriene A4 (LTA4)

Leucotriene A5 (LTA5)

Leucotriene B4 (LTB4) Leucotriene B5 (LTB5)

Peptidoleucotrienes LTC4 LTD4 LTE4 LTC5 LTD5 LTE5

glutathione

H2O Lipoxin A Lipoxin B

Figure 1. Pathways of conversion of 20:4(n-6) and 20:5(n-3) to eicosanoids. The major source of n-3 EFA in fish diets is marine fish oil (MFO) and the partial substitution of MFO with vegetable and animal lipid sources in fish diets

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affects the tissue and cellular lipid composition. Diets containing high levels of n-3 and n-6 fatty acids from fish and vegetable oils modify the fatty acid composition of cell phospholipids of Atlantic salmon (Bell et al. 1993) and halibut leucocytes (Table 1). Total n-3 and n-9 fatty acid concentrations increase significantly in liver, heart and muscle of halibut and Atlantic salmon fed flaxseed and canola oil, respectively. Recently we have separated major eicosanoids in several tissues of Atlantic salmon, haddock and halibut by a newly developed HPLC method using a reversed-phase C18 column, a linear gradient mobile phase and diode-array detector. Their identities have been confirmed by LC-MS with negative-ion electrospray ionization. LTB5 and LTB4 were found to be the most abundant leucotrienes generated by these fish followed by prostaglandins and hydroxy fatty acids respectively. 11-HETE and 12-HETE were the most common hydroxy fatty acids in the foregoing fish species. The changes in fatty acid composition of phospholipids affected the synthesis of eicosanoid precursors in halibut leucocytes. When the dietary intake of n-6 fatty acids increased, a higher level of AA-derived eicosanoids was observed. Studies conducted on the effect of n-3 and n-6 fatty acids on the immune responses in fish are preliminary and often inconclusive. Essential fatty acid deficiency in rainbow trout reduces the in vitro killing of bacteria by macrophages and reduces antibody production (Kiron et al., 1995). The increased activity of head kidney macrophages has been associated with higher levels of dietary n-3 fatty acids in catfish (Sheldon and Blazer, 1991). The impacts that dietary fatty acids have on the immune responses are complex and depend on several factors that influence eicosanoid production including competition between n-3 and n-6 fatty acid during metabolism for chain elongation and desaturation, the cell types involved, and the sources of fatty acids in the diet. Although the nature of dietary lipids and the concentration of essential fatty acids have direct effects on eicosanoid metabolism, the regulation of the immune system by their direct effects on cells such as macrophages and lymphocytes or their indirect effects via cytokines requires further studies. Recent advances in our understanding of PUFA metabolism and immunology provide an opportunity to further investigate the role of n-3 and n-6 PUFAs in defense mechanisms of fish and to prevent diseases.

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Table 1. Fatty acid composition of head kidney leucocytes from halibut fed diets containing anchovy oil, vegetable oils and poultry fat. Diets Fatty Acid anchovy flaxseed canola sunflower poultry 14:0 2.0±0.5 1.6±0.2 1.4±0.1 1.3±0 1.2±0.2 16:0 12.8±0.9 13.4±0.7 12.2±0.3 12.2±1.0 14.9±0.5 18:0 7.3±0.7 9.8±0.4 7.4±0.3 8.3±0.7 8.9±0.1 Total saturates1 23.8 26.6 23.1 24.6 27.6 16:1 3.4±0.6 2.7±0.2 2.7±0.1 2.3±0.1 3.4±0.3 18:1 n-9 9.1±0.3 11.6±0.9 14.2±0.8 10.3±0.7 14.3±0.3 18:1 n-7 3.2±0.5 2.6±0.2 2.9±0.2 2.6±0.2 3.4±0.2 20:1 1.1±0.3 1.3±0.2 1.6±0.1 1.2±0.2 1.5±0.1 22:1 0.9±0.7 0.4±0.1 0.3±0.04 0.3±0.04 0.2±0 24:1 0.8±0.1 0.8±0.1 0.9±0.1 1.0±0.01 1.6±0.04 Total monoenes1 20.2 21.1 24.0 18.9 25.5 18:2 n-6 1.4±0.2 7.0±0.9 6.7±0.2 18.4±1.7 7.4±0.4 18:3 n-6 0.1±0.08 0.04±0.04 0.05±0.01 0.01±0.02 - 20:2 n-6 0.2±0.1 0.5± 0.04 0.7±0.02 1.9±0.23 0.7±0.07 20:3 n-6 0.1±0.07 0.04±0.04 0.1±0.01 0.05±0.01 0.2±0.01 20:4 n-6 2.8±0.9 1.7±0.03 2.1±0.1 1.6±0.2 2.7±0.2 22:2 n-6 1.8±1.7 1.5±0.5 1.5±0.2 1.5±0.5 0.7±0.1 22:5 n-6 0.4±0.1 0.2±0.02 0.3±0.03 0.2±0.03 0.3±0 Total n-61 6.8 11.0 11.4 23.6 12.0 18:3 n-3 0.3±0.04 5.9±1.1 1.3±0.06 0.4±0.1 0.4±0.04 18:4 n-3 0.4±0.05 0.14±0.06 0.2±0 0.1±0.01 0.2±0.02 20:3 n-3 0.2±0.2 1.8±0.3 0.4±0.02 0.1±0.05 0.1±0.01 20:4 n-3 0.3±0.1 0.1±0.02 0.1±0.01 0.1±0.01 0.2±0.01 20:5 n-3 9.9±2.5 6.0±0.6 7.6±0.4 5.4±0.4 7.7±0.2 22:4 n-3 1.3±1.5 1.1±0.4 1.1±0.1 0.9±0.4 0.4±0.1 22:5 n-3 2.0±0.6 1.2±0.1 1.4±0.1 1.2±0.1 1.7±0.1 22:6 n-3 15.8±4.1 9.1±1.3 12.9±1.2 10.1±1.8 12.6±0.3 Total n-31 30.1 25.4 25.0 18.4 23.3 Total PUFA1 52.4 49.5 50.2 53.6 44.0 n-3/n-6 4.4 2.3 2.2 0.8 1.9 1 Includes all the fatty acids identified in the respective group

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References NRC (National Research Council), 1993. Nutrient requirements of fish. National

Academy of Sciences, Washington, DC, 114 p. Balfry, S.K. and D.A. Higgs. 2001. Influence of dietary lipid composition on the

immune system and disease resistance of finfish. In: Nutrition and Fish Health (eds. C. Lim and C.D. Webster), pp. 213-234, The Haworth Press Inc., New York.

Higgs, D.A. and F. M. Dong. 2000. Lipids and fatty acids. In: Encyclopedia of

Aquaculture (ed. R.R. Stickney), pp. 476-496, John Wiley & Sons, Inc., New York.

Kiron, V., H. Fukuda, H., T. Takeuchi, T. Watanabe. 1995. Essential fatty acid

nutrition and defence mechanisms in rainbow trout Oncorhynchus mykiss. Comp. Biochem. Physiol. 111A: 361-367.

Bell, J.G., D.R. Dick, A.H. McVicar, J.R., Sargent and K.D. Thompson. 1993.

Dietary sunflower, linseed and fish oils affect phospholipid fatty acid composition, development of cardiac lesions, phospholipase activity and eicosanoid production in Atlantic salmon (Salmo salar). Prostaglandins, Leukotrienes and Eicosanoid Fatty Acids 49: 665-673.

Sheldon, W.M., Jr. and V.S. Blazer. 1991. Influence of dietary lipid and

temperature on bactericidal activity of channel catfish macrophages. J. Aquat. Anim.Health 3: 87-93.

Acknowledgements The review contains research information from a project (AP 7) supported by AquaNet, a NSERC’s Network of Centre of Excellence in Aquaculture. The support of the National Research Council, Department of Fisheries and Oceans and AquaNet is gratefully acknowledged.

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FISH PROTEOMICS AND NUTRITION

Houlihan D.F1, Martin S.A.M1, Médale, F.2 & Kaushik, S2 .

1Department of Zoology, University of Aberdeen, Aberdeen AB24 2TZ, UK

2Fish Nutrition Laboratory, INRA-IFREMER 64310, Saint Pée sur Nivelle, France

EXTENDED ABSTRACT ONLY- DO NOT CITE The proteome is the expressed protein complement of a genome in a tissue. Protein extraction followed by high resolution 2 dimensional electrophoresis, coupled with gel image analysis allows hundreds of protein to be monitored in parallel, permitting a global picture to emerge of changes in protein profile under different metabolic states. Protein spots are then subjected to further analysis leading to protein identification. A rainbow trout proteome map is being developed in which all proteins are recorded in terms of molecular weight, isoelectric point (pI) and abundance. Protein spots that are found to be of interest are excised from the gel and subjected to trypsin digestion and separation of peptides by mass spectrometry. These peptide mass fingerprints are then used to search the Genbank data base for protein identification. A proteomics approach has been used to study the protein profiles of livers of rainbow trout following different nutritional challenges. Dietary changes will result in a multitude of metabolic and gene expression responses fish, identifying proteins that are altered in their expression pattern will help us have an insight into the biochemical responses of this tissue. Initial experiments were performed on fish that were starved for a period of two weeks, this revealed a panel of proteins that were altered in expression, some of these proteins are directly involved in protein breakdown. This approach supplemented information from transcriptome analysis by showing which proteins were actually increased in amount. A second project concerns the substitution of fish meal protein with plant derived proteins (soybean); a twelve week growth trial was performed. The growth rate of the fish fed the two diets were not different, however utilisation

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of ingested protein was different, with consumption and protein synthesis greater in fish fed the diet with a high proportion of soybean meal (SBM). For both this trial proteomic analysis of the liver has been carried out. During these studies ~800 proteins were analysed for expression pattern, some proteins were up or down regulated as a result of the nutritional change. Currently eighteen proteins have been positively identified from trypsin digest fingerprints using the MASCOT search program. In conclusion we demonstrate how proteomics coupled with genome characterisation could help understand dietary responses in fish and in the development of alternative feed sources. Acknowledgement This research was funded by EU (Q5RS-2000-30068) PEPPA References Martin, S.A.M., P.Cash, S. Blaney & D.F. Houlihan 2001. Proteome analysis of

rainbow trout (Oncorhynchus mykiss) liver proteins during short term starvation. Fish Physiology and Biochemistry 24 (3): 259-270

Carter C, D. Houlihan, A. Kiessling, F. Médale and M. Jobling 2001.

Physiological effects of feeding. In Food intake in fish (Edited by Houlihan, Boujard & Jobling) Blackwell Science, UK

DIGESTIVE ENZYME EXPRESSION IN THE EXOCRINE PANCREAS

DURING THE ONTOGENY OF THE WINTER FLOUNDER

(PSEUDOPLEURONECTES AMERICANUS).

H.M. Murray AquaNet, Memorial University of Newfoundland, St. John’s, Nfld. Canada

Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, N.S., Canada, B3H 3Z1

Phone: (902) 426-4319 Fax: (902) 426-9413

E-mail: [email protected]

J.C. Perez-Casanova Department of Biology, Dalhousie University, Halifax, N.S., Canada, B3H 4J1

Institute for Marine Biosciences, National Research Council of Canada

J.W. Gallant, S. Douglas, and S.C. Johnson Institute for Marine Biosciences, National Research Council of Canada

EXTENDED ABSTRACT ONLY-DO NOT CITE

Introduction One of the major challenges to successful rearing of marine fish is providing high quality nutrition at reasonable expense, especially during early larval life. Live feeds such as rotifers and Artemia give the best larval growth and survival, however these feeds are difficult and expensive to produce at commercial levels. Considerable effort has been put into the development of relatively cheap, high quality formulated diets. Unfortunately, these diets generally do not support the same level of larval growth and survival as live feeds. This may be due to improper diet formulation stemming from a lack of information on the dietary requirements for many species. To determine specific dietary requirements in part requires an understanding of digestive physiology. We are using histological and molecular techniques to describe the functional development of

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the exocrine pancreas in the winter flounder with specific reference to trypsinogen, amylase and bile salt activated lipase (BAL). Methods

Fish Rearing

Winter flounder larvae were reared at the Institut des Sciences de la Mer de Rimouski (ISMER), Quebec, Canada.

In situ Hybridisation

Sectioning

Winter flounder larvae at hatch, 5, 10, 20, and 35 days post-hatch (dph) and newly metamorphosed were fixed in 10 % formalin for 6-8 hours, dehydrated through ethanol series, cleared in xylene, infiltrated in paraffin and embedded for sagittal section. Seven micron sections were cut from one to two blocks and placed on silane coated microscope slides, dried briefly and then baked overnight at 60 oC. Fish from duplicate blocks were serial sectioned and stained in haematoxylin and eosin for general histology.

Probe Preparation

Probes specific for winter flounder trypsinogen, amylase and BAL were prepared by in vitro transcription of linearized cDNA clones using the DIG RNA labelling kit (Roche Applied Science, Laval, PQ, Canada) and T3 and T7 RNA polymerase. Following synthesis, probes were hydrolysed to 250 bp, precipitated and then resuspended in in situ hybridisation buffer (Murray et al., 2002). The specificity of the antisense (AS, cRNA sequence) probes was verified using the sense (S, mRNA sequence, control) probes.

