Consellería do Mar Centro de Investigacións Mariñas Annual report 2008 Technical data

Edit XUNTA DE GALICIA Consellería do Mar CIMA (Centro de Investigacións Mariñas)

Coordination Alejandro Guerra (CIMA)

Composing IMPRONTA GRÁFICA, s.l

Printing LUGAMI A.G., s.l.

Dep. Legal C-2747-2009

Translation Andrea Martínez Beiras Annual report 2008 Centro de Investigacións Mariñas

Index

13 1.- INTRODUCTION

17 2.- CENTRO DE INVESTIGACIÓNS MARIÑAS (CIMA, Marine Research Centre)

19 2.1. STRUCTURE AND FUNCTION

20 2.1. CIMA STAFF 2.2.1. STAFF EMPLOYED AT THE CIMA OF VILANOVA DE AROUSA 2.2.2. STAFF EMPLOYED AT THE MARINE CULTURES CENTRE OF RIBADEO

24 2.3. RUNNING EXPENSES OF THE CIMA 2.3.1. CIMA’S EXPENSES I.- DISTRIBUTION OF CIMA’S BUDGET BY CHAPTERS II.- SOURCE OF FUNDS TO FINANCE THE INVESTIGATION

25 3.- SCIENTIFIC ACTIVITY

27 3.1. SUMMARIES OF THE RESEARCH PROJECTS

A.- PROJECTS IN WHICH THE CHIEF RESEARCHER BELONGS TO THE CIMA

3.1.1 AREA OF AQUACULTURE

PROJECTS -Culture and management of the perlemoen abalone (Haliotis tuberculata spp). -Reuse of wastewater of marine and auxiliary culture establishments located in terrestrial area. -Culture of new species of bivalve molluscs of interest in hatcheries. -Towards an integrated management of Ensis stocks (TIMES). -ALBA: Improvement of the pullet carpet shell clam (Venerupis pullastra, Montagu, 1803). -Optimization of the shell clam intensive culture and identification of the genetic markers for the follow up of the res- tocking. -Culture and management of the sea urchin (Paracentrotus lividus, Lamarck, 1816). Subproject I A: Determination of the reproductive cycle and biochemical composition of gonads of the sea urchin Paracentrotus lividus, Lamarck, 1816, in natural populations of Galicia. Culture of the sea urchin in laboratory: production of juveniles for repopulation. -Proposals and improvements for the design and control of the restocking plans and evaluation of the impact of esca- pes of fish and crustaceans. -Integrated Aquaculture: Experimental experience for the development of multitrophic culture system. -Optimization of the octopus (Octopus vulgaris) grow-out. Compound feed development (Galicia).

ACTIONS -Prevention and protection study techniques of the principal predator species of the natural shoals bivalve molluscs’ resources and culture areas at the Galician coast and estuaries. -Integral microbiological control in molluscs hatcheries. -Study of operability of the tubort (Psetta maxima) restocking and the European Lobster (Homarus gammarus) at the Coast of Galicia. -Start-up and validation of a grow-out system of clam at the pontoon of the dock of O Vicedo (Lugo). -Follow-up and validation of mollusc minihatcheries. -Grow-out and culture of Queen Scallops (Aequipecten opercularis) and King Scapllop (Pecten maximus).

9 3.1.2. AREA OF PATHOLOGY

PROJECTS -Offsprings production of the corresponding seeds to F3 Generation, in a program of genetic selection to produce a stock of flat oyster Ostrea edulis tolerat to the bonamiosis and with a high rate of survival. -Gene identification of resistance/susceptibility as potential indicators of disease in the flat oyster (Ostrea edulis) and in the Pacific (Crassostrea gigas) -Neoplasic disease in marine bivalves of commercial interest. -Perkinsosis in the Spanish Coast: characterization of the taxonomic variants of the parasite, of its live cycle, and its immunological answer to the host.

ACTION -Tumour alterations that affect the golden carpet shell, the flat oyster, and the cockle in Galicia. Characterization of the disease and effects on the populations.

3.1.3. AREA OF COASTAL OCEANOGRAPHIC PROCESSES

PROJECTS -Miltids Culture: expansion and sustainability. Subproject: Evaluation of the impact of the methods and levels used for the toxin control in mussels. -Reduction of the type PSP and DSP toxins incorporation in the rafts of mussels. -Comparison of methodologies for the determination of paralyzing toxins in bivalves related to the Paralytic Shellfish Poisoning (PSP). Uses in Spanish aquaculture. Subproject: Comparison of methodologies for the determination of paralyzing toxins in bivalves related to the Paralytic Shellfish Poisoning (PSP). Uses in Galician aquaculture.

ACTIONS -Investigation action about toxicological episodes of phytoplankton: in bivalve molluscs found in Galicia. (EPITOX Action). Sub Project 6- Ligaments of the DSP toxins in the cells of the mollusc, acceleration of the elimination of the toxins and the ASP through non digestible substances and analytic aid towards the epi- demiology and genetic sub projects. -Toxic episodes of phytoplankton origin in the bivalve mollusc of Galicia. Sub project 8: Production of fami- lies of the mussels in hatcheries for genetic studies. tóxicos de origen fitoplanctónico en los moluscos bival- vos de Galicia. Subproyecto 8: reducción en criadero de familias de mejillón para estudios genéticos

3.1.4. AREA OF MARINE RESOURCES

PROJECTS -Incorporation fo immunodetection tecniques in the monitorization of the mussel larvae in Galician coastal waters. I. Adaptation of the sampling protocols and larva determination. -Study of the detoxification and grow-out of the King scallop Pecten maximus (Linnaeus, 1758) in the Vigo Ria. -King Scallop Phase 1: Study of the stock populations of King Scallop (Pecten maximus, L., 1758) in Ares, - Arousa and Vigo Rias. -King Scallop Phase 2: Evaluation of the stock populations of King Scallop in Pontevedra, Muros and O Ferrol Rias and study of the dynamic of populations in Vigo and Ares Rias.

ACTIONS -Geographic Information System Oriented towards the Management of Marine Resources (SIGREMAR). -Action Plan for the recovery of the shellfish bed “Lombos do Ulla” and “O Bohído” (Arousa Ria).

10 87 B.- PROJECTS WITH PARTICIPATION OF RESEARCHERS AND/OR CIMA STAFF

-IDENTILARVAS. Genetic characterization of species of bivalves’ mollusc and PCR in real time design systems for its application in the detection and identification of larvae in our planktons. -Optimization of industrial production techniques of the flat oyster (Ostrea edulis). -Action investigation about toxic episodes of phytoplankton origin in mollusc bivalves of Galicia (Epitox Action). Subproject 1. Epidemiological detection aspects of intoxications effect of cooking n toxicity and the size of the portion of bivalves. -Action investigation about toxic episodes of phytoplankton origin in the mollusc bivalves of Galicia (Epitox Action). Subproject 3. Improvement of the control system of bivalves affected by biotoxins through the develop- ment and implementation of dynamic models. -Analysis and depuration of lipophilic toxins, bitter domoic acid that allows the exportation of Chilean wedge clams (Mesodesma donacium) and clam (Mulinia edulis) to European markets from the Coquimbo Bay. -Exploration and follow up of harmful microalgae for the activity of culture of pectinide (II, III and IV Region). -SEMAUMEX: Developmenta of an automatic splitting system of the mussel. -Analysis of evidences of the impacts due to the climate change in Galicia. -Study and characterization of intracellular prokaryote Rickettsia type and other potntially pathogenous oxidati- ve bacterias for the clam. -Study of environmental factors and potential pathogens that affect the grooved carpet shell clam culture (Ruditapes decussatus) in the Pontevedra Ria. -Design and application of the recirculation systems of the larval and grow-out phase of the marine fish and con- tinental aquaculture. -Analysis and study of culture factors and conditions of industrial production of the Senegalese sole (Solea sene- galensis). Subproject: Improvement of the parameters in the reproduction of the Senegalese sole (Solea senega- lensis). -Pigment characterization and light regulation in the dynoflagellates. -Characterization of the quality of the breaded fish. Subproject: Characterization of the quality of the breaded fish. -Feasibility study of the culture of the Pacific Oyster or Giant Oyster (Crassostrea gigas) in the Galician Rias.

