Dr. Pantelis Katharios

Institute of Aquaculture Hellenic Centre for Marine Research

FP7-PEOPLE-MC-IRSES 2010

Part 1: HCMR and Institute of Aquaculture Part 2: Aquaculture in Greece Part 3: Phage therapy and the AQUAPHAGE

project

HCMR was formed in June 2003 after the merge of NCMR and IMBC IMBC HCMR NCMR

5 institutes Oceanography / Fisheries / Aquaculture

Mar. Biol. + Genetics / Inland waters

- About 450 people staff - Various infrastructures

Greek National R.T.D. Mission

Institute of Oceanography Open seas and deep basins Coastal zones and shelf Seas Operational Oceanography

Institute of Marine Biological Resources Fisheries Coastal zone management

Institute of Marine Biology and Genetics Marine Biodiversity Genetic analysis

Institute of Inland Waters Fresh water-Lakes, rivers, groundwater

Institute of Aquaculture

43 people staff 34 permanent 13 researchers

Institute of Marine Biology of Crete

Heraklion and Gournes (Crete)

National Center of Marine Research (Institute of Living Resources)

Agios Cosmas (Athens)

Early fish life + larviculture Hatcheries + Technology Physiology + Metabolism Behaviour + Development

Nutrition + food formulation Pathology

Quality of flesh

A complementarity Rearing +Feeding Feed and Quality

AQUALABS 1 1500 m2 of modern & well equipped infrastructures - Experimental facilities - labs and offices (1st floor) + 1 responsible Researcher + 1 Technician / zone For research on larviculture Microalgae and food chain

2- Mesocosm hatchery (480 m2) - 7 tanks 40-m3 - 2 closed systems (2 tanks each) - Photo/thermo regulated

1- Intensive hatchery (477 m2) - 8 duplicate closed systems (16 tanks 500-L) - 3 triplicate closed systems (9 tanks 2-m3) - 16 triplicate closed systems (48 tanks 50-L)

3 - Solar microalgae production 187 m2 - 5 pilot photobioreactors (> 2-m3 & > 10-m2) - 5 small photobioreactor for stem cultures - 20 columns for classical bloom cultures

4 - Zooplankton production 178 m2 - 2 automatic systems for production of rotifers - 24 experimental columns for artemia & rotifer - Small tanks

1 4

2 3

AQUALABS 2 1700 m2 of experimental and rearing structures for - brood stock management - fish behaviour and feeding - Fry weaning and nursery + offices and labs + 1 researcher + 1 technician / zone

1- Brood stock management (500 m2) - 7 tanks 20-m3 + 3 tanks 60-m3 +1 tank 360-m3 - 3 closed systems with 4 tanks 5-m3 each - 3 opened systems with 4 tanks 2-m3 each - photo / termo regulation+eggs collectors - small facilities for eggs/prelarvae rearing -

2- Nursery and pregrowing (500 m2) - 18 tanks 10-m3 in open or closed system - 12 tanks 2-m3 in open system (2 waters) - 3 recirculating systems - wet lab

3- behavior and feeding (350 m2) - 16 closed systems with 3 tanks 500-L each - 3 with possibility of quartantine for wild fish - self feeding system / camera

4 - well equipped laboratories (200 m2) - Physiology (reproduction) - nutrition and metabolism - behavior

1 3 2

AGIOS COSMAS INFRASTRUCTURES 500 m2 of offices and laboratories

+ 200 m2 of experimental rearing structures

LIVE BROOD STOCKS 7 families & 16 species

Polyprion americanus

Scientific name Stocks Fishes Weight(Kg)