Hybridization and marker detection

Hybridization conditions were modified from Murray et al., (2002). Briefly, deparaffinised, rehydrated and equilibrated tissue sections were hybridised with 50 µl of probe overnight at 45º C in a closed humid incubation chamber. Digoxigenin was detected using sheep anti-DIG-alkaline phosphatase conjugated antibodies following a 30 minute block in 0.1 M Tris pH 7.5, 150

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mM NaCl containing 1% BSA and 10% lamb serum. Antibody was diluted to 1:250 with the above buffer and 500 µl was added to each slide. Slides were incubated for 30 minutes at room temperature in a humid chamber. Slides were then washed for a further 30 minutes in fresh buffer without lamb serum. Antibody was detected by incubating slides for 10-15 minutes in a chromogenic buffer containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. The reaction was stopped in 1x fish saline, and slides were rapidly dehydrated through ethanol series, cleared in xylene and mounted in cytoseal for observation using differential interference contrast microscopy. Images were captured using an Optronics VI-470 camera and Simple PCI software and grouped into plates using Adobe Photoshop 6.0.1. Results The pancreas was first identified in winter flounder larvae shortly following hatch and appears as a small compact structure situated just dorsal and slightly posterior to the liver (Figure 1A). As the fish develop toward metamorphosis the pancreas becomes diffuse, eventually spreading throughout the mesentery surrounding the stomach, the upper intestine and later the pyloric caecae (Figure 1B). Hybridization conditions and reaction times were identical for all probes. Winter flounder trypsinogen, amylase and BAL all showed expression from about 5 dph (mouth opening) (Figure 2 ACE). Sense controls consistently showed no staining reaction (not shown). Trypsinogen gave the strongest reaction of all probes and remained as such through metamorphosis (Figure 2 AB). Amylase produced a strong reaction at mouth opening but gradually decreased in fish of later stages (Figure 2 CD). BAL gave a light reaction at mouth opening but gradually became more intense towards metamorphosis (Figure 2 EF). Conclusions In winter flounder the development of the pancreas through ontogeny is similar to that described for the Japanese flounder by Kurokawa and Suzuki (1996). In situ hybridization results for trypsinogen, amylase and BAL

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Figure 1. Sagittal sections through winter flounder larvae at mouth opening (A) and at metamorphosis (B) showing distrubution of the pancreas (arrows). stom, stomach; esoph, esophagus; intest,intestine; H, hindgut; M, midgut; F, foregut. Ys, yolksac. Scale bar = 100 µm.

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Figure 2. Sagittal sections through winter flounder larvae at mouth opening (A,C,E) and at metamorphosis (B,D,F) following in situ hybridization with cRNA probes for either trypsinogen, (A,B) amylase (C, D), or bile salt activated lipase (E,F). Note specificity of the hybridization reaction for the pancreas (arrows) and the differences in the distribution of the pancreas between a 5-dph fish and one at metamorphosis. Asterisk shows the position of the liver relative to the pancreas. Scale bar = 100 µm.

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show that the exocrine pancreas actively expressed these genes at the time of mouth opening. The expression of trypsinogen at such an early age suggests that winter flounder are prepared to digest proteins long before the stomach is functional. The intense chromogenic reaction associated with the trypsinogen probe relative to the others, suggests that this gene is important in digestion at the start of exogenous feeding. The strong reaction for amylase transcripts at mouth opening suggests that this enzyme is important in early development. BAL is expressed at low levels at mouth opening with an increase in reaction intensity through ontogeny. Expression of these genes at mouth opening indicates that at the start of exogenous feeding, this species has potential for digesting a wide range of nutritionally important items including proteins, lipids and carbohydrates. We are presently verifying this expression data with biochemical analysis. References Kurokawa, T. and Suzuki, T. 1996. Formation of the diffuse pancreas and the

development of digestive enzyme synthesis in larvae of the Japanese flounder Paralichthys olivaceus. Aquaculture. 141; 267-276.

Murray, H.M., Hew, C.L., Kao, K.R. and Fletcher, G.L. 2002. Localization of

cells from the winter flounder gill expressing a skin type antifreeze protein gene. Can. J. Zool. 80; 110-119.

Acknowledgements - AquaNet - Institut des Sciences de la Mer de Rimouski - National Research Council-Institute for Marine Biosciences

INDUCTION OF DIGESTIVE ENZYMES IN THE BRAZILIAN

CATFISH (Pseudoplatystoma coruscans)

Lícia Maria Lundstedt Department of Genetics and Evolution, Federal University of São Carlos

Rod. Washington Luís, km 235; CEP: 13565-905; CP 676. São Paulo. Brazil Phone/Fax: 54 (16) 260-8376 / 54 (16) 260-8377

e-mail: [email protected]

José Fernando Bibiano Melo* and Gilberto Moraes** Department of Genetics and Evolution, Federal University of São Carlos

e-mail: *[email protected]; ** [email protected]

Keywords. Digestive enzymes; Protease; Trypsin; Chymotrypsin; Lipase; Amylase; Pseudoplatystoma coruscans

Introduction Growth reflects the metabolic interactions and adjustments, which are sustained by the nutritional status. This is a function of feeding and is straightly related to digestion. Fish digestive processes are scarcely known and this is a reason for the many problems raised in fish feeding (Dabrowski and Glogowski, 1977). Knowing digestive enzyme profile can be the clue to explain the digestive processes. The ability of fish and other vertebrates to use nutrients depends on some factors such as the synthesis of appropriate enzymes, the enzyme production in suitable amounts and the enzyme distribution along the gut lumen. Usually, distribution and activity of digestive enzymes along the gut change with feeding habits (Tengjaroenkul et al., 2000). Nowadays, the understanding on how diets control the digestive enzyme expression becomes a relevant tool on the optimization of macronutrient level of feeding. Protein and lipids are pivotal in the diet composition for the most carnivorous fish (Chou et al., 2001). The absence or unbalance of any nutrient leads to metabolic rearrangement to compensate or replace it. The possibility of carbohydrates to replace protein is limited and, in carnivorous fish, it is controversial. However, proper adjustments can improve the use of dietary carbohydrate. The efficiency of energy retention and the best use of proteins are

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observed in trout fed on proper diets (Suarez et al., 1995). A few factors can account for such effect but adaptive changes of the digestive enzyme spectrum associated with the feeding, are the main. High protease activities in carnivorous fish and high carbohydrase activities in omnivorous and herbivorous one, has been reported (Ugolev and Kuz’mina, 1994). The background on the enzyme specific activities is fundamental in animal comparative biochemistry. The total hydrolytic capacity of the digestive tract is a clue to evaluate the animal potential for the best use of the feeding. However, this demands enzyme analysis of all gut sections as the best way to assess the whole digestive capacity (Buddington et. al, 1997). Considering the gut responsiveness to macronutrient levels versus the establishment of the best concentration, or the best ratio among them, we proposed to optimize the feeding for the Brazilian catfish Pseudoplatystoma coruscans. This is a carnivorous teleost fish, appreciated by the fillet quality, as sportive fishing and by the large aquaculture potential in spite the culture technology is not well established. The activity of digestive enzymes of the Brazilian catfish was quantified at the long of the gut, and the adaptive responses to dietary protein were studied. Materials and Methods Juveniles of P. coruscans (70 ± 9g) of both sex were maintained in 2,000L fiberglass tanks with flow-through re-circulated and filtered water at 23.5 ± 1.2ºC, pH 6.62 ± 0.11, D.O. 6.8 ± 0.49 mg/l, alkalinity 95.0 ± 2.96 Siemens/cm2, ammonia 14.44 ± 1.38 g/L and hardness 38.0 mg/L. Thirty six fish were divided into four tanks and artificially reared were fed on four dietary levels of crude protein (table 1). Feeding corresponding to 8% of the biomass was offered twice a day, during 28 days. The feeding was ceased twenty-four hours before sampling. At the time of tissue collection, six fish from each treatment were sampled, anesthetized (Benzocaine 100mg/L of water), and killed by cervical separation. The digestive tract was excised and kept frozen at –20ºC for further digestive enzyme analysis. Tissue extracts: samples from stomach, anterior intestine, medium and posterior one, were homogenized with a Potter-Elvehjem tissue-cell disrupter under ice-bath in a v:v glycerol : buffer 0.02M Tris/0.01M phosphate pH 7.0. The extracts were centrifuged at 11,000 g for 3 min and the supernatants (crude extracts) were used as enzyme source.

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Enzyme assay: the unspecific proteolytic activity was determined by 1% casein hydrolysis (Kunitz, 1947) modified by Walter (1984). The pH values were previously optimized for each gut fraction, 0.2M glycine/HCl buffer pH 2.0 for stomach, 0.1M Tris/HCl buffer pH 7.5 to anterior intestine, and 0.1M Tris/HCl buffer pH 9.0 to medium and posterior intestine. Reaction was done at 25ºC for 1 h, stopped by 15% TCA, and the optical density of the supernatant was recorded at 280nm against tyrosine as standard. Specific activity is expressed as µmol of hydrolyzed substrate.min-1.mg-1 of protein (U/mg protein). The protease trypsin and the chymotrypsin were assayed by specific methods as Hummel (1959). Trypsin substrate was 1.04 mM TAME.HCl (α-p-Toluenesulphonyl-L-arginine methyl ester hydrochloride) in 0.01M CaCl2.H2O/0.2 M Tris/HCl buffer pH 8.1. The temperature reaction was 25ºC and the optical density was followed at 247nm for 20 seconds. Chymotrypsin substrate was 1mM BTEE (N-benzoyl-L-tyrosine ethyl ester) into methanol 2:3 (v:v). The reaction was performed into 0.1M CaCl2.H2O/0.1 M Tris/HCl buffer pH 7.8 at 25ºC, and followed for 40 seconds at 256nm. Both activities were respectively expressed in µmol of arginine.min-1.mg-1 of protein (U/mg protein) and nmol of tyrosine.min-1.mg-1 of protein (mU/mg protein). Amylase was assayed into 0.2M citrate/phosphate buffer pH 7.0 with 5% starch solution as substrate (Bernfeld, 1955). The reaction was incubated at 25ºC for 30 minutes and stopped with 5% ZnSO4, 0.3N Ba(OH)2. Free glucose was determined by Park-Johnson method (1949) at 690nm. Specific activity was expressed in µmol of reducing sugars.min-1.mg-1 of protein (U/mg protein). Lipase determination was adapted from Albro et al. (1985). Reactions were incubated with 0.4 mM ρ-nitrophenyl myristate in 24 mM ammonium bicarbonate, pH 7.8, with 0.5% Triton X-100. The optical density was registered at 405 nm for 30 min. One unit was defined as µmol of substrate hydrolyzed per min and expressed per mg of protein (U/mg protein). Protein was determined by the method of Lowry et al. (1951), with bovine albumin as standard.

The statistical test applied to analyze the data set was the non-parametric procedure of Kruskal-Wallis followed by post-test of Dunn’s with significant level of α=0,05. All values are reported as mean ± s.e.m. (n = 6)

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Table 1: Ingredient and approximate composition of the experimental diets.

Experimental Diets Ingredient, percent 20% CP 30% CP 40% CP 50% CP Fishmeal 15,58 34,75 55,00 74,50 Meat meal 11,00 10,00 8,00 6,50 Corn 59,4 41,00 22,03 3,18 L-Lysine 0.45 0,28 - - Vegetable Oil 1 7,35 7,40 7,5 7,85 Cellulose 4,75 5,10 6,00 6,50 Feeding stimulants 2 0.40 0.40 0.40 0.40 Vitamin-mineral Premix 3 0,50 0,50 0,50 0,50 Vitamin C 0.05 0.05 0.05 0.05 Pellet binder 4 0.50 0.50 0.50 0.50 Antioxidant 5 0.02 0.05 0.02 0.02 Total 100,00 100,00 100,00 100,00 Dry Matter (%) 90,98 91,57 92,15 92,78 Energy (kcal/kg) 4086,75 4086,99 4079,35 4087,37 Crude Protein (%) 20,07 30,01 40,03 50,07 Crude Fiber (%) 6,29 6,01 6,19 6,02 Crude Fat (%) 13,36 13,25 13,1 13,21 Calcium (%) 2,76 3,86 4,87 5,91 Phosphorus (%) 1,54 2,09 2,6 3,12 Lysine (%) 1,36 1,99 2,58 3,36 Methionine+cistina (%) 0,72 1,06 1,41 1,75 Vitamin C (%) 0,04 0,04 0,04 0,04 Starch6 36.23 25.01 13.44 1.94 1 Canola oil 2 Betaine 3 Suprevit Peixes (Supremais), 1,000g: Vit. A 1200,000 IU; Vit. D3 200,000 IU; Vit. E 12,000 IU; Vit. K3 2,400 mg; Vit. B1 4,800 mg; Vit. B2 4,800 mg; Vit. B6 4,000 mg; Vit. B12 4,800 mg; Folate 1,200 mg; Calcium pantotenate 12,000 mg; Vit. C 48,000 mg; Biotin 48 mg; Choline 65.000 mg; Niacin 24,000 mg; Iron 10,000 mg; Copper (Cu) 600 mg; Manganese 4,000 mg; Zinc 6,000 mg; Iodine 20 mg; Cobalt 2 mg; Selenium 20 mg. 4 Carboxymethylcellulose 5 BHT (3,5-di-ter-butyl-4-hydroxytoluene) 6Value established considering the cornstarch content as 61%.