113 C.- PROJECTS AND RESEARCH LINES PROMOTED AND/OR FINANCED THROUGH THE DXIDP

-Genetic markers for the identification of the “Mexillón de Galicia”. -INDAQUA: Selection of indicators, determination of reference values, design of programs and protocols, and mea- sures for environmental studies in marine aquaculture. -Sanitary Management in Aquaculture (GESAC). Adaptation to the new ground rules. Characterization and standar- dization of conditions of animal health in marine aquaculture: creation of epidemiologic maps and strategies of ela- boration for the design of a net for epidemiologic surveillance. -Adjustment of an intensive culture system of octopus paralarvea, Octopus vulgaris, and importance of the bioche- mical composition in the larval diet.

125 3.2. DOCTORAL THESIS AND ADVANCED STUDIES DEGREES (DEA) CARRIED OUT AT THE CIMA.

131 3.3. ADVISORY SERVICES FOR THE SECTOR AND THE ADMINISTRATION

139 3.4. SCIENTIFIC AND TRAINING ACTIVITIES CARRIED OUT BY THE STAFF 3.4.1. ORGANIZATION, CHAIRMANSHIP AND PARTICIPATION IN SCIENTIFIC MEETINGS 3.4.2. DIRECTION OF DOCTORAL THESES AND INTERN TRAINING 3.4.3. COURSES GIVEN 3.4.4. PARTICIPATION ON EXAMINING BOARDS TO JUDGE FOR PHDs, DIPLOMAS OF ADVANCED STUDIES AND MAS- TER’S DEGREES

11 3.4.5. EDITION OF SCIENTIFIC PUBLICATIONS 3.4.6. REVIEW OF ARTICLES IN SCIENTIFIC PUBLICATIONS

149 3.5. STAFF TRAINING 3.5.1. VISITS TO OTHER CENTRES 3.5.2. COURSE ATTENDANCE

152 3.6. COLLABORATION AGREEMENTS

153 3.7. OUTSIDE VISITORS

154 3.8. PUBLICATIONS 3.8.1. ARTICLES IN JOURNALS LISTED IN THE SCI 3.8.2. ARTICLES IN JOURNALS NOT LISTED IN THE SCI 3.8.3. CONTRIBUTIONS TO BOOKS

159 4. SCIENTIFIC MEETINGS AND TRAINING ORGANIZED BY THE CIMA

161 4.1. SEMINARS

163 5. ANNEX -Abbreviations used -Summary of 10 years of activity in the CIMA

12 1. INTRODUCTION

One more year I have the opportunity of presen- Communities and with Galician research organiza- ting this annual report and making some thoughts tions. From 10 National Plans which are operative at about all the work we have done and the work that a national level, the researchers from the CIMA and we have in our way. Since 1998, when the first edi- the Galicia Fishing Administration coordinate four tion was published, there are summarizing docu- of them and they participate in other five which are ments about the activities developed every year in run from other Autonomous Communities. the CIMA, which are very useful to spread the rese- arch activities and to assess with objectivity the We also participate in several projects of Galicia CIMA work and its contribution on the marine rese- official anouncements through the PGIDT and arch area. Throughout these years, and analyzing other national, European, etc. This participation the annual reports’ data, we can observe a strong net in projets led to 15 specific agreements in colla- and clear link between the world of researching, boration with numerous organizations. They lay represented by the CIMA, the fishing productive sec- down the type of commitment, and the research tors, the shellfish gathering and the aquaculture, nature and targets. The attendance and presenta- through an applied research focused on improving tion of different works in scientific congresses and the socioeconomic reality of these sectors in the meetings was significant last year. We presented 20 Autonomous Community of Galicia. works in national meetings and 13 in international activities. We published 25 articles, 15 of them in This annual report has a dobble function: It is use- journals listed in the SCI and four contributions in ful to show the work developed to the outside and books. also inside the centre. It is an analysis and review tool, in which it is possible to make the necessary The training work is based mainly in the supervi- adjustments. sion of PhD thesis and vocational interns. This labour is completed with courses, more over the The sciencific activity in 2008 was carried out Interuniversity Program of the USC and UDV, from through 50 research projects, whose funding comes the EGAP, etc. We must highlight that this year our from official announcements of autonomic, national staff has been increased with three researches and or international levels. This funding is complemen- four lab analysts in different activity areas. ted with the one from the CPAM through the so- called research actions. The effort that this publication involves for 11 years is related to the purpose of bringing closer and This year research actions have increase in number releasing to society the research work, pointing out and funding, and most of them are joined to speci- where this institution spends the budget for investi- fic agreements, where many researchers and CIMA gation and which is the purpose and the results.To staff collaborate, and also numerous organizations this effect, at the end of the publication, it is indica- and external research groups that develop interes- ted the evolution of the most significant sign of the ting research lines for the Galician Fishing CIMA production since the beginning of the annual Administration. Researchers and other entities take reports publication. part in these actions, such as the USC through the Aquaculture Institute, the Universities of Vigo and A Finally, I would like to thank all the staff for their Coruña, the IIM (CSIC), the IEO from Vigo and A effort and involvement on their job, which makes Coruña, the CETMAR, the Consello Regulador do possible the making-up of this annual report. Mexillón, etc. During the last years, Galicia was the Community that has received most of the funds for the financing of JACUMAR Plans. Research lines, mostly of applied and productive nature, are deve- loped through these National Plans, in collaboration Fátima Linares Cuerpo with research groups from other Autonomous Director-General of Innovation and Fisheries Development

15

2. CENTRO DE INVESTIGACIÓNS MARIÑAS

CENTRO DE INVESTIGACIÓNS MARIÑAS

2.1 STRUCTURE AND FUNCIÓN

The CIMA belongs to the Regional Ministry of of commercial interest in order to set up strate- Fishing and Maritime Affaire, and comprises the gies that will effectively combat these alterations CIMA of Corón in Vilanova de Arousa leading to a reduction in the pathological effects. (Pontevedra) and the Marine Cultures Centre in Ribadeo (Lugo). The Basic function of the CIMA is Area of Coastal Oceanographic Processes. The to carry out research aimed at achieving the ratio- objective of this area is to shed light on the nal management of renewable marine resources aspects of oceanography related to the produc- within the geographic scope under the jurisdic- tion of fishery and shellfishery resources in tion of the Administration of Galicia. Galicia. The lines of research are mainly related to toxic episodes caused by phytoplankton. The scientific activity is divided into four areas: The CIMA, as an institutional body, acts as an Area of Marine Resources. The objective is to advisor to the Galician fisheries administration on gain knowledge on the biology, ecology and matters under their jurisdiction as well as on population dynamics of marine species having issues raised by the different sectors via the commercial interest in order to improve the Directorate-General and CPAM. The CIMA is also management of these natural resources. involved in training new researchers (with tutors- hip and direction of doctoral theses) and lab tech- Area of Aquaculture. The objective of this area is nicians, through grant programs. The research to develop and improve methods of rearing species work is disseminated at different types of scienti- considered to be marketable. There are two basic fic meetings (conferences, seminars, forums, lines of research: mollusc culture and fish farming. workshops, etc.) and in a number of different scientific publications. Area of Pathology. The objective is to study the pathological alterations affecting bivalve molluscs