Seriola dumerilii

Umbrina cirrosa Argyrosomus regius

2 + 2 8 - 30 12 + 6 1 + 1 2-5 / 15-30 10 + 5

2 0.5 15 1+2 0.3 - 2 50+25

Pagrus pagrus

Diplodus vulgaris

Sparus aurata 1+4 0.5 - 4 100 1+2 0.4 - 2 50

1 0.3 50 Diplodus sargus

Pagellus erythrinus Dentex dentex

1+2 0.4 - 1.5 50

1+2 2 - 5 50+40 1+1 0.3-0.5 50

Dicentrarchus labrax

Sciaena umbra

Puntazzo puntazzo

1+3 0.5- 5 75

1+2 1 - 6 40

1+2 0.2 - 0.5 50+ 50

Family

Polyprionidae Carangidae Sciaenidae

Sparidae

Dicentrarchidae Centrolophidae Epinephelidae

Schedophilus ovalis 1 2 - 8 5 Epinephelus marginatus

1 2 - 13 100 + 6

Epinephelus aeneus 3+1

5 - 17 4

Eggs

+ +

+ ++

+ ++++

+++ +++

+++ ++

+ ++

++++ 0

± ±

F(sel.)

0

0 0 0

0

0-1-2

0 0

0-1

0-1

0

0-1-2 0

0-1

0

0

Larval culture + Early life stages Mesocosm Technology Fish reproduction

Built as a commercial fish farm Large Scale Facilities

RESEARCH PRIORITIES

1- Fry production 1 - Control of reproduction 2 - Biology and fish development 3 - Improving hatchery technologies 4 - Ontogeny of feeding behavior 5 - Mastery of larval food chain technologies (phyto - zoo) 6 - Early technological pathology and micro-parasitism

2- On-growing 1- Fish nutrition 2- Fish pathology stress and immunology 3- Fish flesh quality 3- Three collaborative axes 1- Aquaculture and Environmental Interaction (HMCR + Univ.s) 2- Brood stock genetic improvement (HMCR + Univ.s) 3- Biotechnology (microalgae), and molecular biology (Univ.s)

4- Three basic technological / market / environment objectives 1- New species for diversification 2- low input & automatic systems of production 3- Product quality

◦ New species, mainly fast growers (Dentex, Seriola, Polyprion,

Tunas) ◦ Mechanisms of fast growers metabolism (comparison with slow

growers) ◦ Role of environment in fish reproduction ◦ Disease prevention (alternative treatments) ◦ Immunology ◦ “Megacosm“ technology (lagoon management, tuna ranching etc) ◦ Artificial reefs, FADs ◦ Aquariology

FINEFISH (malformations/skeletal deformities) AQUAMAX (fish nutrition) SELFDOTT (blue fin tuna) FASTFISH (stress) SEACASE (extensive/coastal aquaculture) BIOEXPLORE (Microalgae Biotechnology)

0

40000

80000

120000

160000

1991 1994 1997 2000

PRODUCTION SEA BASS & SEA BREAM

Mediterranean Greece

SEA BASS & SEA BREAM SECTOR DISTRIBUTION IN MEDITERRANEAN

CROATIA

PORTUGAL

MOROCCO

TURKEY

ITALY MALTA

TUNISIA

FRANCECYPRUS

GREECE

SPAIN

260

39 66

4

49

43

2

3

19

175

• Adequate Geography & Climate • Adequate Strategical /Political • & Environmental choices • Adequate R & D supply • Dynamism of farmers

• Matching technique / species with environment • Low input & density sea cage ongrowing • Low input & density hatchery

HIGH MARKET COMPETITIVITY

- 310 site licences (264 farms, 39 Hatcheries, 7 other) - 269 enterprises (235 active) (87.4 %) - 7 in stock market - 15 among the 100 biggest Greek food industries

- 9500 employements (6000 direct + 3500 indirect) - 127 farms & 16 hatcheries in islands (46.1 %)

- 100 000 tonnes production + 301 000 000 fry - 92 % bass & bream + 8 % new species

- 440 MEuro income - 250 MEuro export

A Greek success story ==> The youngest sector of primary production ==> The first sector of product exportation

Gilthead seabream (Sparus aurata) European seabass (Dicentrarchus labrax) Rainbow trout (Onchorynchus mykiss) European eel (Anguilla anguilla) Sharpsnout seabream (Diplodus puntazzo) Common pandora (Pangelus erythrinus) White seabream (Diplodus sargus) Atlantic bluefin tuna (Thunnus thynnus thynnus) Common sole (Solea solea) Flathead grey mullet (Mugil cephalus) Common dentex (Dentex dentex)