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Results The water quality was preserved to restrict the biochemical results as a consequence of nutritional changes. Hydrolysis of protein, carbohydrate and lipid was detected at the long of the Brazilian catfish gut (Table 2). In the stomach, unspecific proteolytic activity is very high compared to the other sections. Trypsin and chymotrypsin activity were slightly higher in the stomach than intestine. Stomach proteases were not responsive to the feeding protein contents and the highest activities for unspecific proteases were detected into acid pH. As well as the dietary protein concentration were changed, the carbohydrate contents also varied. The distinct values of protein corresponded to different amounts of starch. The concentration of 13 and 25% (30 and 40% of crude protein respectively) resulted in the maximum amylase activity, which was differentially responsive to such content on diets. The higher activities were observed in the stomach. Lipase activity was also observed in stomach. Unspecific proteases of intestine were few expressive and were not detected in the medium section. Chymotrypsin and trypsin were detected in the whole intestine. Amylase activities of intestine were very low compared to stomach. Lipase was more active in the intestine and the highest activity was observed in the medium gut.

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Table 2: Mean ± s.e.m. enzyme activity in the gastrointestinal sections of the Brazilian catfish (Pseudoplatystoma coruscans).

Treatments

(%CP) S A.I. M.I. P.I.

Unspecific protease activity (U/mg protein) 20 52.2 ± 17.5 0.60 ± 0.017* - 17.5 ± 14.96 30 79.2 ± 15.5 1.03 ± 0.022* - 3.9 ± 3.87 40 52.6 ± 9.1 0.43 ± 0.022 - 7.9 ± 5.83 50 60.7 ± 17.2 0.46 ± 0.059 - 12.6 ± 9.24

Trypsin (U/mg protein) 20 3.6 ± 1.43 0.24 ± 0.11 0.75 ± 0.23 0.60 ± 0.50 30 3.6 ± 1.40 0.24 ± 0.04 0.56 ± 0.22 0.53 ± 0.45 40 2.7 ± 1.12 0.33 ± 0.18 0.61 ± 0.25 0.41 ± 0.24 50 4.2 ± 1.47 0.41 ± 0.13* 0.22 ± 0.08* 0.19 ± 0.09

Chymotrypsin activity (mU/mg protein) 20 1.2 ± 0.43 0.48 ± 0.31 0.83 ± 0.17 1.18 ± 0.41 30 1.4 ± 0.38 0.40 ± 0.09 1.34 ± 0.33* 0.97 ± 0.18 40 1.5 ± 0.39 0.58 ± 0.27 1.51 ±0.18* 1.21 ± 0.23 50 1.5 ± 0.45 0.62 ± 0.11 0.97 ± 0.16 0.96 ± 0.18

Lipase activity (U/mg protein) 20 3.8 ± 2.31 5.53 ± 1.04 12.52 ± 1.42 4.96 ± 2.18 30 1.9 ± 0.77 6.68 ± 1.36 15.05 ± 5.31 8.04 ± 1.24 40 3.1 ± 0.61 5.89 ± 2.34 28.14 ± 20.66 9.11 ± 7.08 50 3.9 ± 1.19 14.49 ± 4.80 26.04 ± 13.28 5.78 ± 1.37

Amylase activity (U/mg protein) 20 0.01 ± 0.010 0.023 ± 0.008 0.013 ± 0.005 0.012 ± 0.008 30 0.18 ± 0.016* 0.053 ± 0.010 0.022 ± 0.014 0.029 ± 0.019 40 0.14 ± 0.020* 0.032 ± 0.007 0.021 ± 0.009 0.011 ± 0.004 50 0.03 ± 0.010 0.027 ± 0.008 0.020 ± 0.016 0.003 ± 0.002

CP: crude protein percent; S: stomach; A.I.: anterior intestine, M.I.: medium intestine; P.I.: posterior intestine. * Statistically different (P < 0.05).

38

Discussion The digestive pattern of the Brazilian catfish P. coruscans is quite particular compared to other species. The highest proteolytic activity occurring in acid pH of the stomach highlights the role of this structure in the digestive process of the species. The unresponsive peptic activity to the dietary protein in P. coruscans was similar to Colossoma macropomum (Kohla et al. 1992), Tilapia mossambica (L.) (Nagase, 1964), and Brycon cf. melanopterus (Reimer, 1982). High proteolytic acid activities are largely reported among fish. Hidalgo et al. (1999), studying digestive enzymes of fish with different nutritional habits, reported the highest proteolytic activity in the stomach of eel at pH 1.5 and nearly nil at neutral and alkaline one. Considering the unspecific proteases, this pattern is quite similar to the Brazilian catfish. Unexpectedly, trypsin and chymotrypsin activity were present in the stomach of P. coruscans. Alkaline proteases were previously reported in the gastric juice of other species (Kuz’mina, 1991). The unresponsiveness of proteases to dietary protein emphasizes the conclusion upon the best use of protein by the species is independent of diet. Moreover, their feeding habits tell us about the time of residence of the prey should be longer in the stomach. In spite of the proteolytic activity be present at the long of the intestine it was very low compared to the stomach. Considering the short size of this structure and the enzyme picture, we suppose the main role of the intestine in the Brazilian catfish is the nutrient uptake. Digestion of carbohydrates is usually disregarded in carnivorous fish. Amylase is not considered fundamental in fish digestive process. Few non-proteolytic enzymes, as amylase, are reported in the gastric juice of Clupea harengus and Dorosoma cepidianun (Hoar et al., 1979). However, Sabapathy and Teo (1993) studding digestive enzymes in the gut of the omnivorous fish Siganus canaliculatus and the carnivorous Lates calcarifer, reported lower amylase in the carnivorous one, in spite of both were able to digest carbohydrate and proteins. Seixas Filho et al. (1999) reported amylase in the digestive juice of tropical freshwater fishes, as the omnivorous “piracanjuba” Brycon orbignyanus and “piau” Leporinus friderici, and the carnivorous Brazilian catfish Pseudoplatystoma coruscans. Curiously, the enzyme activity of the Brazilian catfish was higher than “piracanjuba”. Raising levels to a maximum value around 13-25% of starch increased the amylase activity. That is a considerable amount of starch in carnivorous fish diets and can be understood considering the role of carbohydrates in the energy supply of the species. Cahu and Zambonino Infante, (1994) studying early weaning of sea bass larvae verified, in contrast to trypsin, amylase specific activity was stimulated by the dietary change. This

39

enhancement was induced by the starch content of diet. Kawai and Ikeda (1973) in De Silva and Anderson (1995) and Plantikow (1981 in Kohla et al., 1992) reported amylase increase in rainbow trout fed on high protein levels and constant carbohydrate. Dabrowski et al. (1992) reported ten-times higher amylase in the charr Salvelinus alpinus, as the feeding was high protein content (65-70%). Conversely, Das and Tripathi (1991) observed higher amylase in Ctenopharyngodon idella fed on low protein levels. The Brazilian catfish amylase is responsive to starch instead the glycogen, as proposed by Sabapathy and Teo (1993) for many carnivorous species. Other factors than starch can influence the amylase activity in fishes as the degree of gut stuffing (Bitterlich, 1985; Kohla et al., 1992), the nutritional condition (Vonk and Western, 1984; Munilla-Morán and Stark, 1990), the feeding habit (Fish, 1960; Sabapathy and Teo, 1993), the age (Munilla-Morán and Stark, 1990; Kuz’mina, 1996), the structural complexity of the carbohydrate (NRC, 1993), the temperature and season of the year (Kuz’mina et al., 1996). It is important to consider that higher contents of starch impair the carbohydrate hydrolysis. Perhaps, such particular characteristic is responsible for the relative intolerance of carnivorous fish by dietary carbohydrate. Amylase of the Brazilian catfish may also works hydrolyzing glycogen and other oligosaccharides in a lesser specificity. As a general rule both proteolytic and amylolytic activities are related to the feeding habit, and usually protease content of carnivorous fish is higher than omnivorous. However, the differences between proteases are lower than that for carbohydrases as amylase (Kuz’mina, 1991; De Silva and Anderson, 1995). The content of fats seems to be also important in the Brazilian catfish diets. Fishes are assumed to consume fat-rich food. Thus, the occurrence of lipase in the digestive tract of fishes is justified (Chakrabarti et al., 1995). The lipase activity is present in the whole sections of the gut of P. coruscans. It is interesting to see that the highest activity is observed in the middle gut. This is probably the digestive step when the largest fat content can be accessed. The Brazilian catfish P. coruscans is unresponsive to the dietary protein levels but responds to carbohydrate. Considering the large protein range assayed we can assume that the feeding optimization is completely dependent on the best ratio protein/energy/carbohydrate.

40

Acknowledgements This work was sponsored by FAPESP, CNPq. The authors are thankful to the colleagues of the Adaptive Biochemistry Lab for suggestions and support in the technical development of experiments. References Albro, P.W.; Hall, R.D.; Corbett, J.T. and Schroeder, J. 1985. Activation of

nonspecific lipase (EC 3.1.1.-) by bile salts. Biochemica et Biophysica Acta 835: 477-490.

Bernfeld, P. 1955. Amylases α e β: colorimetric assay method. In: Methods in

Enzymology. Ed. Colowich, S.P. & Kaplan, N.O., Vol. 1. New York: Academic Press Inc.

Bitterlich, G. 1985. Digestive enzyme pattern of two stomachless filter feeder,

silver carp, Hypophthalmichthys molitrix Val., and bighead carp, Aristichthys nobilis Rich. J. Fish. Biol. 27: 103-112.

Buddington, R.k.; Krogdahl, A and Bakke-Mckellep, A.M. 1997. The intestine

of carnivorous fish; structure and functions and the relations with diet. Acta Physiol. Scand. Suppl 638: 67-80.

Cahu, C.L. and Zambonino Infante, J.L. 1994. Early weaning of sea bass

(Dicentrarchus labrax) larvae with a compound diet: effect on digestive enzymes. Comp. Biochem. Physiol. 109A: 213-222.

Chakrabarti, I.; Gani, M.D.A.; Chaki, K.K.; Sur, R. and Misra, K.K. 1995.

Digestive enzimes in 11 freshwater teleost fish species in relation to food habit and nich segregation. Comp. Biochem. Physiol, 112: 167-177.

Chou, R.L.; Su, M.S. and Chen, H.Y. 2001. Optimal dietary protein and lipid

levels for juvenile cobia (Rachycentron canadum). Aquaculture 193: 81-89.

41

Dabrowski, K. and Glogowski, J. 1977. Studies on the role of exogenous proteolytic enzymes in digestion processes in fish. Hydrobiologia 54: 129-134.

Dabrowski, K.; Krumschnabel, G.; Paukku, M. and Labanowski, J. 1992.

Ciclic growth and activity of pancreatic enzimes of Arctic charr (Salvelinus alpinus L.) alevins. J. Fish. Biol. 40: 511-521.

Das, K.M. and Tripathi, S.D. 1991. Studies on the digestive enzymes of grass

carp, Ctenopharyngodon idella (Val.). Aquaculture 92: 21-32. De Silva, S.S. and Anderson, T.A. 1995. Fish nutrition in Aquaculture.

Chapman & Hall. Aquaculture 1 Series. 319p. Hidalgo, M.C.; Urea, E. and Sanz, A. 1999. Comparative study of digestive

enzymes in fish with diferent nutritional habits. Proteolytic and amylase activities. Aquaculture 170: 267-283.

Hoar, W.S.; Randall, D.J. and Brett, J.T. 1979. Fish Physiology, Vol 8:

Bioenergetics and growth. Academic Press. 786p. Hummel, B.C.W. 1959. A modified spectrophotometric determination of

chymotrypsin, trypsin, and thrombin. Canadian Journal of Biochemistry and Physiology. 37(12): 1393-1399.

Kohla, U.; Saint-Paul, U.; Friebe, J.; Wernicke, D.; Hilge, V.; Braum, E. and

Gropp, J. 1992. Growth, digestive enzyme activities and hepatic glycogen levels in juvenile Colossoma macropomum Cuvier from South America during feeding, starvation and refeeding. Aquaculture and Fisheries Management, 23: 189-208.

Kuz’mina, V.V. 1990. Characteristics of enzymes involved in membrane

digestion in elasmobranch fishes. I.D. Papanin Institute of Biology of Inland Waters, Academic of Science of the USSR, Borok, Yaroslavl’ Province. Translated from Zhurnal Évolyutsionnoi Biokhimii i Fiziologii, Vol. 26 (2): 161-166.

Kuz’mina, V.V. 1991. Evolutionary features of the digestive-transport function

in fish. Plenun Publishing Corporation. Translated from Zhurnal Évolyutsionnoi Biokhimii i Fiziologii, Vol. 27 (2): 167-175.

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Kuz’mina, V.V. 1996. Influence of age on digestive enzyme activity in some freshwater teleosts. Aquaculture 148: 25-37

Kuz’mina, V.V.; Golovanova, I.L. and Izvekova, G.I. 1996. Influence of

temperature and season on the some characteristics of intestinal mucosa carbohydrases in six freshwater fishes. Comp. Biochem. Physiol. 113B: 255-260.