Centro de Investigacións Mariñas Pedras de Corón s/n. Apartado 13. 36620 Vilanova de Arousa (Pontevedra). Spain. Phones: +34 886 20 63 64 +34 986 50 01 61 Fax: + 34 986 50 67 88 e-mail: [email protected]

Centro de Cultivos Mariños Muelle de Porcillán s/n. 27700 Ribadeo (Lugo). Spain. Phones: +34 982 88 94 03 +34 982 88 94 01 Fax : +34 982 88 94 19 e-mail: [email protected]

web: http://www.cimacoron.org

CONSELLERÍA DO MAR Annual Report CIMA 2008 19 CENTRO DE INVESTIGACIÓNS MARIÑAS

2.2. CIMA STAFF

Director: Guerra Díaz, Alejandro [email protected] Manager: Villanueva Rodríguez, Marta [email protected]

2.2.1. STAFF EMPLOYED AT THE CENTRO DE INVESTIGACIÓNS MARIÑAS OF VILANOVA DE AROUSA.

Researchers Dr Rodríguez Moscoso, Mª Eugenia3 Dr Abollo Rodríguez, Elvira1 [email protected] [email protected] Mrs Sánchez Mata, Adoración1 Dr Blanco Pérez, Juan Carlos [email protected] [email protected] Mr Santos Piñeiro, Ignacio Dr Cao Hermida, Asunción7 [email protected] [email protected] Mrs Varela Manso, Mª Obdulia5 Dr Carballal Durán, Mª Jesús [email protected] [email protected] Dr Villalba García, Antonio Mr Cerviño Eiroa, Antonio [email protected] [email protected] Dr Zapata Gago, Manuel Mr de Coo Martín, Alberto [email protected] [email protected] Mrs Díaz Costas, Seila María5 Post-graduate fellows [email protected] Mr Aranda Burgos, José Andrés Dr Fuentes González, José Miguel Mrs Domínguez Pérez, Laura [email protected] Mrs Escudeiro Rossignoli, Araceli Mr García Fernández, Antonio Mr Fernández Boo, Sergio [email protected] Mrs Martín Gómez, Laura Dr Guerra Díaz, Alejandro Mrs Mauriz Pereira, Aida [email protected] Mrs Ruiz Pérez, Maite Dr Guerrero Valero, Salvador [email protected] Lab analysts Dr Iglesias Estepa, David7 Mrs Blas Rodríguez, Carmen6 [email protected] Mr Cid González, Emilio5 Mrs Linares Cuerpo, Fátima2 Mr Fariña Iglesias, Juan Antonio [email protected] Mrs Fernández Besada, Mercedes Dr López Gómez, Mª del Carmen Mr Fernández Abuín, Isidro5 [email protected] Mr Fernández Franco, Bruno5 Dr Mariño Balsa, José Carlos5 Mr Giráldez Rivero, Ramón [email protected] Mrs Graña Pereira, Mª Isabel5 Dr Molares Vila, José Mrs Gregorio Chenlo, Mª Victoria [email protected] Mrs Mariño Cadarso, Mª Carmen Dr Montes Pérez, Jaime Mrs Martín Sánchez, Helena6 [email protected] Mrs Otero Otero, María5 Mr No Couto, Edgar7 Mrs Paz Márquez, Marta6 [email protected] Mr Pazos Pazos, Juan Carlos5 Mrs Outón Caamaño, Mª José Mrs Pazos Sieira, Gema5 [email protected] Mrs Santamaría Búa, Iria5 Mrs Pérez Acosta, Carmen Mrs Soto Fraga, Nerea5 [email protected]

20 CONSELLERÍA DO MAR Annual Report CIMA 2008 CENTRO DE INVESTIGACIÓNS MARIÑAS

Lab assistants Computer Expert Mrs Campaña Ferro, Emilia Mr Miguéns Ramos, Juan Luis6 Mrs Cores González, Mª José [email protected] Mrs Meléndez Ramos, Mª Isabel Mrs Penas Pampín, Elena Administrative assistants Mr Lago Torrado, José Vocational interns [email protected] Mrs Álvarez Fernández, Mª Jesús Mrs Vilas Fandiño, Milagros7 Mrs Ancosmede Garduño, Cristina [email protected] Mrs Balsa Freire, Sonia Mrs Caamaño Pérez, Lucinda Mrs López Souto, Mª Jesús [email protected] Mr Ramos Caamaño, José Manuel Mrs Rodríguez Galvan, Beatriz Ancillary employees - Clerks Mr Agra Carregal, Luís Manuel General Maintenance Mr Martínez Crespo, José Manuel Mr López Ares, Hermes6 Mr Rey Coredoira, Paulino

Head of the administrative dept. Cleaner Mr García Paz, Gonzalo Mrs Mourgan Barral, Domitila6 [email protected]

Library Mrs Álvarez Francisco, Mª Esther7 [email protected]

1Ramón and Cajal Program 2In a special administrative situation 3Carries out the activity in a different administrative unit 4Parga Pondal Program 5Temporary staff from CPAM associated with rese- arch projects 6Temporary staff 7Temporary civil servant 8Substitute

CONSELLERÍA DO MAR Annual Report CIMA 2008 21 CENTRO DE INVESTIGACIÓNS MARIÑAS

2.2.2. STAFF EMPLOYED AT THE MARINE CULTURES CENTRE IN RIBADEO

Researchers Mrs Costa Costa, Diana Mrs Fernández Álvarez, Aurora Mrs Rodríguez Castro, Belén [email protected] Dr Martínez Patiño, Dorotea General Maintenance [email protected] Mr Loureiro Barcón, José Mª Dr Nóvoa Vázquez, Susana [email protected] Senior administrative Mrs Ojea Martínez, Justa Mrs Neira Páez, Concepción [email protected] [email protected] Mrs Cerviño Otero, Ana 5 [email protected] Administrative Mrs Cruzado Estévez, Ana Post-graduate Fellows Mrs Louzán Pérez, Andrea Ancillary employee - Clerk Mrs Loureiro Rodríguez, Mercedes Lab Analysts Mr Álvarez Llamas, Luis Manuel Cleaner Mrs Álvarez Padín, Alicia5 Mrs Fernández Goás, Carmen6 Mrs Bouzamayor Yáñez, Mª Victoria6

Lab Assistants Mrs Cotarelo Jardón, Mª José Mr Páez Pérez, Francisco Javier Mrs Ruiz Misioné, Mercedes

Vocational Interns 5Temporary staff of the CPAM, associated with rese- Mrs Brión Bello, Rosario arch projects. Mrs Cardoso Loureiro, Iria 6Temporary staff

22 CONSELLERÍA DO MAR Annual Report CIMA 2008 CENTRO DE INVESTIGACIÓNS MARIÑAS

STAFF EMPLOYED AT THE CIMA IN 2008

CONSELLERÍA DO MAR Annual Report CIMA 2008 23 CENTRO DE INVESTIGACIÓNS MARIÑAS

2.3. RUNNING EXPENSES OF THE CIMA

2.3.1. CIMA’S EXPENSES

2.3.2.- FUNDS SOURCE TO FINANCE THE RESEARCH

24 CONSELLERÍA DO MAR Annual Report CIMA 2008 3. SCIENTIFIC ACTIVITY

SCIENTIFIC ACTIVITY

3.1. SUMMARIES OF THE RESEARCH PROJECTS

A.- PROJECTS IN WHICH THE CHIEF RESEARCHER BELONGS TO THE CIMA

3.1.1. AREA OF AQUACULTURE

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Culture and management of the perlemoen abalone Guerra Díaz, A. Haliotis tuberculata spp. Project coordinated by the AC of Canary Islands

START: November, 2006 RESEARCHERS: FINISH: November, 2008 CIMA: Varela Manso, M.O.; Martínez Patiño, D.; Montes Pérez, J.; Ojea Martínez, J.; Nóvoa Vázquez, S. FINANCIAL ASSISTANCE: IGAFA: Lastres Couto, M.A.; Andrés Rivas, M.C. National Plan for development of Marine Cultures JACUMAR RESEARCH SUPPORT: CIMA: Fernández Besada, M.; Meléndez Ramos, M.I.; Penas Pampín, E.