92%

Hatcheries (inland facilities) Floating cages (sea water)

14-19 months 350 g

…..here the size counts !!!!! Fish species Egg size

(mm) Larva size (mm)

Atlantic salmon 5-6 15-25

European sea bass 1.2-1.4 7-8

Gilthead sea bream

0.9-1.1 3.5-4.0

Yolk sac reserves Atlantic salmon: 3 weeks Sea bream: 3 days

Mouth size at first feeding Particle size Salmon: 1mm Sea bream: 0.1mm Digestive system Salmon: Stomach, enzymes, digestion Sea bream: Intestine, exogenous enzymes

Rotifers Artemia

Microalgae culture Rorifers culture Artemia culture

The first 15-20 days fish live and feed in a “soup” filled with micro-organisms in a delicate equilibrium

Survival rate at the end of larval rearing 10-50%

Cause of mortality: Nutritional imbalance, malformations, cannibalism, bacterial infection

Source of bacteria: water, live feeds Pathogenic bacteria: V. anguillarum,

Photobacterium damsela subsp. piscicida, Taenacibaculum maritimus

Aquaculture: an industry of >65 bn $ and 8.8% annual growth

Greatest economical losses due to disease Bacterial diseases are common and often

catastrophic Treatment of bacterial disease is based

almost exclusively in antibiotics

The problem The solutions

Overuse/abuse Resistant strains of fish pathogens Resistant strains of

human/terrestrial animal pathogens

Destruction of environmental microflora

Destruction of fish intestinal microflora

Residues Environmental impact Cost (of drugs and mortalities) Image of the industry

Good Management Practices (Easy to say…hard to apply)

Vaccines (only few, stage?) Probiotics Alternative treatments

(oregano oil etc) Nutrition Bacteriophages

Viruses that infect bacteria Lytic and temperate Highly host specific (strain-specific) Self propagated Self restricted Tools for molecular biology Biotechnological applications Phage therapy both in Veterinary and Human

Medicine (mostly in Former USSR)

Earlier than antibiotics Replaced and eventually forgotten after the

triumph of penicillin Re-emerged again following the first

antibiotic resistant bacteria

Science report (2002): “Stalin’s forgotten cure”

Biological way to confront bacterial disease (organic aquaculture)

No residues No multiple doses required Do not destroy intestinal microbiota Do not destroy environmental microflora They can be used in “delicate” environments such

as hatcheries

Specificity (cocktails?) Risk for mutations (can be overcome with

lytic phages) Immune reaction (possible due to dead

bacterial cells) Resistance It’s cheap, rather easy and not patentable (no

interest by the pharmaceutical industry)

Japan (1999, Nakai et al. Dis. Aquat. Org) in Lactococcus garviae

Thailand (2005, Pasharawipas et al Virus Res) Vibrio harveyi in shrimps

Denmark, Canada, Chile, Belgium, Greece

National project 2003-2006 Collaboration with G. Eliava Institute of

Bacteriophages, Microbiology and Virology, Tbilisi, Georgia

Aim Vibrio harveyi (4 lytic phages) First positive results from in vitro and in vivo

Continuation of collaboration

Network for the development of phage therapy in aquaculture

FP7 Marie Curie, IRSES-2010 Linking European Research with Third

Countries Staff exchanges Network Training, experience exchange,

workshops/seminars

Institute of Aquaculture, HCMR, Greece (coordinator)

Department of Biotechnology, Agricultural University of Athens, Greece

Marine Biological Laboratory, University of Copenhagen, Denmark

Zoology Department, Trinity College Dublin, Ireland G. Eliava Institute of Bacteriophages, Microbiology

and Virology, Tbilisi, Georgia INTA, Universidad de Chile

Staff exchanges Common project Phage isolation for Vibrio and Flavobacterium Sea breams, salmon, trout Phage diversification Phage lytic enzymes Storage and delivery methods Infectivity studies

Large scale infectivity tests (proof of concept) Phage lytic enzymes Immunogenic lysates

Thank you for your attention