Lovell, T. 1988. Nutrition and feeding of fish. New York: Chapman & Hall,

259p. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L. and Randall, R.J. 1951. Protein

measurement with the Folin phenol reagent. J. Biol. Chem. (193): 265-275.

Munilla-Morán, R. and Stark, J.R. 1990. Metabolism in marine flatfish. VI

Effect of nutritional state on digestion in turbot, Scophthalmus maximus (L.). Comp. Biochem. Physiol. 95B: 625-634.

NRC 1993. Nutrient requirements of fish. Washington: National Academy

Press. 114p. Park, J.T. and Johnson, M.J. 1949. A submicro determination of glucose. J.

Biol. Chem. 181: 140-151 Raul, F.; Noriega, R.; Doffoel, M.; Grenier,J.F. and Haffen, K. 1982.

Modifications of brush border enzyme activities during starvation in the jejunum and ileum of adult rats. Enzyme 28: 328-335.

Nagase, G. 1964. Contribuition to the physiology of digestion in Tilapia

mosambica: digestive enzymes and the effects of diets on theis activity. Zeitschrift für Vergleichende Physiologie 49: 270-284.

Reimer, G. 1982. The influence on diet on the digestive enzymes of the

Amazon fish Matrincha, Brycon cf melanopterus. J. Fish Biol. 21: 637-642.

Sabapathy, U. and Teo, L.H. 1993. A quantitative study of some digestive

enzymes in rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer. J. Fish Biol. 42: 595-602.

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Seixa Filho, J.T., Oliveira, M.G.A.; Donzele, J.L.; Gomide, A.T.M. and Menin,

E. 1999. Atividade de amilase em quimo de três espécies de peixes Teleostei de água doce. Ver. Bras. Zootc. 28(5): 907-913.

Suarez, M.D.; Hidalgo, M.C.; García Galego, M.; Sanz, A. and De la Higuera,

M 1995. Influence of the relative proportions of the energy yelding nutrients on the liver intermediary metabolism of the European ell. Comp. Biochem. Physiol. 111A(3): 421-428.

Tengjaroenkul, B.; Smith, B.J.; Caceci, T. and Smith, S. A. 2000. Distribuition

of intestinal enzyme activities along the intestinal tract of cultured Nile tilapia, Oreochromis niloticus L. Aquaculture 182: 317-327.

Ugolev, A.M. and Kuz’mina, V.V. 1994. Fish enterocyte hydrolases. Nutrition

adaptations. Comp. Biochem. Physiol. 107A: 187-193. Vonk, H.J. and Western, J.R.H. 1984. Comparative biochemistry and

physiology of enzymatic digestion. London: Academic Press. Walter, H.E. 1984. Proteinases: methods with hemoglobin, casein and azocoll

as substrates. In Bergmeyer, H.U. (Ed.). Methods of Enzymatic Analysis, vol. V. Verlag Chemie, Weinheim, pp. 270-277.

AQUACULTURE OF TAMBAQUI AND ITS VITAMIN C

REQUIREMENTS

Rodrigo Roubach

Aquaculture Department Instituto Nacional Pesquisa da Amazônia/INPA CP 478, CEP 69011-970, Manaus, AM, Brasil. Tel. +55-92-643-1894 / Fax. +55-92-642-1384

[email protected]

Alzira Miranda de Oliveira Laboratory for Ecophysiology and Molecular Evolution

Instituto Nacional Pesquisa da Amazônia/INPA [email protected]

Nívea Geovana Feitosa de Oliveira

INPA/BADPI [email protected]

Edsandra Campos Chagas

EMBRAPA, Amazônia Ocidental [email protected]

Adalberto Luís Val

Laboratory for Ecophysiology and Molecular Evolution Instituto Nacional Pesquisa da Amazônia/INPA

[email protected]


Vitamin requirements in fish vary quantitatively between different species due to several factors, e.g. sex, age, weight, size, nutrition, temperature, toxicity and metabolic functions (growth, stress responses and resistance to diseases) (Lovell, 1989). Ascorbic acid (vitamin C) is a water soluble vitamin, heat sensible, composed of six carbon atoms and structurally related with the glucose and other hexoses (Lovell, 1989). Recent experiments had shown that addition of vitamin C in tambaqui diet promotes greater resistance to stress, favoring growth and a better weight increase in these animals (Chagas, 1998).

45

Amongst the factors that directly affect fish survival, temperature is a major one, since it also affects growth and reproduction. Temperature is the biggest limiting factor in the biological processes, from simple chemical reactions to the ecological distribution of one given species. In the Amazon basin, air temperature determines, to a large extent, the average water temperature of the great rivers, lakes and flooded plains. During wider climatic variations in the region, average temperature of Amazonian waters oscillates between 25 and 34°C (Araújo-Lima & Goulding, 1998). Native fish of the Amazon, in general, need waters with temperature between 25 and 30°C for the accomplishment of its vital functions. Therefore suden changes in water temperature can cause stress, leaving individuals vulnerable to diseases. Temperature variations exert considerable effect on physiological processes and especially on fish metabolism. In general, a rise of 10°C in temperature provokes an increase of 2 to 3 times the normal oxygen consumption. Colossoma macropomum, known as tambaqui, belongs to the Serrassalmidae family and the Characiformes order. It is widely distributed in the northern part of South America. Currently, it is the main species raised at local commercial aquaculture in Central Amazon region. Due to its great regional economic importance and its great potential for aquaculture, tambaqui is a species that continues to draw attention from researchers and fish farmers (Val & Honczaryk, 1995; Araújo-Lima & Goulding, 1998). A 60-day feeding trial was conducted in individual tanks system to evaluate the effect of ascorbic acid (vitamin C) on growth and hematology of Colossoma macropomum under different temperatures and to evaluate if dietary vitamin C (0 and 500 mg/kg) positively influences in the physiology and growth response of the experimental animals to such conditions. Duplicate groups of 10 fish each with a initial weight of 8.7 g were reared at four temperatures (24, 26, 30 and 32ºC) and were fed practical (fish meal + soybean meal) type diets supplemented with two levels of vitamin C (0 and 500mgAA/kg). Fish exposed to 30ºC presented a better growth and weight gain (Table 1). Ascorbic acid level did not influence those parameters at this temperature. However, best feed conversion was observed on the group that received the vitamin C suplementation in the diet.

46

Table 1. Vitamin C supplementation effect on the growth parameters of tambaqui, Colossoma macropomum, under different temperatures (n=12; mean ± SD). (* and **) indicates significant (P<0.05) statistical differences between temperatures.

Temperature (°C) 24 26 30 32

Vitamin C (mg/kg)

Parameters

0 500 0 500 0 500 0 500

Initial weight (g)

21.9 ± 5.5

19.5 ± 5.5

19.7 ± 0.8

21.8 ± 1.1

20.0 ± 1.1

24.8 ± 1.3

24.9 ±3.3

27.9 ± 3.3

Initial length (cm)

10.7 ± 1.0

9.9 ± 0.9

8.5 ± 0.1

8.9 ± 0.1

8.7 ± 0.1

13.0 ± 0.4

11.4 ± 0.6

11.6 ± 0.8

Final weight (g)

24.5 ± 5.7

23.3 ± 5.4

41.8 ± 2.5

46.9 ± 2.4

55.8 ± 4.1

64.6 ± 4.7

35.1 ± 5.4

42.7 ± 7.9

Final length (cm)

11.6 ± 0.9

11.3 ± 0.8

10.6 ± 0.2

11.3 ± 0.3

13.5 ± 0.4

13.0 ± 0.4

13.3 ± 0.9

14.2 ± 0.8

Weight gain (%)

11.48* 19.5* 112.1* 115.8* 178.4* 159.9* 41.0* 53.0*

Size increase (%)

8.58** 14.1** 24.74** 27.0** 56.17** 47.8** 16.7** 22.4**

Feed conversion

7.2 3.4 1.8 1.7 1.6 1.4 2.6 1.8

Total biomass (g)

292.88 279.6 441.4 503.8 714.6 794.2 421.2 512.4

All treatments presented significant differences (p<0.05) in growth after 60 experimental days. Vitamin C did not influence fish blood parameters analysed (hematocrit, hemoglobin, circulanting red cells, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration) (data not shown) at the lowest tested temperature (24ºC) or highest (30 and 32ºC) compared to the ambient water temperature of 26 ºC. Vitamin C effect on the blood parameters observed in this work with Colossoma macropomum is similar to those previously described with other important aquaculture species of the Serrasalmidae family.

47

Fish exposed to 30ºC and fed diet supplemented with vitamin C showed lower glucose levels than fish fed diets without vitamin C. While fish exposed to 32ºC showed higher glucose levels for the group fed vitamin supplemented diets, which suggests that at this temperature the diet supplemented with 500mgAA/kg vitamin C was too high. Therefore, for the metabolic processes, vitamin C is important for these species regulatory processes affected by temperature variation.

Temperature (ºC)

Glu

cose

(mg/

dl)

40

60

80

100

120

140

160

1800mgAA/Kg500mgAA/Kg

24 26 30 32

ae

b

c

d

f

g

* *e

A

48

Adalberto Luís Val

Where is the legend of these figures?

References Araujo-Lima, C.A.R.M. & Goulding, M. 1997. So fruitful a fish. Ecology,

conservation, and aquaculture of the amazon's tambaqui. Biology and resources management in the tropics. Columbia University Press, New York, New York, USA.

Chagas, E.C. 1998. Efeito do ácido ascórbico (vitamina C) sobre a resistência ao

estresse hipóxico em Colossoma macropomum (Cuvier, 1818). Monografia. Fundação Universidade do Amazonas.

Lovell, R. T. (1989). The nutrients. In: Nutrition and feeding of fish. New York,

AVI – VNR. v. 2, p. 11- 71. Martins, M. L. (1995). Effect of ascorbic acid deficiency on the growth, gill

filament lesions and behavior of pacu fry (Piaractus mesopotamicus Holmberg, 1887). Braz. J. Med. Biol. Res., v. 28, p. 563 - 568.

Val, A.L.; Almeida-Val, V.M.F. & Affonso, E.G. (1990) Adaptative features of

Amazon fishes: Hemoglobin, hematology, intraerythrocytic phosphates and whole blood bohr effect of Pterygoplichthys multiradiatus (Siluriformes). Comp. Biochem. Physiol. 97B, 3;p.435-440.

Val, A. L. & Honczaryk, A. (1995). Criando peixes na Amazônia. Manaus,

INPA. 160p. (Eds.).

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50

EFFECTS OF PHOTOPERIOD MANIPULATION ON REPRODUCTIVE

CYCLICITY IN HADDOCK (MELANOGRAMMUS AEGLEFINUS)

D.J. Martin-Robichaud Fisheries and Oceans Canada

St. Andrews Biological Station 531 Brandy Cove Rd.

St. Andrews, New Brunswick, Canada, E5B 2L9

506-529-8854, Fax 506-529-5862 [email protected]

D.L. Berlinsky

Department of Zoology University of New Hampshire

Durham, NH USA 03824 603-862-0007, FAX 603-862-3784

[email protected]

Haddock (Melanogrammus aeglefinus) is an important, temperate, groundfish harvested commercially in the North Atlantic. As landings have declined over the last 40 years, haddock aquaculture is being developed to take advantage of high market prices and to diversify existing aquaculture operations. Haddock are group synchronous, serial spawners. Each female releases 8-10 batches of eggs spontaneously in communal spawning tanks over 4-6 weeks. For many temperate marine species, photoperiod, acting through the hypothalamic-hypophysial-gonadal axis, is the main environmental cue that influences endogenous, circannual, reproductive cycles. (Reviews: Bye, 1990; Bromage et al., 2001). For this reason, photomanipulation of reproductive cycles is routinely used to alter the spawning period of cultured fish for efficient hatchery production. The purpose of this study was to gain a basic understanding of haddock reproductive cycles and to determine the effects of photoperiod manipulation on gonadal development and circulating steroid hormone levels. Wild haddock broodstock (56-73 cm FL) were tagged and held in two 6-m tanks supplied with flow-through seawater at ambient temperatures. The photoperiod

51



of one tank was advanced by 2 months (advanced phase-shifted photoperiod; ADV) while the other was maintained on a simulated natural photoperiod (SNP). At 6-8 week intervals, from Nov 1996 to May 1998, plasma samples, gonadal biopsies and ovarian ultrasound measurements were collected from a subset of fish in each tank. Following histological preparation, follicle diameters were measured and developmental stages assessed. Levels of estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were determined by radioimmunoassay. The spawning period of haddock maintained on the ADV commenced 9 and 6 weeks ahead of the SNP broodstock in 1997 and 1998, respectively, and the duration of spawning was prolonged. Plasma E2 levels started to increase in September and August for the SNP and ADV groups, respectively, peaking at about 3 ng/ml during spawning. Similarly, follicle size and gonad indices increased in the fall, as E2 stimulated vitellogenesis. E2 levels decreased abruptly during the last half of the spawning season (Fig. 1). Male testosterone levels, like E2 in females, peaked at 1.0-1.5 ng/ml during the spawning season and dropped after spawning to low or non-detectable levels. Plasma levels of 11-ketotestosterone in males were low or non-detectable throughout the year. Haddock exhibit similar patterns of ovarian growth and seasonal steroid production as other temperate, batch-spawning teleosts, such as Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) (Hansen et al., 2001; Methven et al., 1992). Levels of estradiol increase around the autumn equinox when day length and temperature are declining. Estradiol promotes oocyte growth through vitellogenin incorporated into the oocytes. Peak estradiol levels correspond with spawning, and fluctuate as sequential oocyte clutches mature and ovulate (Methven et al., 1992). This study demonstrates that haddock respond readily to photomanipulation and maintain normal, phase-shifted, seasonal cycles. The extended spawning duration, characteristic of phase-shifted stocks, is likely due to individual responsiveness to environmental cues and persistent endogenous rhythms.