INTERNS: Ruiz Pérez, M.; Romanos Mondragón, L.; Balsa Freire, S.

SUMMARY This National Plan JACUMAR includes research hatcheries seed productions; neither other relevant groups of the Autonomous Communities of Galicia, pathologies were detected. Asturias and Canary Islands. The Autonomous Community of Galicia takes part in the following Reproduction and biochemical composition: From lines: Assessment of the resource, biochemical analy- a total of 540 specimen sampled and analyzed all sis, pathological analysis and culture. through the research action, samples gathered through diving by apnea at Illa de Arousa (Ria de Assessment and resource condition. The extrac- Arousa), Cies (Ría de Vigo) and Fisterra have been tion of the perlemoen abalone at Galicia started in processed monthly. From each sample of 20 speci- the 80´s, its commercial exploitation was forbidden mens, the following biometrical data have been during the period of 1993 and 2002 when higher considered: length, width, total weight, shell levels of PSP have been detected as regards to the weight, meat weight and conical appendix weight. stated legal limit. This situation was repeated since The meat condition index (wet meat weight / total December 2005 till June 2008 (last date of available weight) has been calculated and the gonadal condi- data). During this second period, 16 analyses about tion index (conical appendix weight / wet meat the toxin level have been carried out, at Rias de Vigo weight). and Pontevedra and Fisterra, all of them with positi- ve results for PSP and higher than the allowed limits For the observation of the gonadal development for its commercialization. and pathological analysis, it has been taken from each specimen a transversal cut of the conical The species is present all along the coastal strip of appendix, gill and mantle and was processed Galicia, with a higher incidence in Rias Bajas (South through histological techniques. For the biochemical of Galicia). No parasites from the Haplosporidium analysis, in each monthly sampling, the adductor type have been detected from the hystopathological muscle and the conical appendix of eight individuals analyses carried out in wild populations, which (four males and four females) have been freeze- during these years affected a great number of the dried after their ultra-freezing at -80°C.

CONSELLERÍA DO MAR Annual Report CIMA 2008 27 SCIENTIFIC ACTIVITY

Three wild populations have been studied (Illas condition index and the histological preparations Cies, Illa de Arousa and Fisterra). Only from the of the gonads show some individuals under matu- population of Illas Cies monthly and continuous rity phase in March-April, maximum of gonadal ple- information was obtained during three years (2006- nitude in June-July with partial spawning and a 2008). As depicted in the figure, a standard of progressive decrease until October when, between gonadal development is repeated during the three 50% and 60% of the individuals, have released years, with light variations possibly associated to most of the gametes, and the gonad is more redu- the environmental conditions. The GCI gonadal ced.

Figure. Evolution of the Gonadal Condition Index (GCI), in mean value ± SD in the populations of the Illes Cíes (Ria of Vigo).

The results indicate that the evolution of the repro- systems and fixation plates inside the larval tanks ductive cycle is similar and repeats itself in the three themselves. analyzed years. The gametes release starts in spring in some individuals, continues in summer and takes part The diet with weaning feed in specimens’ form 5 mostly in autumn. Individuals in proliferative phase can mm of size was effective as an alternative to the diet be observed in this time of the year. The gonadal cycle with phyto-colonized plates, with high survivals and and the reproductive activity evolve in parallel with the growths comparable to the ones obtained with the water temperature, being more intense during the classic diets. months in which it rises and less when it drops. The system of parent stocks conditioning with diffe- Culture.- A main aspect, which limits the industrial rent photo periods (darkness, normal photo period production of the perlemoen abalone seed, is the high and advanced photo period), at constant temperatu- mortality observed at the moment of the larvae re, allowed daily adequate intakes in the three tested attachment, and also the diet change that has to take conditions, being during the darkness photo periods place afterwards. In this sense, several research lines in which a higher feed consumption took place. have developed directed to improve the larval culture techniques as well as the handling and post larval diet The isolation and selection of native benthic dia- and the grow-out-fattening of the seed. toms allowed high indexes of phytocolonization and feed availability in the systems used for the larvae The handling of the larval cultures of Haliotis tuber- attachment. culata was carried out using directed open flow

28 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Reuse of wastewater of marine and auxiliary culture Guerra Díaz, A. establishments located in terrestrial area. Project coordinated by the AC of Galicia

START: 2006 RESEARCHERS: FINISH: 2009 CIMA: Guerrero Valero, S.; López Gómez, C.; Montes Pérez, J.; Nóvoa Vázquez, S. FINANCIAL ASSISTANCE: USC: Microbiology Dpt.: López Romalde, J. National Plan for development of Marine Cultures JACUMAR RESEARCH SUPPORT: CIMA: Meléndez Ramos, M.I.; Penas Pampín, E.; Fernández Besada, M.; Otero Otero, M.

INTERNS: Balsa Freire, S.; Ruíz Pérez, M.

SUMMARY This National Plan includes the following research AQUACULTURE CLUSTER. The seed grow-out results groups of the Autonomous Communities: Galicia, obtained from these locations are compared to the Asturias and Andalusia. ones performed in parallel trials carried out in a tra- ditional seed-farm (REMAGRO S.A.) in the Arousa The dumping of water into the sea with high con- Ria and close to both fish farms. centration of nutrients, coming from effluents of the in land farms of fish culture, is a matter that the Pathological and microbiological analysis of the aquaculture industry and the fishery administration grown-out seed in the different systems and feed itself try to manage adequately in order to produce sources. At the end of the grow-out period, or befo- the lowest impact in the marine environment. In re anomalous mortality events, samples are collec- order to “reduce” and reuse the organic matter con- ted in order to determine the presence of pathoge- tent of the effluents, two investigation lines are nic organisms and estimate the general histological opened: on one side to analyze the use of small condition, assessing possible differences inferred by bivalve mollusc seed as “biofilter” of small organic the grow-out conditions. Particularly, smear analysis particles (<20 µm), aspect that is assessed as an alter- are carried out for the identification of trematode native for the seed grow-out and that it is a molluscs parasites, thioglycollate techniques for Perkinsus culture phase that is not sufficiently developed, at and classical histology for the rest of the patholo- an industrial scale and efficiently in Galicia. In para- gies. In relation to the microbiological analysis, all llel, a second research line is developed that analy- the isolated corresponding to the initial samples zes the gross particulate that the effluent drags (time 0) and part of the isolated of the final samples along (more than 95% of the total), for farming (time 1) have been characterized from the different and/or forestry uses. locations. The bacteriological study showed an increase of the total number of bacteria in the three Seed grow-out, fish-farm effluent versus traditio- species of analyzed clam. As regards the number of nal seedbed. The grow-out trials are performed in vibrios it can be observed a similar dynamic to the two land fish-farms. The turbot farm, of the com- one of the total number of bacteria in general. pany INSUIÑA S.L., deriving by gravity a part of the effluent with pre-filtered water (with rotary filter Use of effluent mud in farm and industrial activi- <500 µm) to pools of 7 m2 adapted for such purpo- ties. For the analysis of the composition and possible se, also in the effluent of mixed farm of fishes of the usefulness of the effluent mud, as fertilizing ele-

CONSELLERÍA DO MAR Annual Report CIMA 2008 29 SCIENTIFIC ACTIVITY

ment in arboreal and forestry species, the mud, if a good maintenance and control of the faci- Edaphology Department receives collaboration lities is performed. The process of compost of this from the Forestry Technical Engineer School of mud is being performed at ECOCELTA S.A., company Pontevedra. The mud characterization shows that it dedicated to the elaboration of compost and worm is a waste with a very low dry material content; it humus. Initially, two types of complementary mate- shows a mean humidity content in the region of rial are being used; an experience with mud and 85%, this will condition the application techniques, sawdust of Pinus pinaster and another where the as well as its transportation. As regards to its chemi- mud is complemented with rests of fruit, sawdust cal properties, they present a moderate alkaline pH. and dung of chicken farm. The compost of this mud The mean content of organic material is high, in the has also been tried through worms, but it was not region of 33,8%. The relation C/N, slightly low, indi- feasible due to the low content of salts. In order to cates that the mineralization of organic matter sum up this section it has to be pointed out that in given by the muds, might be relatively fast, as it spite of the saline nature of this mud, it has been might be expected due to the origin of the organic considered as a whole as valid material to be used in carbon. The level of asimilable phosphorus is rele- tasks for the recovery of degraded soils. In order to vant, versus the levels of degraded or impoverished make this destination possible, the mud should be soils. It presents very low quantities of potential treated in order to reduce the humidity, to increase toxic materials, values. far from the maximum limits the C/N relation and to reduce its content of salts; all allowed by the European legislation for the applica- this is possible through a compost procedure with tion of residual mud to the soil. The concentration complementary material. of heavy metals has a trace character, which does not suppose any risk, but it plays an important role Results diffusion. The partial results of this project as micronutrient support. are collected in the communications submitted to the XI National Congress of Aquaculture and X and The presence of Salmonella and E. Coli is null, as it XI Forum of Marine Resources and Aquaculture of has been expected, given the low probability of the the Galician Rias. presence of pathogenic organisms in this sort of

30 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Culture of new species of bivalves molluscs of interest Martínez Patiño, D. in hatcheries. Project coordinated by the AC of Galicia

START: October, 2006 RESEARCHERS: FINISH: December, 2009 CIMA: Carballal Durán, M.J.; Cerviño Eiroa, A.; da Costa González, F.; García Fernández, A.; López FINANCIAL ASSISTANCE: Gómez, C.; Nóvoa Vázquez, S.; Ojea Martínez, J. National Plan for development of Marine Cultures IGAFA: Andrés Rivas, M.C.; Lastres Couto, M. JACUMAR RESEARCH SUPPORT: Álvarez Llamas, L.M.; Bouzamayor Yáñez, V.; Cotarelo Jardón, M.J.; Meléndez Ramos, M.I.; Páez Pérez, J.; Penas Pampín, E.; Ruíz Misioné, M.; A. Álvarez Padín.

INTERN: Louzán Pérez, A.

SUMMARY This National Program of Marine Cultures coordi- the gonadal maturity condition of the wedge shell: nated by the Autonomous Community of Galicia, in E0 of sexual rest, E1 of the gametogenic develop- collaboration with research groups of the ment starting, E2 advanced gametogenesis, E3: Autonomous Communities of Andalusia and maturity and spawning and E4 of post-spawning. Catalonia, has as objective the culture of new bival- The wedge shell shows a maturity period and ve mollusc species of commercial interest. The inves- sawing from March to August. A short rest period tigation lines are focused on the production of seeds can be observed in this species that takes place in of these bivalve molluscs in Farms. The Autonomous September. The evolution in the contents of car- Community of Galicia, at the Centre of Marine bohydrates keeps an inverse relation with the game- Cultures of Ribadeo (CIMA): performs the Sub-pro- togenic cycle, it falls as it advances. As the carbohy- ject I: “Wedge shell culture (Donax trunculus) and drates fall, the lipid component increases. The pro- sword razor shell (Solen marginatus)”. teins do not show variations that might be related directly to the cycle. The following research lines have been developed during 2008: Spawning has been observed between the months of March and June. Growth and survival controls Culture of wedge shell: Since the year 2006, have been carried out during the larval and postlar- monthly samplings have been performed at the val development. The individuals reach the attach- Valdoviño bed (Ria de Cedeira). In 2008 they are ment with a size of 270 µm after 27 days of culture; continued in order to complete two years of study. the larval survival was of 10%. The postlarvaes are The objective is to know about the gametogenic cultured in cylinders of 150 µm of mesh. After five cycle of the species and its culture in farm for the months of farm culture, the wedge shells move to obtaining of the seed with repopulation aims. A the natural environment in containers without subs- gonadal histological study has been carried out as trates separated by sizes 5.3 mm and 2.98 mm and well as analysis of the majority biochemical compo- distributed in batches of known density. The survival sition of gonads and the rest of the individuals’ from attachment to grow-out in the natural envi- body. Five stages have been described as regards to ronment is of 37%.In December, with a survival of

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65%, the final sizes that have been obtained are of acids. The big size of the ovocytes of the S. 6.3 mm and 3 mm after nine months of culture in Marginatus (150 µm) allows storing a great quantity the external environment during winter. The culture of neutral lipids (reserve), making possible the experience will continue during the next year until attachment achievement in 8-9 days. During the the seed reaches the susceptible size for re-culture in embryogenesis, a drop in the reserve lipids can be the natural environment. observed. From the larvae D up to the pediveliger a mixotrophic phase is present in which the larvae’s A hystopathological study is conducted in the keep on using the reserves coming from the ovocy- same natural bed in each season in order to detect tes and feeding themselves externally at the same the presence of parasites and evaluate the possible time. pathological alterations. A histological cut of 5 µm was done from each wedge shell that included all Experiences are being carried out with the seed of the organs and it was stained with hematoxylin and the S. marginatus with systems suspended in the eosin for its observation at the optical microscope. pontoon of the port of Ribadeo (Lugo). PVC tubes of The results indicated that the wedge shells were 30 cm of diameter, perforated and covered with a parasited by gregarines, rickettsias, ciliates and mesh of 1 mm are used. Seed of 5 mm have been metacercarias of trematoda. The infection intensity arranged using two densities (low and high) and is low in all the performed samplings. two substrates (fine and coarse sand). The highest survival corresponds to the fine sand, regardless of Culture of the European razor clam: The survival the density. The growths reached up to present are of the bivalves if very much influenced by the stored similar among the different treatments. reserves that can be used in the embryogenesis and attachment. For this, the biochemical evolution of A hystopathological study of the S. marginatus most of the components has been studied all along has been conducted in two sampling points: Cabo the larval development of the Solen marginatus in de Cruz (Arousa Ria) and Redondela (Vigo Ria). The two spawning in duplicate. The ovocytes show high most important pathologies that have been detec- protein content and also an important load of lipids. ted are: Marteilia spp and organisms of the rickett- During the embryo development, the proteins are sian type in digestive glands, besides a possible dis- used from the larvae D up to the attachment ener- seminated neoplasia. getic reserves are accumulated indistinctly in order to overcome the metamorphosis. The Marteilia spp, appears in the bed of Cabo de Cruz. The presence of plasmoids type Moreover, following these larval developments, Haplosporidium spp can be observed in the bed of samples have been collected for the analysis of fat Vigo Ria.