52

Figure 1. Seasonal profiles of mean plasma estradiol levels • (—) , follicle diameters • and ovarian indices (open squares) of haddock maintained under

53

54

SNP and 2 month advanced photoperiods. Shaded bars indicate spawning intervals. Acknowledgements Thanks are extended to Melissa Rommens for her dedicated technical support, Drs. Tillmann Benfey (University of New Brunswick) and Craig Sullivan (North Carolina State University) for supplying 11-KT antisera and radiolabelled ligand. Partial funding for this study was obtained from the Canada/New Brunswick Aquaculture Development Program for Non-traditional Species. References Bromage, N.R., M. Porter and C. Randall. 2001. The environmental regulation

of maturation in farmed finfish with special reference to the role of photoperiod and melatonin. Aquaculture 197: 63-98.

Bye, V.J. 1990. Temperate Marine Teleosts. In Reproductive Seasonality in

Teleosts: Environmental Influences (eds A. Munro, A. Scott and T. Lam) pp. 126-141. CRC Press, Boca Raton, FL.

Hansen, T., O. Karlsen, G.L. Taranger, G-I. Hemre, J.C. Holm and O.S. Kjesbu.

2001. Growth, gonadal development and spawning time of Atlantic cod (Gadus morhua) reared under different photoperiods. Aquaculture 203: 51-67.

Methven, D.A., L.W. Crim, B. Norberg, J.A. Brown, G.P. Goff and I. Huse.

1992. Seasonal reproduction and plasma levels of sex steroids and vitellogenin in Atlantic Halibut (Hippoglossus hippoglossus). Can. J. Fish. Aquat. Sci. 49: 754-759.

55

THE INFLUENCE OF ALTERNATE SUPPLEMENTAL DIETARY

LIPIDS ON THE GROWTH AND HEALTH OF ATLANTIC SLAMON

IN SEAWATER

Shannon BalfryFaculty of Agricultural Sciences, University of British Columbia

2357 Main Mall, Vancouver, B.C., Canada V6T 1Z4Telephone: 604-666-0034; Fax: 604-666-3497

Email: [email protected]

Dave Higgs, Nancy RichardsonFisheries and Oceans Canada, West Vancouver Laboratory

4160 Marine Drive, West Vancouver, B.C., Canada V7V 1N6

Santosh LallInstitute for Marine Biosciences, National Research Council of Canada

1411 Oxford Street, Halifax, N.S., Canada B3H 3Z1


Introduction

Marine fish oils (MFOs) from such sources as anchovy and herring havetraditionally been used in diets for cultured fish to provide fish with energy,essential fatty acids, and other nutritional factors. However, these oils are ingreat demand world-wide and as a result of their limited supply, they arebecoming increasingly more costly. To maintain and enhance the economicviability of aquaculture it has become necessary to find suitable, less expensivesources of lipid. The goal of this research project was to identify economical,alternate plant and/or animal lipid sources that would satisfy the nutritionalrequirements of Atlantic salmon in sea water for growth and health, whilesimultaneously providing a quality marketable product. When MFOs are replaced with other lipids it is essential to provide the fish withthe correct amounts and balance of fatty acids to ensure high growth ratewithout compromising fish health. Fish cannot synthesize n-6 and n-3

56

polyunsaturated fatty acids (PUFAs), and therefore to maintain optimal growthand health, these fatty acids must be included in the diet in the correct amounts,forms, and ratios. In general, the fatty acid composition of the fish tissuesmirrors that of the diet. The fatty acid composition of cell membranes isparticularly important because these fatty acids serve as progenitors for a groupof immunomodulatory compounds call eicosanoids. It has been reported thatdiets high in n-6 PUFAs result in relatively higher levels of the pro-inflammatory eicosanoids (2-series prostaglandins and 4-series leukotrienes andlipoxins) derived from the highly unsaturated fatty acid (HUFA), arachidonicacid (Balfry and Higgs 2001). Alternatively, diets high in n-3 PUFAs producerelatively higher levels of the anti-inflammatory eicosanoids (3-seriesprostaglandins and 5-series leukotrienes and lipoxins) derived mainly from theHUFA, eicosapentaenoic acid.

This research project was designed to examine the effects of partially replacinga MFO (i.e., anchovy oil) with vegetable oils (sunflower oil, flaxseed oil) and/oranimal lipids (poultry fat). Various parameters of growth performance and dietutilization, as well as hematology and immune response were measured inreplicated groups of Atlantic salmon reared on one of eight differentexperimental diets. The fatty acid composition of the diets and fish muscle wereexamined, along with the eicosanoids produced from stimulated leucocytes. Methodologies

The feeding trial was performed at the West Vancouver Laboratory, usingAtlantic salmon (Salmo salar, mean starting weight 102.3 g). Fish were rearedin outdoor 4000-L tanks supplied with a continuous flow of aerated ambientseawater. Eight diets were prepared whereby supplemental anchovy oil in thebasal diet was partially replaced with flaxseed oil, sunflower oil, or poultry fat,and diets that contained blends of each vegetable oil with poultry fat (see Table1). The eight dietary treatment groups were assigned to triplicate groups of 40fish using a randomized complete block design. All fish groups were fed theirprescribed diet by hand twice daily to satiation for 20 weeks. At the end of thisperiod various indices of growth performance, diet absorption and utilizationwere determined viz., specific growth rate, feed efficiency, protein efficiencyratio, gross energy utilization, whole body and muscle proximate composition,and diet digestibility. Health assessments were performed by measuring severalhematological variables including: erythrocyte numbers, hematocrit,hemoglobin, mean erythrocyte volume (MEV), mean erythrocyte hemoglobin

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content (MEHC), differential leucocyte numbers, mean erythrocyte hemoglobin,and erythrocyte fragility. The immune responses of the different dietarytreatment groups were compared using various tests that examined the relativeactivity of the non-specific cellular and humoral defense systems (serumlysozyme activity, serum bactericidal activity, respiratory burst activity ofperipheral blood leucocytes and head kidney leucocytes). The fatty acidcompositions of the diets and muscle samples from the fish were analyzed usinggas chromatography. Eicosanoid production of calcium ionophore-stimulatedhead kidney leucocytes was examined by liquid chromatography andelectrospray mass spectrophotometry. The results were analyzed by randomizedblock ANOVA to test for significant diet-related differences.

Table 1. Percent composition of the supplemental lipid sources used in thepreparation of the experimental diets. A total of 150g of supplemental lipid wasadded to each kg of diet on an air-dry basis. All diets contained equivalentconcentrations of protein (501.5 g/kg) and lipid (241.4 g/kg) on a dry weightbasis. 1/

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8

AnchovyOil

100 25 25 25 25 25 25 25

PoultryFat

0 75 0 0 50 50 25 25

FlaxOil

0 0 75 0 25 0 50 0

Sunflower Oil

0 0 0 75 0 25 0 50

1/ The composition of the basal diet was as follows (g/kg air-dry basis): Peruvianfish meal 693.3, wheat flour 124.5, spray-dried blood meal 25.5, vitamin premix4.7, mineral premix 1.7, supplemental lipid 150.4.

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Results and Conclusions

Following the 20-week feeding trial, there appeared to be no significant adverseeffects of the different lipid sources on any aspect of growth performance.Comparisons made between the groups fed the different diets revealed that thespecific growth rate and final weight of fish fed the high flaxseed diet (Diet 3),were significantly higher than those noted for the groups fed the control diet(Diet 1).

The use of alternate lipid sources did not compromise any of the aforementionedimmune response variables, as no significant diet-related differences weredetected. Mortality during the 20 week feeding trial was low (<1%) with no dietdifferences. The various hematological measurements were all within normalranges for cultured Atlantic salmon (Sandnes et al., 1988), indicating that thenutritional requirements were met in all diet groups. Hence, the health of allgroups of fish was not compromised by any of the diet treatments. Somevariations in hematocrit, MEV, MEHC, and erythrocyte fragility were detectedin fish fed Diets 4 and 8 (high and mid sunflower oil diets), relative to those fedthe other diets

In summary, our results to date, reveal that significant cost saving can bepotentially realized by using the aforementioned alternate lipids as sources ofsupplemental lipids for Atlantic salmon. This is especially true in the case ofpoultry fat (presently about half the cost of fish oil) and the blends of poultry fatwith the vegetable oils.

References

Balfry, S.K. and D.A. Higgs. 2001. Influence of dietary lipid composition on theimmune system and disease resistance of finfish. In: Nutrition and FishHealth. Lim, C. and C.D.Webster (Eds.) Haworth Press In., New York.Pp. 213-234.

Sandnes, K., Lie, O. and R. Waagbo. 1988. Normal ranges of some bloodchemistry parameters in adult farmed Atlantic salmon, Salmo salar. J.Fish Biol. 32: 129-136.

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Acknowledgements

This project was funded through AquaNet, National Center of Excellence(awarded to Drs. Higgs and Lall). The authors are grateful for the technicalassistance of Mahmoud Rowshandeli, Janice Oakes, Jill Sutton, IndraWeerasinghe, and Joyce Milley

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EFFECTS OF ß-AGONIST FEEDING ON RAINBOW TROUT

(ONCORHYNCHUS MYKISS) MUSCLE GROWTH

AND MYOSATELLITE CELLS

Haude Levesque Department of Biology, University of Ottawa

Ottawa, ON K1N 6N5 Canada Tel: (613) 562 5800 x 6009 Fax: (613) 562 5486


Thomas Moon Department of Biology, University of Ottawa

Ottawa, ON K1N 6N5 Canada Tel: (613) 562 5800 x 6002



The adrenergic system, characterized by the catecholamine hormones adrenaline and noradrenaline, integrates and modulates many aspects of vertebrate, including fish, metabolism. β2-Adrenergic agonists (β2-AA; synthetic catecholamine analogues signaling through the β2-adrenergic receptor) increase muscle growth in mammals (Maltin et al., 1992) and act as repartitioning agents by re-directing nutrients from adipose tissue to muscle (Roberts and McGeachie, 1992). β2-Agonists also increase weight gain, feed utilization, lean body mass and protein accretion in mammals. In this study, we examined the effects of ractopamine (RACT) and clenbuterol (CLEN), two β2-adrenergic agonists of the phenethanolamine group, on both rainbow trout muscle growth and myosatellite cell proliferation and differentiation. Currently, ractopamine is commercially used in swine farming, and has previously been tested in rainbow trout (Vandenberg and Moccia, 1998) and channel catfish (Ictalurus punctatus) (Mustin and Lovell, 1993) where it has demonstrated lower effects than reported in other vertebrates. Clenbuterol increases myosatellite proliferation in mice (Roberts and McGeachie, 1992) and muscle fiber hypertrophy in Lister rats (Maltin et al., 1992), but its effects on fish myosatellite cells and muscle growth remain unknown.