32 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Towards an integrated management of Ensis stocks Martínez Patiño, D. (TIMES). Project coordinated by David Roberts (Northern Ireland). START: March, 2007 FINISH: June, 2008 RESEARCHERS: da Costa González, F.; Nóvoa Vázquez, S.; Ojea FINANCIAL ASSISTANCE: Martínez, J. Unión Europea (Interreg IIIB: Programa Transnacional de la Zona Atlántica) RESEARCH SUPPORT: Álvarez Llamas, L.M.; Bouzamayor Yáñez, V.; Cotarelo Jardón, M.J.; Páez Pérez, J.; Ruíz Misioné, M.

SUMMARY The reduction of the razor clam and sword-razor was moderately higher in the case of the seed kept shell farms represents a reduction in an important in substrate, but the survival was remarkably higher economical activity in different regions of the in the case of the seed kept without substrate. Atlantic area. In this context, the SHARE project arose continuing with the TIMES project, which The effect of the different diets in the growth and focused on the environmental impact of the fishing survival of the recently attached postlarvaes has techniques of the razor clams and sword-razor been proved with Ensis arcuatus. The diets that have clams, on the development of the culture techni- been tried are as follows: diet 1: I. galbana, P. luthe- ques and on molecular markers for the assessment ri, C. calcitrans and T. suecica, diet 2: C. calcitrans; of the genetic variability of the different stocks. diet 3: I. galbana and C. calcitrans and diet 4: fasting With the purpose to reply to these demands, a study during 4 days and then they had been fed with the is being developed whose aim is to determine the diet 1. The survival of the postlarvaes was low in all most adequate methods for the obtaining of the the cases. farm spats. A postlarvaes diet test has been performed with In this project, a scanning study of the Ensis siliqua this species with three different portions (1, 2 and has been performed about the embryology and lar- 3% of dry weight) using a mixture of I. galbana, P. val development at the optical and electronic lutheri, C. calcitrans and T. suecica. It starts with pos- microscope. The trochophore phase of this species is tlarvaes of 1.5 mm of length, reaching 3.8% at the reached after 12 hours of post-fertilization (hpf). end of the experience (after 15 days) in the portion Aftr 24 hpf the larvae D veliger presents a totally of 3%, which was the one that proved the highest developed digestive apparatus. The recent attached growth. In the portions of 2 and 3%, the highest sur- postlarvaes have a size of 427 µm after 14 days of vivals are achieved, both close to 100% in both culture. cases.

The spats phase has also been studied with the An experience with different diets has been two species. With the seed E. siliqua, the maintenan- carried out with E. arcuatus seed of 40 days: diet 1: ce without substrate and with a fine grain substrate Tetraselmis suecica and Skeletonema costatum; 2: has been proved. For this, lots of 38.000 spat indivi- Isochrysis galbana, Pavolva lutheri and T. suecica; 3: duals of 2.5 mm of length have been placed in I. galbana, P. lutheri, S. costantum and T. suecica; all duplicate in a raft of 1.000 L of volume and were of them with the same initial portion of 120 cells kept there during 15 days. The growth of the seed equivalent per µL. Better growths have been obser-

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ved with the diets 2 and 3, though the survival was with optimal conditions where the resource disap- of 100% in the three tested diets. peared.

Different experiences have been carried out in the Besides, with E. arcuatus seed of 11 mm of length, natural environment until the seed reaches the com- tests have been performed in containers hanging in mercial size. Thus, in collaboration with the the pontoon of the port of O Vicedo (Lugo). The Fihsermen´s Confraternity “San Cipriano” of Aldán- system consists of perforated bottles of 5 L of volu- Hio (Ria de Vigo) the culture of 9.600 units of E. sili- me, covered with a mesh and full of substrate. The qua seed of 15 mm of length and of 37.200 units of seed was kept with two sorts of sand (fine and coar- E. arcuatus of 8 mm, has been performed, both in se) and at two densities (low and high) in duplicate. subtidal. Moreover, in collaboration with the confra- The experience lasted 1 month, with similar survivals ternity, different grow-out systems in E. arcuatus among systems and always higher than 86%. have been proved. For such purpose, it has been arranged the seed of 13 mm of length in duplicate Also in the case of the E. siliqua, experiences are in two different containers (oyster baskets with a being conducted with hang systems in the pontoon fine mesh in order to retain the sand and trays) and of the Ribadeo port. The containers have been two sorts of substrates (fine and coarse sand). improved in order to facilitate its filling, cleaning and periodical sampling. These are PVC tubes of 30 In collaboration with the Fishermen’s cm of diameter; perforated and covered with a Confraternity “San Pedro” of Ribeira (Ria de mesh of 1 mm. Seeds of 10 mm have been placed, Arousa), 10.500 units of E. arcuatus seed of 15 mm using two densities and two substrates as in the pre- of length have been cultured in a limited area of a vious experience. The seed is controlled monthly in natural bed without mesh cover. During this expe- order to obtain growth and survival results. The best rience, the growth and survival will be controlled in results have been obtained using fine sand and low a zone of razor clam natural production, therefore, density.

34 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: ALBA: Improvement of the pullet carpet shell clam Martínez Patiño, D. Venerupis pullastra (Montagu, 1803). RESEARCHERS: START: December, 2006 Cerviño Otero, A.; da Costa González, F.; Nóvoa FINISH: December, 2009 Vázquez, S.; Ojea Martínez, J.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: Program of Marine Resources Álvarez Llamas, L.M.; Bouzamayor Yáñez, M.V.; Dirección Xeral de Investigación, Desenvolvemento Cotarelo Jardón, M.J.; Páez Pérez, F.J.; Ruíz Misioné, e Innovación M. Consellería de Innovación e Industria PGIDITO6RMA50801PR INTERN: Brión Bello, R.

SUMMARY This is a project coordinated between the Marine The control of the seed produced at the hat- Research Center of Ribadeo (CIMA) and the Genetics chery was continued in the grow-out phase in the Department of the University of A Coruña. It compri- natural environment, taking samples in order to ses two subprojects aimed to improve the production carry out the biochemical analyses and the histolo- of the pullet carpet shell clam (Venerupis pullastra, gical follow-up of the first reproductive maturity; Montagu, 1803), an autochthonous species of great likewise, the growth and mortality control was commercial interest. This species has a wide maturity done. and spawning period, without showing a defined rest period, which means that spawning in the hatchery, From the twelve lots of stock parents, 8 spaw- can be obtained all year round. However, these are nings were obtained. The mean size of the ooctyes different in the different seasons of the year and was of 73 µm, ranging the quantity of ooctyes per according to the origin of the parent stocks. spawning between 13 and 2.5 millions. After two days, the obtained larvae D, reached a size in the During the year 2007, parent stock coming from region of 100-105 µm. The duration of the larval the natural beds of Galicia: Barallobre, Vilaxoán and culture was relatively fast, after 14 days the appe- Cangas have been collected; and during 2008 parent arance of the foot in the first larvae could be stocks have been collected from other three beds: O observed and after 18 days the larvae reached the Barqueiro, Camariñas and O Grove. The samplings attachment with a mean size between 210-230 were performed in each season of the year (spring, µm. After 40 days the post larvae reached already summer, autumn and winter). At its arrival at the the sieve of 400 µm with an approximate size of Center, its gametogenic condition has been assessed 600 µm. through the conventional histological techniques and its reserves with biochemical analytics (carbohy- The subproject that is carried out by the rese- drates, lipids and proteins) and fat acids. When the arch group of CIMA at Ribadeo, is focused on stu- spawning took place, samples of the ooctyes have dies aimed at the improvement of the intensive been taken in order to obtain the data about size, farm culture, to procure an optimum quality and dry weight and quantity, as well as for its later an appropriate quantity of spat. Taking into analytics of biochemical composition. During the lar- account that the rational management of a val development, growth and survival controls were resource requires knowing the genetic variability performed, besides controlling several factors as degree and the populations structure of the the supplied diet or the culture temperature. parent stock beds, samples of these parent stocks

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used at the hatchery have been delivered to the 2009, and when they reach the size for the perfor- Genetics Department of the UDC, with the aim of mance of the study, a sample of the spat obtained in assessing the genetic variability present in the these spawnings will be delivered. The last aim is to natural beds of the Galician coast. study if there are differences between the genetic variability of the parent stocks and the seed that The mentioned department will also perform a gave origin. complementary project that will provide genetic cri- teria to contribute to the sustainable harvesting of During the next year, the analytics will conclude the Venerupis pullastra and to the rational transfer and the data treatment will be undertaken, as well of the hatchery-reared spat stocks. During 2008, as the study of the sexual maturing of the seed lots parent stock lots have been delivered from which derived from the spawning obtained during this spawnings have been obtained and during this year year will be performed.

36 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Optimización del cultivo intensivo de la almeja e iden- Martínez Patiño, D. tificación de marcadores genéticos para el seguimiento Project coordinated by the AC of Galicia de las repoblaciones. RESEARCHERS: START: June, 2008 CIMA: Cerviño Otero, A; da Costa González, F; FINISH: June, 2010 Nóvoa Vázquez, S; Ojea Martínez, J., Cerviño Eiroa, A. FINANCIAL ASSISTANCE: National Plan for development of Marine Cultures RESEARCH SUPPORT: JACUMAR CIMA: Álvarez Llamas, L.M.; Bouzamayor Yáñez, M.V.; Cotarelo Jardón, M.J.; Páez Pérez, F.J.; Ruíz Misioné, M.

INTERN: Brión Bello, R.; Costa Costa, D.

SUMMARY The Autonomous Communities of Galicia, • Identification of the microbiota associated to Andalusia, Asturias, Cantabria and Catalonia take the mortalities present during the different part in this National Plan. The optimization of the phases of the hatchery culture. shell clam culture is set out; investigation lines will be developed aimed to clear up the aspects that The project started by the middle of the year there- block the spat production in the hatcheries, espe- fore preliminary experiences have been undertaken cially the grooved carpet shell clam (Tapes decussa- with the aim of determining a work protocol to tes). Different aspects will be tackled as: the selec- follow during the duration of the project. Parent tion of parent stocks in order to achieve a good qua- stocks of two shell clam species: grooved carpet shell lity of the spawning; the relation of the biochemical clam and short necked clam have been brought from compositions and fat acids of the oocytes obtained the natural environment during June and from the in these spawnings and the good larval develop- same origin (Palmeira, Arousa Ria). No fitting-out was ments, the search of the diets to grant good results carried out as they arrived already matured from the in the seed production and finally, the monitoring natural environment. The biometric data were obtai- of the flora associated to the different culture pha- ned for the estimation of the gonadal condition ses at the hatchery, preferably in the larval culture index and a histological and biochemical test has and in the metamorphosis phase, in which impor- been done of the parent stocks at their arrival at the tant mortalities take place. All this aspects are of hatchery. Four spontaneous spawnings have been maximum interest in order to obtain a better quality obtained from the short necked clam while only one seed and in enough amounts to satisfy the market. from the grooved carpet shell clam. The larval survi- val of the grooved carpet shell clam spawning was of The objectives that are pointed out with the per- 5% and variable survivals between 1.5 and 43% have formance of the project are as follow. been obtained from the short necked clam; the survi- val increased from the first to the last spawning. a)Optimization of the intensive culture of the Samples of the ovules were collected or the larvae for shell clam in the hatchery the biochemical composition study in fat acids as the spawnings quality criterion. • Determination of the spawnings quality crite- ria. A diet test has been undertaken with one of the • Diet tests in the larval cultures. spawnings of the short necked clam. Two diets have

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been tested: one with Isochrysis, Phaeodactylum and b) Follow-up of the repopulations and incidence Tetraselmis, and the other with Monochrysis, in the natural environment. Skeletonema and Tetraselmis. Both diets were also tried with and without antibiotics. The larval survi- • Identification of genetic markers in the shell val values have been appropriate in all the cases (33- clam. 51%), having more influence the use or not of the • Genetic characterization of the natural beds. antibiotic than the diet itself. • Comparison of the genetic variability of the natural bed with the seed obtained from A microbiological control protocol has been deter- parent stock from the natural environment. mined adapted to the farm itself. Water samples have been taken in the different circuits (parent During this year, the performed work was focused stocks, larval, seed and greenhouse), as well as sam- on the foreseen tasks for the achievement of the ples from the phytoplankton, from the parent stocks first objective: identification of the micro-satellite of from the larval developments. The samples are genetic markers, by means of the construction of cultured in Agar Marino and TCBS. At the arrival of gene library enriched in micro-satellite sequences the parent stocks, they present a high bacterial load and the use of primers to set out the other objecti- and after a month this load reduces itself both in the ves. grooved carpet shell clam and in the short necked clam. The oxidative bacteria predominate in the lar- The developed work led to the identification of 10 val cultures of the grooved carpet shell clam with micro-satellite polymorphic loci with a number of antibiotics, while in the cultures without antibiotic alleles between 3 and 11, which might allow under- the fermentative ones predominate. Vibrios associa- taking the foreseen genetic analyses in the other ted to the larvae can be observed in the short nec- objectives. Besides other 11 loci amplify satisfacto- ked clam, increasing in general as the attachment rily by CRP. gets closer.

38 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Culture and management of the sea urchin Subproject I A: Ojea Martínez, J. (Paracentrotus lividus, Lamarck, 1816) Project coordinated by the AC of Galicia Subproject I A: Determination of the reproductive cycle Subproject I B: Rey Méndez, M. SCIENTIFIC ACTIVITY and biochemical composition of gonads of the sea urchin Paracentrotus lividus, Lamarck, 1816, in natural popula- RESEARCH COORDINATOR: tions of Galicia. Culture of the sea urchin in laboratory: CPAM: Catoira Gómez, J.L. production of juveniles for repopulation. Subproject I B: Genetic characterization of the sea RESEARCHERS: urchin Paracentrotus lividus, Lamarck, 1816, in natural CIMA: Martínez Patiño, D.; Nóvoa Vázquez, S. populations of Galicia, Asturias and the Canary Islands. Experience of juvenile cultures in cages in rafts. . RESEARCH SUPPORT: USC: Duarte Hernández, A. START: January, 2006 FINISH: December, 2009 INTERNS: Costa Costa, D.; Brión Bello, R. FINANCIAL ASSISTANCE: National Plan for development of Marine Cultures JACUMAR

SUMMARY Several Autonomous Communities take part in 61.97 mm and 86.87 gr for Fisterre. The highest this project: Asturias, Canary Islands and Galicia. The values were obtained in Reinante with a mean of termination date is scheduled to take place on 66.78 mm of size and 109.83 gr of weight followed December 2008, but a year extension has been by Cedeira with 63.60 mm and 96.15 gr. Several requested that has been accepted, therefore it will gonadal condition indexes are estimated with the end in December 2009. It is a project coordinated by biometric data; all of them relate the gonad fresh Galicia and two subprojects are carried out in this weight with other parameters as: the total fresh Community. In one of them (Subproject 1 A), the weight, the shell weight and the size of the sea Principal Investigator is of CIMA. urchins. The obtained values are different according to the used parameter, but the evolution all along Subproject I A. During this year, the second year of the year is the same for the three of them. The mean monthly samples of the sea urchin natural popula- values obtained for the GCI (gonad fresh tions have been completed in four points of Galicia; weight/total fresh weight *100) during these two two of them in the Cantabrian area (Cedeira and years of sampling were of 6.36, 6.11, 7.97 and 9.8 for Reinante) and two others in the Atlantic area the urchins populations of Aguiño, Fisterre, (Aguiño and Fisterre). The gametogenic cycle study Reinante and Cedeira, respectively. The histological is completed during two years. The obtained biome- cuts of the gonad have been performed in the tric data (diameter, height, total fresh weight, fresh monthly samplings of the four studied populations. gonad weight, etc.) follow the same guideline than These preparations have still to be studied in order those obtained in 2007, being the Cantabrian popu- to determine the different stated of the gametoge- lations the ones that present the highest values both nic cycle. The biochemical analyses of the gonads in size of the urchins and fresh weight. In Aguiño have been carried out corresponding to the year and Fisterre, the size and the mean weight of the 2007, the mean annual values of the sugars range sea urchins in these two years of samplings is in the between 12.98% of Cedeira and 17.14 of Fisterre. region on 61.75 mm and 82.54 gr for Aguiño and For the proteins, the mean annual values range bet-