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Rainbow trout (80 fish per 110-115 L tank) were fed the following three experimental diets: commercial diet sprayed with 10 ppm ractopamine, 10 ppm clenbuterol or carrier (sham) for 10 to 12 weeks. Fish were ~ 6 g at the beginning of the experiment and ~ 30g at 12 weeks. After exposure, 12 fish per group were sacrificed and blood, liver, white and red muscle, gut and kidney were sampled for metabolic enzyme and metabolite assays; liver and visceral lipid were removed and weighed to calculate hepatosomatic index (HSI) and lipid somatic index (LSI). Ten additional fish per group were used for BrdU (bromo-deoxy-uridine) injection to characterize in situ myosatellite cell proliferation. The remaining fish were used for myosatellite cell extraction according to Fauconneau and Paboeuf (2000) to characterize in vitro proliferation and differentiation. Data were analyzed with ANOVA followed by Tukey-Kramer test and T-test with the JUMP program. Clenbuterol- and RACT-treated fish had significantly higher condition factor (CF) compared with sham fish (Table 1) and RACT-treated fish had a lower visceral lipid content (Table 1). No significant differences were found in HSI Table 1: Condition factor (mean + SE) at the beginning of the experiment (CFi) and at the end of the exposure (CFe), hepatosomatic index (HSIe) (mean + SE) and lipid somatic index (LSIe) (mean + SE) at the end of the exposure; n represents the number of fish. CF, HIS and LSI have been calculated respectively by the following equations [(weight • length-3) x 100, (liver weight•total weight-1) x 100 and (weight of abdominal cavity lipids• total weight-1] x 100. **, indicates significant differences compared to the sham (α = 0.05)

Treatment n CFi CFe HSIe LSIe SHAM 12 1.55+0.05 1.11+0.02 1.15+0.19 1.84+0.09 RACT 12 1.23+0.05 1.35+0.04 ** 1.39+0.09 1.42+0.10 ** CLEN 12 1.34+0.16 1.42+0.06 ** 1.12+0.13 1.65+0.11

between treatments. Preliminary results indicate that the activities of alanine aminotransferase (ALT), pyruvate kinase (PK) and isocitrate dehydrogenase (IDH) in the liver were higher in fish treated with 10 ppm RACT compared with sham fish (Table 2). There were no differences in phosphoenolpyruvate

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Table 2: Specific activity (µmol•min-1•mg-1 protein) of pyruvate kinase (PK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME) and isocitrate dehydrogenase (IDH) in liver (mean + SE, n = 8). **, indicates significant differences compared to the sham (α = 0.05); *, indicates significant differences compared to the sham (α = 0.1)

Treatment SHAM CLEN RACT PK 1.60+0.21 1.69+0.22 2.19+0.19 ALT 1.71+0.17 1.98+0.22 2.65+0.18 **AST 3.39+0.29 2.67+0.13 ** 3.54+0.19 G6PDH 2.02+0.15 1.41+0.10 ** 1.93+0.14 ME 0.46+0.09 0.39+0.02 ** 0.47+0.05 IDH 1.05+0.06 0.87+0.07 * 1.25+0.07 *

carboxykinase (PEPCK), aspartate aminotransferase (AST), glutamate dehydrogenase (GDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose-6-phosphate (G6PDH), or malic enzyme (ME) activities in the liver between sham and RACT-treated fish. The activities of AST, G6PDH, ME and IDH in the liver were lower in fish treated with CLEN compared with sham fish (Table 2). There were no differences between sham and CLEN-treated fish for LDH, MDH, GDH, ALT, PEPCK and PK in the liver. Extraction yield of myosatellite cells were 2.9 x 104 for the sham group, and 1.2 x 105 and 6.4 x 104 cells•g-1 muscle for the RACT- and CLEN-treated fish, respectively. The higher CF and myosatellite cell extraction yield suggest that both β2-AAs administrated to rainbow trout for 10 to 12 weeks significantly increase muscle growth. The differences seen in the action of RACT and CLEN on liver metabolic enzymes suggest a different mode of action of those two β2-AAs. Ractopamine treatment increased AST, an enzyme implicated in protein metabolism and PK, a glycolytic enzyme, whereas CLEN treatment decreased G6PDH, ME and IDH, three enzymes implicated in lipid synthesis.

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References Fauconneau, B., and Paboeuf, G. 2000. Effects of fasting and refeeding on in

vitro muscle cell proliferation in rainbow trout (Oncorhynchus mykiss). Cell Tiss. Res. 301: 459-463.

Maltin,C.A., Delday,M.I. and Reeds, P.J. 1992. Satellite cells in innervated and

denervated muscles treated with clenbuterol. Mus. Nerve 15: 919-925. Mustin, W.T. and Lovell, R.T. 1993. Feeding the repartitioning agent,

ractopamine, to channel catfish (Ictalurus punctatus) increases weight gain and reduces fat deposition. Aquaculture 109: 145-152.

Roberts, P. and McGeachie, J.K. 1992. The effects of clenbuterol on satellite

cell activation and the regeneration of skeletal muscle: an autoradiographic and morphometric study of whole muscle transplants in mice. J. Anat. 180: 57-65.

Vandenberg, G.W., Leatherland, J.F. and Moccia, R.D. 1998. The effects of the

beta-antagonist ractopamine on growth hormone and intermediary metabolite concentrations in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquacul. Res. 29: 79-87.

Acknowledgements We would like to thank Dr. B. Fauconneau and G. Paboeuf for help in the myosatellite cell extraction method. These studies were supported by grants to the authors from AquaNet Canada (AP13), NSERC and OGSST.

DIET INFLUENCES PROTEOLITIC ENZYME PROFILE

OF THE SOUTH AMERICAN CATFISH RHAMDIA QUELEN

Lícia Maria Lundstedt1, José Fernando Bibiano Melo2 , Cristiano Santos Neto3 and Gilberto Moraes4

Department of Genetic and Evolution, Federal University of São Carlos Rod. Washington Luís, km 235; CEP: 13565-905; CP 676. São Paulo. Brazil

Phone/Fax: 54 (16) 260-8376 / 54 (16) 260-8377 e-mails: [email protected], [email protected] 2,

[email protected], [email protected] 4 Keywords: Fish digestion, Dietary protein, Digestive tract, Protease, Trypsin, Chymotrypsin, Rhamdia quelen. Introduction The enzyme gut profile of fishes can be adapted to the type of feeding and ratio of nutrients. To improve fish digestions and nutrient uptake the range of responses included as adaptive, must be established (Senger et al., 1989) Glass et al. (1989) assume the knowledge upon the exact amount and specificity of digestive enzymes as a tool to predict precisely the digestive process in fish. Among the several fishes many of them are particularly important for the zoo-technical characteristics. We are studying the South American catfish “jundiá” Rhamdia quelen, which display several farming qualities. Eleven species are presently included in the Rhamdia genus, a neotropical Siluriform-Pimelodidae reported from Mexico to Argentina (Silfvergrip, 1996). This species easily accept artificial feeding since the hatching, presenting large survival rates and fast growth and development. (Piaia and Radünz Neto, 1997; Cardoso et al., 1999; Melo et al., 2002). Nowadays, no information concerning digestion of the species from the genus Rhamdia is disposable. Considering that “jundia” is omnivorous and presents good use of numerous kinds of diets, we supposed that the digestive tract is responsive to the dietary levels of protein. The digestive acid and alkaline unspecific proteases, plus trypsin and chymotrypsin were assayed in “jundia” fed on different dietary protein.

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Materials and methods Forty juveniles of “jundia” R. quelen, from the same strain weighing 40.98 ± 13.32 g and ranging 16.92 ± 1.44 cm were brought to the 2,000L tanks supplied with filtered, aerated and temperature-controlled water in a closed system. The fish were fed on isocaloric diets with 3,500 Kcal / kg of digestible energy for 25 days. The net protein content was 20, 27, 34 and 41 per cent (Table I). The pellet diets were offered twice a day “ad libitum”. At the end of the feeding period the fish were killed and the digestive tract were excised and separated into stomach, anterior, medium and posterior intestine. The gut sections were frozen at –20o C for further analysis. The experimental design considered randomly incomplete blocks with 10 repetitions per treatment. The data analysis was done by MANOVA using loge transformed values to preserve normality and the means were compared by the test of DUNCAN. Acid and alkaline proteases were previously assayed in the different sections of the gut to optimize the pH reaction, temperature, incubation time, aliquot of enzyme and substrate concentration. Enzyme reaction mixture for acid proteases was 1% casein in 0.1M glycine/ HCl pH 2.0 with proper aliquot of enzyme homogenate. After incubation for 30 min at 35 oC the reaction was stopped by 15% TCA solution. The reaction mixture was centrifuged at 3,000 g for 3 min and the optical density of the supernatant was determined at 280nm (Sarath, 1989). Enzyme reaction mixture for alkaline proteases was 1% casein in 0.1M Tris / HCl pH 8.5 with proper aliquot of enzyme homogenate. After incubation for 30 min at 35o C the reaction was stopped by 15% TCA solution. The samples were kept at 35 oC by one hour and centrifuged for 10 min at 1,800 g (Walter, 1984). Tyrosine was used as standard for acid and alkaline proteases. Protease activities are expressed in µmol of hydrolyzed substrate/min/mg of protein. Both trypsin and chymotrypsin proteolytic activities were determined as Hummel (1959). The reaction mixture for trypsin was 1,04mM TAME (p-toluenesulfonyl-L-arginine ethyl ester), 0.01M CaCl2 in 0.2M Tris pH 8.1. The product formation was continuously determined at 247nm. One unit of enzyme was established as the hydrolysis of 1 µmol of TAME/min. The reaction mixture for chymotrypsin was 0.001M BTEE (N-benzoil-L-tyrosine ethyl ester), CaCl2 0,10M in 0,10M TRIS- HCl pH 7.8. The product formation was followed at 256 nm. Trypsin activity was expressed in nmol of arginine min/mg/protein and chymotrypsin in nmol of tyrosine min/mg/ of protein.

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Protein content of the enzyme source was determined by the method of Lowry et al. (1951), using albumin as standard.

Table I: General composition of the experimental diets (g/100g dry diet).

Ingredient (%) Experimental Diets (net protein %)

20 27 34 41 Fish meal 14.2 24.2 19.2 35.2 Soybean 8 11 11 24 Yeast 8 8 20 15 Wheat 14 14 14 10 Corn 38 30 26 8 Canola oil 17 12 9 7 Vitamin Premix 0.2 0.2 0.2 0.2 Mineral Premix 0.1 0.1 0.1 0.1 Vitamin C 0.05 0.05 0.05 0.05 Salt 0.5 0.5 0.5 0.5

Total 100 100 100 100 Dry Matter (%) 90.06 8.81 8.61 9.74 Gross energy (kcal /kg) 4419 4610 4504 4438 Crude Protein (%) 19.37 26.33 33.20 40.14 Crude Fiber (%) 3.24 3.24 3.14 3.92 Crude Fat (%) 18.57 14.76 11.98 9.70

Results and discussion

The increase of dietary protein levels induced the enhancement of acid protease activity in the stomach of “jundia” (table II). Different response is observed in Colossoma macropomum, as the feeding animal or vegetal protein increase results in steady acid protease activity (Kohla et al., 1992).

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The anterior, medium and posterior sections of intestine were biochemically differentiated concerning alkaline proteases. Differentiated responses to protein levels were also observed in every sections of intestine. Alkaline protease activities in the anterior and medium sections of the intestine were maxim for 27%. Decrease of enzyme activity for higher dietary protein suggests regulation of enzyme synthesis by the protein content or derivatives present in the diet. Another possibility is a limited nutrient uptake capacity of intestinal mucous membrane. Different responses are reported for fish alkaline proteases versus dietary protein content. While this enzyme activity decreases in Rutilus rutilus (Hofer and Uddin, 1985) a converse response is reported in rainbow trout (Kawai and Ikeda, 1973). Comparison of the bowel sections for a protein level, the alkaline protease activity was higher in the anterior one. In the posterior section the enzyme activity remained increasing until 34% of protein. This should be attributed to a lesser amino acid uptake in that intestine section. The amino acid absorption in the proximal intestine of Ctenopharygodon idella is about 40-50% but the posterior section absorbs 25% (Stroband and Van Der Veen, 1981). The presence of trypsin and chymotrypsin in the stomach of “jundia” testifies the enzyme plasticity of this structure. Those enzymes were responsive to dietary protein and were reduced for protein contents greater than 27%. The responsiveness of the anterior and medium intestine was distinct. Activities of chymotrypsin and trypsin increased in the anterior until 27% of dietary protein, keeping constant toward 41%. The highest activities were also observed in the anterior section of the intestine. There is a clear interaction (p < 0.01) among the changes of the enzyme gut profile of jundia and the dietary protein levels. This fact emphasizes the adaptive responses of the gut in the species. Alkaline digestive enzymes, in spite of decreasing towards the end of the bowels, are distributed along all the gut of “jundia”. Digestive enzymes of eleven freshwater teleosts species, with different feeding habits, are distributed along all the gut Chakrabarti et al. (1995). However, differently of “jundia” the guts were not differentiated and no digestive strategies were observed, as occur in vertebrates. Alkaline proteases (trypsin and chymotrypsin) were reported at the long of the digestive tract of Siganus canalicutus and Lates calcarifer, including esophagus, stomach, pyloric cecum and liver (Sabapathy and Teo, 1993). The South American catfish “jundia” was responsive to feeding protein content with evident adaptive changes of digestive enzyme profile. Stomach plays a

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special role in digestion of the species presenting proportional responsiveness to the protein content and the anterior intestine seems to conclude the hydrolysis of protein. The long of medium and terminal intestine are probably important in absorptive processes.

Table II: Activities of digestive enzymes of Rhamdia quelen fed on distinct protein levels.

Dietary protein level (%) 20 27 34 41 Acid protease Stomach 0.96d 1.29c 1.43b 1.62a Alkaline Protease Anterior 1.17b 1.40a 1.36a 1.26a Medium 0.70b 1.11a 1.09a 0.97a Posterior 0.61c 0.94b 0.98a 0.87b Trypsin Stomach 0.18a 0.02a -0.32b -0.42b Anterior 0.31b 0.50a 0.51a 0.32b Medium -0.26c -0.01b 0.16a 0.05b Posterior 0.10b 0.20a 0.10b 0.08b Chymotrypsin Stomach 3.00a 2.91a 2.57b 2.53b Anterior 2.73b 3.06a 3.11a 3.00a Medium 1.49b 2.46a 2.71a 2.68a Posterior 1.58c 2.23a 2.30a 2.32a

Enzyme activity values are expressed as loge. Different letters in the line means statistically different (P < 0,01). Acknowledgements The authors thank the colleagues of the Biochemistry Adaptive Lab for suggestions and experimental support. This work was sponsored by FAPESP and CNPq. References

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Cardoso, A.P.; Medeiros, T.S. and Radünz Neto, J. 1999. Criação de larvas de jundiá Rhamdia quelen alimentadas com rações contendo fígado bovino ou de aves. Resumo: Anais da XXXVI Reunião Anual da Soc. Bras. de Zool. Porto Alegre-RS. pp.316.

Chakrabarti, I.; Gani, Md. A.; Chaki, K.K.; Sur, R. and Mirsa, K.K. 1995.

Digestive enzymes in 11 freshwater teleost fish especies in relation to food habit and niche segregation. Comp. Biochem. Physiol. 112 A: 167-177.

Glass, H.J.; Macdonald, N.L.; Moran, R.M. et al. 1989. Digestion of protein in

different marine species. Comp. Biochem. Physiol. 91 (3): 607-611. Hummel, B.C.W. 1959. A modified spectrophotometric determination of

chymottypsin, trypsin, and thrombin. Canadian journal of biochemestry and physiology. 37 (12): 1393-1399.

Hofer, R. and Nasir Uddin, A. 1985. Digestive processos during the

development of the roach, Rutilus rutilus. J. Fish Biol. 26: 683-689. Kawai, S. and Ikeda, S. 1973. Studies on digestive enzymes of fishes. III

Development of the digestive enzymes of rainbow trout after hactching and effect of dietary chang on the activities of digestive enzymes in the juvenile stage. Bull. Jap. Soc. Scient. Fish. 39: 817-823.

Kohla, U.; Saint-Paul, U.; Friebe, J.; Hilge, V. and Gropp, J. 1992. Growth,

digestive enzyme activities and hepatic glycogen levels in juvenile Colossoma macropomum Cuvier from south america during feeding, starvation and refeeding. Aquaculture and Fisheries Management. 23: 189-208.

Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. 1951. Protein

measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Melo, J.F.B.; Radünz Neto, J.; Da Silva, J.H.S. and Trombetta, C.G. 2002.

Desenvovolvimento e composição corporal de alevinos de jundiá Rhamdia quelen alimentados com dietas contendo diferentes fontes de lipídios. Ciência Rural. 32 (2): 323-327.

Piaia, R.; Uliana, O.; Felipetto, J. and Radünz Neto, J. 1997. Alimentação de

larvas de jundiá Rhamdia quelen, com dietas artificiais. Rev. Ciência e

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Natura. Santa Maria, RS. 19: 119-131. Sarath, G.; De La Motte, R.S. and Wagner, F.W. 1989. Protease assay methods.

IN: proteolitic enzymes. A Pratical approach (edited by Beynon, R.J. and Bond, J.S.). 22-55.

Sabapathy, U. and Teo, L.H. 1993. A quantitative study of some digestive

enzymes in rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer. J. Fish Biol. 42: 595-602.

Segner, H.; Rosch, R.; Schmidt, H. and Von Poeppinghausen, K.J. 1989.

Digestive enzymes in larval Coregonus lavaretus L. J. Fish Biol. 35: 249-263.

Silfvergrip, A.M.C. 1996 A sitematic revision of the neotropical catfish genus

Rhamdia (Teleostei, Pimelodidae). Department of Zoology, Stockholm University and Department of Vertebrat Zoology, Swedish Museum History. Stockholm, 156 p.

Stroband, H.W.J. and Van Der Veen, F.H. 1981. Localization of protein

absorption during transport of food in the intestine of the grasscarp, Ctenopharyngodon idella (Val.). The Journal of Experimental Zoology. 218, p. 149-156.

Walter, H.E. 1984. Proteinases: methods with hemoglobin, casein and azocoll as

substrates. In: Bergmeyer, H.U. (Ed). Methods of enzimatic analysis, Vol. V. Verlag Chemie, Weinheim, 270-277.

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72

CLONING AND CHARACTERIZATION OF GLUCOSE

TRANSPORTERS FROM COD (Gadhus morhua) HEART

Jennifer R. Long, Memorial University of Newfoundland,

Ocean Sciences Centre, St. John’s, NF, A1C 5S7,

(709) 737-2519, Fax: (709) 737-3220, [email protected]

William R. Driedzic, Memorial University of Newfoundland,

Ocean Sciences Centre, St. John’s, NF, A1C 5S7,

(709) 737-2519, Fax: (709) 737-3220, [email protected]

EXTENDED ABSTRACT ONLY- DO NOT CITE We are currently studying the effects of temperature and hypoxia on growth rates and survival of cod (Gadhus morhua). Of particular interest is cardiac performance under these conditions. In cod heart, glucose is an essential metabolic fuel for the production of ATP and the maintenance of contractile function under both aerobic and particularly anaerobic conditions. Glucose transport across the membranes of animal cells is mediated by facilitative glucose transporters (GLUTs). Thirteen of these transporters have been identified in humans and are expressed in a tissue specific manner. Of particular interest to the heart are GLUT-1 and GLUT-4. GLUT-1 is the basal glucose transporter and is induced in other species under hypoxic conditions. GLUT-4 is the insulin sensitive glucose transporter. GLUT-1 has recently been cloned from carp (Cyprinus carpio) (Teerijoki et al., 2001) and rainbow trout (Oncorhynchus mykiss) (Teerijoki et al., 2000). A GLUT molecule with high sequence similarity to GLUT-4 has also recently been cloned from brown trout (Salmo trutta) (Planas et al., 2000). We have identified two glucose transporters in cod heart by reverse transcription - polymerase chain reaction (RT-PCR). Primers were designed based upon

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consensus sequences in conserved areas from other species. A phylogenetic tree was generated using AlignX (Informax Inc.) and indicates that the first is most likely GLUT-1 and the second, GLUT-4. Of the sequence data generated thus far, cod GLUT-1 has approximately 82%, 79% and 76% nucleotide identity to rainbow trout, carp and human GLUT-1, respectively. Cod GLUT-4 has approximately 73%, 68% and 70% nucleotide identity to human GLUT-1 and human and brown trout GLUT-4, respectively. Cod GLUT-1 and GLUT-4 have approximately 73% nucleotide identity. Northern blot analysis indicates that the mRNA transcript for cod GLUT-1 is approximately 6 kB in length and is highly expressed in brain, gill, heart and kidney. The 5’ untranslated region (UTR) has been cloned by 5’ Rapid Amplification of cDNA Ends (RACE) and is 215 bp. The open reading frame (ORF) is estimated to be 1500 bp based on the published sequences of GLUT-1s from other species. Approximately 1300 bp of ORF sequence has been identified thus far. A cod heart cDNA library has been constructed and is being screened with a probe generated from the known cod GLUT-1 ORF sequence to obtain the remaining ORF sequence and that of the 3’UTR. The ORF of cod GLUT-4 is also estimated to be about 1500 bp. Approximately 1200 bp of ORF sequence has been generated. The cod heart cDNA library will also be screened to obtain the remaining GLUT-4 sequence data. References Planas, J.V., Capilla, E. and Gutierrez, J. 2000. Molecular identification of a

glucose transporter from fish muscle. FEBS Lett. 481(3):266-70.

Teerijoki, H., Krasnov, A., Pitkanen, T.I. and Molsa, H. 2000. Cloning and characterization of glucose transporter in teleost fish rainbow trout (Oncorhynchus mykiss). Biochim Biophys Acta. 1494(3):290-4.

Teerijoki H., Krasnov A., Pitkanen, T.I. and Molsa, H. 2001. Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier. Comp Biochem Physiol B Biochem Mol Biol.128(3):483-91.

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Acknowledgements We thank Connie Short for technical support, Robert Evans for guidance with the cDNA library construction and Madonna King and Dr. G. Fletcher for use of equipment. This project was supported by funding from AquaNet and N.S.E.R.C.

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RNA-DNA RATIO IN EXTRACTS OF FISH SCALES

CAN INDICATE FEEDING CONDITION

Todd R. Smith

Marlboro College P.O. Box A

Marlboro, VT 05344 802-258-9254/[email protected]

Lawrence J. Buckley URI/NOAA CMER Program

Graduate School of Oceanography University of Rhode Island

Narragansett, Rhode Island 02882 401-874-6671/[email protected]


Introduction The ratio of RNA to DNA (R/D) has been used as an index of growth and feeding condition of fish (review: Bulow 1987), and R/D in extracts of whole larval or early juvenile fish have been used to examine variability in growth and condition in individuals (review: Buckley et al. 1999). These approaches typically require sacrificing the fish, which precludes repeated sampling. However, a method using teleosts scales, which are covered with live tissue (Kardong 1998), offers a new approach. In the present study we measured the nucleic acid content of extracts of scales removed from juvenile cod (Gadus morhua). Our hypothesis was that R/D in scale extracts would provide a non-lethal, non-surgical method for estimating the recent feeding condition and growth of live fish, which could be performed repeatedly on the same individuals. We performed experiments in which juvenile cod were fed either different quantities, or different types, of food. Scales were periodically removed and assayed for nucleic acids to calculate R/D.

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Methods and Results Assay of RNA and DNA Juvenile cod reared from eggs (Buckley et al. 2000) were anaesthetized with MS-222 (75 mg/L, Sigma), measured (total length) and weighed. Scales or epidermal scrapings were removed from the left side of each fish just above the pectoral fin. Samples were stored in extraction buffer (1 % n-lauroylsarcosine, 5 mM TRIS, 0.5 mM EDTA, pH 7.5) at -80°C. Total RNA and DNA were quantified using the fluorescent dye ethidium bromide (EB) as previously described (Wagner et al. 1998). Briefly, nucleic acids were extracted from tissue with extraction buffer by shaking for 1 hour at room temperature. The samples were centrifuged and the supernatant was retained. EB was added to RNA standards, DNA standards and samples. RNA was estimated from the difference between total fluorescence (prior to an RNase treatment) and fluorescence after treatment with RNase (DNA fluorescence). Statistical tests T-tests, and repeated measures analysis of variance (RM-ANOVA) were performed with StatView 4.51. Significant differences within each sampling period were analyzed with the Bonferroni post-hoc test. Statistical tests were considered significant at P<0.05. Experiment I Extracts from scales of juvenile cod (mean length 18.8 cm) exhibited no significant endogenous or residual fluorescence when compared to the RNA and DNA standards. We concluded that in our samples extracted from scales of juvenile cod, endogenous and residual fluorescence were not a significant source of error in the quantitation of RNA and DNA. Experiment II Juvenile cod (mean length 18.8 cm) were either starved, or fed (2.5% to 7% body weight/day; BW/D). Two replicate tanks were used for starved fish; four for the fed fish, with nine fish in each tank. The fish were sampled every two weeks. After the fourth inventory (day 42), the tanks were divided in half, with four fish on one side and five on the other. One half of the tank was maintained at the original treatment, while in the other half the treatment was reversed. So for one week some of the originally starved fish were then fed (“Starved/Fed”), and some of the originally fed fish were starved (“Fed/Starved”).

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Length of the fed and starved groups was significantly different only on day 42 (by RM-ANOVA and Bonferroni post-hoc test). The mean R/D of fed fish was significantly higher than that of the starved fish on days 15, 28 and 42 (Figure 1). On day 49, mean R/D in the fed treatment was significantly greater than in the Fed/Starved treatment (Figure 1).

RNA/DNA

1.01.52.02.53.03.54.0

-1 4 9 1

TREATMENT SPLIT

Figure 1. Mean RStarved (filled squANOVA, followedifference, P<0.05and Starved vs. Stt-test; #=significan

Experiment III Sixty fish (mean lengcommercial diet at 5%BW/D. Inspection ofdiscernable scales. Mean weight and totcommercial diet (by differed, with fish fed R/D than the fish fed c

*

4 19 24

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/D (±sd) of fares) on dayd by the Bo. Comparisoarved/Fed (t difference

th 2.8 cm) BW/D, an

these fish

al length wt-test; Figurthe commeropepods (Fi

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open square), p<0.05.

were divided three fed

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ere significae 2A & 2B

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79

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44

iment I. 42 we

-hoc tes. Fed/S on day

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ntly gre). R/D

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49 54

Fed (filled circles) andre compared by RM-t; *=significant tarved (open circle), 49 were performed by

ng six tanks: three fed a f mixed copepods at 5% microscope revealed no

ater for the fish fed the in tissue scrapings also significantly higher mean

C OthRmscred Awrein R B

Figure 2. Size and R/D of juvenile cod with no apparent scales: mean weight (A), mean length (B), and mean R/D of epidermal scrapings (C) at inventory 2. Fish were fed a commercial diet or wild-caught plankton; inventory 2 (Invt. 2) was 21 d after inventory 1 (Invt. 1). Means represent 3 tanks (± 1 sd). The treatments were compared by t-test; *=significant difference, P<0 05

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onclusions

ur data indicate that RNA and DNA in scale extracts can be measured and that e R/D of scale tissue is a responsive measure of feeding condition and growth. /D of scale extracts was more sensitive to a change in feeding condition than easurements of fish weight or length. We also calculated R/D in surface rapings from juvenile cod that had no scales. The R/D from this tissue flected differences in diet, indicating that the method is not limited to fish with

iscernable scales.

pplying this fluorescence-based assay of R/D to samples extracted from scales ould be a useful tool for fisheries research, especially in studies that require peated sampling of the same individual. Samples can be obtained quickly from dividual animals, and sample collection is non-lethal and non-surgical.

eferences

uckley, L., E. Caldarone, and T-L. Ong. 1999. RNA-DNA ratio and other nucleic-acid-based indicators for growth and condition of marine

fishes. Hydrobiologia 401:265-277. Buckley, L.J., T.M. Bradley, and J Allen (2000) Production, quality and low

temperature incubation of eggs in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). Journal of the World Aquaculture Society 31:22-29.

Bulow, F.J. 1987. RNA-DNA ratios as indicators of growth in fish. Pages 45-64

in Summerfelt, R.C. and G.E. Hall editors. The Age and Growth of Fish. Iowa State University Press, Iowa.

Kardong, K.V. 1998. Vertebrates: Comparative Anatomy, Function, Evolution,

2nd edition. WCB/McGraw-Hill, Massachusetts. Wagner, M., E. Durbin and L. Buckley. 1998. RNA:DNA ratios as indicators of

nutritional condition in the copepod Calanus finmarchicus. Marine Ecology Progress Series 162:173-181.

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ONTOGENY OF DIGESTION IN LARVAL ATLANTIC COD

(Gadus morhua) AND HADDOCK (Melanogrammus aeglefinus)

J. C. Pérez-Casanova

Department of Biology, Dalhousie University, Halifax, N.S., Canada, B3H 4J1 Institute for Marine Biosciences, National Research Council Canada, Halifax,

N.S., Canada, B3H 3Z1 E-mail: [email protected]

H. M. Murray

AquaNet, Memorial University of Newfoundland Institute for Marine Biosciences, National Research Council Canada

J. W. Gallant, S. Douglas, N. W. Ross and S. C. Johnson

Institute for Marine Biosciences, National Research Council Canada

EXTENDED ABSTRACT ONLY-DO NOT CITE Introduction Two major problems faced in the culture of non-salmonid fish are high mortality during the larval stages and the requirement for use of live feeds such as rotifers and Artemia. High mortality during the larval stage may be due to feeding problems such as poor nutrition. Commercial scale production of live feeds is difficult and expensive and is considered to be a bottleneck for successful fish production. The development of a formulated diet for larval haddock and cod would simplify and reduce production costs. Information on what the larvae are capable of ingesting and digesting during ontogeny is important for the development of such diets. We used biochemical and molecular biological techniques to examine the patterns of digestive enzyme activity throughout development in these species. Methods Biochemical Analysis of Enzymes Activities Triplicate pooled samples of 100-300 non-fed haddock and cod larvae were collected from hatch through to 45 days post hatch (DPH). Larvae were raised

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using standard larval rearing protocols for these species. Average water temperature was 11.9º C for haddock and 10.6º C for cod. Upon collection the larvae were immediately frozen on dry ice and transferred to –80º C for long-term storage. Whole body homogenates were prepared by homogenizing the larvae on ice in 150 mM NaCl. These homogenized samples were aliquoted and stored at –80º C until use. Samples were analyzed for general protease (GP), trypsin, pepsin, α-amylase, general lipase and bile salt activated lipase (BAL) activity using different biochemical techniques (Gawlicka et al. 2000; Parent 1998; Iijima et al. 1998). Enzymes specific activities are reported as units of activity per mg of protein. Samples of live prey were analyzed for digestive enzyme activities and their contribution to larvae digestion was calculated. Analysis of Haddock Bile Salt Activated Lipase Expression by RT-PCR Replicate pools of 20 haddock larvae were taken from hatch through to 45 DPH, rinsed in RNALater (Ambion, Austin, TX, USA), transferred into 1.5 ml Eppendorf tubes containing 0.5-1.25 ml RNALater, and stored at -80° C until used. For each sampling date total RNA was isolated from the homogenate of a single larval pool using the RNA Wiz kit (Ambion, Austin, TX, USA). Samples were treated with a DNA Free Kit (Ambion, Austin, TX, USA). First strand cDNA was synthesized from 1.5 µg of total RNA using the RetroScript kit (Ambion, Austin, TX, USA) and aliquots of the reaction products were subjected to PCR using rTaq polymerase (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and specific primers designed for haddock BAL. Amplification of GAPDH mRNA was performed to confirm the level of expression of a housekeeping gene and provide an internal control. Amplification products were visualized on a 2.3 % agarose gel. Results Expression of BAL Transcripts of BAL were detected at hatch and were present throughout larval development. Expression of BAL in haddock larvae was low at hatch, when compared to the GAPDH controls, and increased from 2 through to 25 DPH. Expression decreased from 30 to 45 DPH (Fig. 1). Specific Activity of Digestive Enzymes

84

The specific activities of digestive enzymes in larval haddock and cod are shown in Fig. 2. Cod and haddock GP activities generally increased during early development (<15 DPH) and then decreased possibly due to increasing levels of protease inhibitors in the homogenates of larger larvae. General protease activity in both species showed an increase in the oldest larvae (>35 DPH).

Fig. 1. Expression of BAL gene in haddock larvae through development. Numbers on top indicate DPH. Expression using BAL primers ( ) ; and expression using GAPDH primers ( ). Controls: single primers (F’ and R’) and both primers without template (F’/R’).

0 2 4 6 8 10 15 20 25 30 35 45 F’ R’ F’/R’

700 bp 600 bp

300 bp

200 bp

Trypsin-like activity was present in both haddock and cod at hatch. Trypsin-like activity of cod generally showed an inverse pattern to that of haddock. Pepsin-like activity was evident in both species at hatch declining until 25 DPH and increasing thereafter. General lipase activity was evident in cod and haddock from hatch. The level of activity remained relatively constant over time with the exception of haddock that showed a marked increase in activity at 35 DPH. General lipase activity in haddock was higher than that of cod at all ages. Activity of BAL was evident in both species at hatch with the activity in haddock being higher than that in cod at all time points. Haddock BAL activity declined over time, whereas cod BAL activity remained relatively constant. There was no α-amylase activity detected in either species, except at 4 DPH for cod and 15 DPH for haddock. At these times large numbers of rotifers, having high levels of α-amylase activity, were present in the gut. With exception of α-amylase activity the enzyme contribution of live feed to the digestive ability of these species is estimated to be less than 17% for cod and 14% for haddock. Conclusions Biochemical assays demonstrated that larvae of both haddock and cod are capable of digesting proteins and lipids from hatch. The activity of trypsin-like

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enzymes and BAL was present at hatch as reported for other species (Oozeki and Bailey 1995). Although pepsin-like enzyme activity was present at hatch, it is possible that much of the activity is the result of the measurement of other protease that function at low pH since the appearance of gastric glands first occurs at 30 DPH in both cod and haddock. The lack of α-amylase activity in

Fig. 2. Specific activities of digestive enzymes in larvae of haddock ( ) and Atlantic cod (----------). A) General Proteases; B) Trypsin-like activity;

0

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C) Pepsin-like activity; D) General lipase; E) Bile Salt Activated Lipase and F) α-amylase. Values are means ± SE of 3 samples.

cod and haddock was not unexpected as these species in their early life history stages feed primarily on zooplankton (Kane 1984). References Gawlicka, A., Parent, B., Horn, M. H., Ross, N., Opstad, I. and Torrissen, O. J.

2000. Activity of Digestive Enzymes in Yolk-sac Larvae of Atlantic Halibut (Hippoglossus hippoglossus): Indication of Readiness for First Feeding. Aquaculture 184; 303 – 314

Kane, J. 1984. The feeding habits of co-occurring cod and haddock larvae from

Georges Bank. Mar. Ecol. 16; 9-20 Iijima, N., Tanaka, S. and Ota, Y. 1998. Purification and Characterization of

Bile Salt-Activated Lipase from Hepatopancreas of Red Seabream, Pagrus major. Fish. Physiol. Biochem. 18; 59 – 69

Oozeki, Y. and Bailey, K.M. 1995. Ontogenetic development of digestive

enzymes activities in larval walleye pollock, Thelagra chalcogramma. Mar. Biol. 122; 177-186

Parent, B. 1998. Determination of activity of digestive enzymes in Atlantic

halibut and winter flounder using spectrophotometry and zymography. Report. National Research Council’s Institute for Marine Biosciences. Canada

Acknowledgements • AquaNet • Heritage Aquaculture • Institute for Marine Biosciences-National Research Council • Dalhousie University • Fundación Pablo García • Steve Leadbeater, Ron Melanson, Laura Garrison and Christine Berrigan

from IMB-NRC • Ben Levy and Debbie van der Meer from Heritage Aquaculture

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CLONING OF RAINBOW TROUT (ONCORHYNCHUS MYKISS)

α-ACTIN AND MYOSIN REGULATORY LIGHT CHAIN 2 GENES

AND α-TROPOMYOSIN 5’-FLANKER.

FUNCTIONAL ASSESSMENT OF PROMOTERS

Aleksei Krasnov, Heli Teerijoki and Hannu Mölsä

Institute of Applied Biotechnology, University of Kuopio, P.O.B. 1627, Kuopio 70211 FINLAND. E-mail: [email protected]

EXTENDED ABSTRACT ONLY - DO NOT CITE Cloning and characterization of fish promoters is important for gene transfer research, studies of cell differentiation and regulation of gene expression. Identification of genomic structure of fish genes provides valuable sequence information and adds to understanding of their molecular evolution. We aimed at cloning of rainbow trout regulatory sequences that direct expression of genes in skeletal muscle. To address this task, we chose α-actin (�-OnmyAct), myosin light regulatory chain (OnmyMLC2) and tropomyosin (�-OnmyTM). Cloning of genomic sequences was performed using the Genome Walker system. In brief, high molecular weight genomic DNA of rainbow trout was digested was restriction enzymes leaving blunt ends, which was followed by ligation to adaptors including primer sites. Combination of universal and gene specific primers was used for PCR amplification of these libraries. Actins are highly conserved structural proteins. Being components of contractile structures and cytoskeleton, actins are distributed ubiquitously. Of three major vertebrate actin groups (α, � and �), αactins are specific for striated muscle. Cardiac isoforms are predominant in heart, whereas skeletal αactins are found in both skeletal and cardiac muscle. We cloned the whole coding part (2.8 kb), 5’-flanker (2.1 kb) and terminator (0.5 kb) of �-OnmyAct. This gene was expressed in both skeletal and cardiac muscle being a predominant isoform in trunk muscle of adult rainbow trout. Its structure of this gene was identical to all known vertebrate skeletal and part of cardiac α-Act genes. The upstream regions

89


of �-OnmyAct included TATA box and a number of putative regulatory motifs (E-boxes and CArG-boxes). These elements were reported in promoters of α-Act genes of zebrafish (Higashijima et al., 1997), medaka (Kusakabe et al., 1999) and channel catfish (Kim et al., 2000). Myosin complex is a hexamer of two heavy and four light chains, of which regulatory chain is required for calcium binding. This gene has been cloned from one teleost species, zebrafish (Xu et al., 1999). We cloned two distinct OnmyMLC2 promoters (1.6 and 1.0 kb) and both included transposon-like sequences. The OnmyMLC2 promoters included TATA box and a number of putative regulatory motifs (E-boxes), their number being less than in �-OnmyAct. Number (7) and length of exons in this gene was typical for vertebrate MLC2. In skeletal muscle, tropomyosin is a dimer of αand �chains mediating interaction between the troponin complex and actin, which is required for regulation of contraction. Unlike actin and myosin, tissue-specific isoforms of mammalian tropomyosin are encoded by a single gene and multiple proteins arise due to usage of different promoters and alternative splicing. We cloned α-OnmyTM promoter (700 bp) which lacked canonical regulatory elements. This was typical for vertebrate αtropomyosins (Wieczorek et al., 1988). For functional assessment, promoters were cloned with LacZ reporter and these constructs were transferred into rainbow trout eggs. All four promoters were able to control expression of reporter which was detected earliest at the stage of somitogenesis. Reporter expression was found mainly in myotomes and heads and but none of these promoters was strictly muscle-specific. Functionality of four promoters and α-OnmyAct terminator was also confirmed in rainbow primary embryonic cell cultures. Three vectors containing �-OnmySkAct terminator combined with �-OnmySkAct, OnmyMLC2 or α-OnmyTM promoter were constructed. We cloned rainbow trout glucose transporter type I (OnmyGLUT1) into these vectors and the transgenes were transferred into rainbow trout eggs. Recombinant OnmyGLUT1 transcripts were detected in embryos. References Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y. and Eguchi, G. (1997). High-

frequency generation of transgenic zebrafish which reliably express

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GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev. Biol. 192, 289-299.

Kim, S., Karsi, A., Dunham, R.A. and Liu, Z. (2000). The skeletal muscle alpha-

actin gene of channel catfish (Ictalurus punctatus) and its association with piscine specific SINE elements. Gene 252, 173-181.

Kusakabe, R., Kusakabe, T and Suzuki, N. (1999) In vivo analysis of two

striated muscle actin promoters reveals combinations of multiple regulatory modules required for skeletal and cardiac muscle-specific gene expression. Int. J. Dev. Biol. 43, 541-554.

Wieczorek, D.F., Smith, C.W. and Nadal-Ginard B. (1988). The rat alpha-

tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing. Mol. Cell. Biol. 8, 679-694.

Xu, Y., He, J., Tian, H.L., Chan, C.H., Liao, J., Yan, T., Lam, T.J. and Gong Z.

(1999). Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle. DNA Cell. Biol. 18, 85-95.

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