CONSELLERÍA DO MAR Annual Report CIMA 2008 39 SCIENTIFIC ACTIVITY

ween 28.14% for Reinante and 32.04% for Cedeira. 14.28 mm and a total fresh weight of 1.36 gr) so as The lipids present lower variations between 13.22 of to grow-out in rafts (3.400 urchins with a mean size Reinante and 15.57 of Fisterre. of 7.25 mm and a total fresh weight of 0.21 gr).

All along the year, the culture of the urchins with Subproject I B. Grow-out tests of juveniles have parent stocks from the different sampling points has been carried out, ones extracted from the natural been carried out continuously, obtaining the larvae environment and others from the Center of Marine both with spontaneous spawnings and performing Cultures of Ribadeo. Diets with different types of the induction of the spawning through injection of algae and fodders have been tried and with diffe- potassium chloride. The mean number of ovules per rent densities of urchins; the diet is “ad libitum”. female that have been obtained ranged between a minimum of 135.000 in October up to maximums of The grow-out of urchin gonads of commercial size 8 millions in spawnings of May and 6 millions of was performed; three types of diets are given: one spawnings at the end of July. The larval survival fodder of own design with 20% of protein, another varies a lot depending overall on the spawning sea- fodder with a 30% of protein and a third group with son of the year; it is low (between 2 and 6%) during a mixture of macrophytes Ulva spp and Laminaria the months of February, March and April to increase spp. towards the summer with maximums ranging bet- ween 30 and 57% in May, June and July. The population structure is studied through the use of microsatellites and molecular markers of During this year, the juveniles used were produced urchins from several geographic points of the at the hatchery both for repopulation in the natural Canary and Peninsular Islands. environment (2.736 sea urchins with a mean size of

40 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Integrated Aquaculture: Experimental experience for Guerrero Valero, S. the development of multitrophic culture system. Project coordinated with the AC of Andalucía

START: January, 2007 RESEARCHERS: FINISH: December, 2011 UC: Cremades Ugarte, J. INTECMAR Pazos González, Y.; Doval González, FINANCIAL ASSISTANCE: Mª.D. National Plan for development of Marine Cultures JACUMAR RESEARCH SUPPORT: UC: Ocaña Falcón, S. UC: Andrade García, M.

INTERN: CIMA: Ancosmede Garduño, C.

SUMMARY The Autonomous Communities of Andalusia, out both in lower volumes (flasks of 6 liters) and hig- Balearic Islands, Catalonia, Canary Islands, Galicia her volumes (tanks of 500 liters). They have a physi- and Murcia take part in this National Plan. cal limit in the flask marked by the size, therefore, when necessary, they have to be passed to higher The principal objectives of this type of cultures volumes. are, on one side the economic one producing more diversification in the production at the same loca- In land experience in closed circuit: The incorpora- tion, and on the other side the environmental tion of a diet macro-algae culture (Saccharina latissi- improvement as the algae act as a biofilter impro- ma) to a production circuit of turbot and sole. This ving the quality of the water after passing through previous experience has been performed in the faci- the principal culture, either of fishes or molluscs. lities of the collaborating company AQUACRIA AROUSA S.L., which works with turbot During the first year, the different experiments (Scophthalmus maximus) and experimentally with have been designed and developed and some pre- sole (both Solea solea and S. selegalensis). The water vious data have been obtained (mainly of the envi- treatment includes the mechanical filtration, biolo- ronmental conditions) that will be useful for the tre- gical filtration, the gas control, the oxygenation and atment of the data to be obtained in the future. The the sterilization (ozone and UV). The plants of three set out experiences try to contemplate all the Saccharina latissima used in this experience have possible forms of marine culture integration and, been produced on the Plant of Algae Cultures of IEO undoubtedly, they are of great interest for the sec- at Santander, using its germplasm bank and through tor as they open new possibilities to the diversifica- the free-living technique. In the future, these see- tion and optimization of the aquaculture produc- dlings will be developed till the adequate size to be tion, while they can mitigate the possible environ- introduced in the system through culture in suspen- mental impact of it mostly. The cultured species sion in the facilities of CIMA. have been selected within those that are of com- mercial interest for Galicia and that can be used In land experience in open circuit: The biofiltra- both in the direct food industry and for the extrac- tion of the effluent solids of a turbot culture plant tion of phycocoloids (especially agar-agar). The in open circuit with suspensivore invertebrates indoor culture of the different species was carried (anemone), filtration molluscs (clams), and food

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macro-algae. This experience is being performed open circuit where the water is oxygenated by in the facilities of the collaborating company means of a venturi. Its current production is of 300 PUNTA MOREIRAS S.L., which works with turbot Tn. (Scophthalmus maximus) and its functioning is in

Figure. Scheme of the integrated culture disposition in open circuit.

The system used for the algae culture (see ted to mussel rafts (see figure) and cages of fish Figure) in a first area includes a part for the cultu- culture (turbot). This experience is being perfor- re of the sedimentivore invertebrates (Anemonia med, on one side, in the administrative concession viridis) and a system for the culture of the filtration of experimental nature for the development of the invertebrate consisting in several grow-out drums marine macro-algae cultures, which the collabora- of grooved carpet shell (Tapes decussate) and short ting company of canned food PORT-MUIÑOS is the necked clam (Ruditapes philippinarum). The beneficiary, located at the marine culture Polygon second area consists of a culture of assimilator Sada II of the Ares and Betanzos Rias (A Coruña) algae in semi open circuit similar to the already and in the cages for the fish culture property of existing at the CIMA, but of a higher escalation, the collaborating company MARCULTURA S.A., which consists of a unit of three tanks of 2000 one which is located at Somorto, Esteiro, belonging to for the culture of the algaes in suspension. The Muros and Noia Ria (A Coruña). The starting hypo- intention is to mainly cultivate Sccharina latissima thesis is that the integration of these cultures will during winter and spring and Codium spp, during entail as benefits the improvement of the water summer and autumn. quality for the reduction of the eutrophization and

figures of dissolved CO2 and the increase and diver- Sea experience: Culture at open sea of the food sification of the aquaculture production of both algae Saccharina latissima and Codium spp, associa- polygons.

42 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Proposals and imporvements for the design and con- CIMA: Mariño Balsa, J.C. trol of the restocking plans and evaluation of the Coordinated by the CA of Galicia impact of escapes of fish and crustaceans.. RESEARCHERS: START: 2008 IGAFA: Pérez Benavente, G. FINISH: 2010 USC: Bouza Fernández, C. IFAPA: Muñoz Pérez, J.L. FINANCIAL ASSISTANCE: IRTA: Rotllant Estelrich, G. National Plan for development of Marine Cultures LIMIA: Pastor Gracia, E. JACUMAR ICCM: González Henríquez, Mª.N.

RESEARCH SUPPORT: Cid González, E.; Fernández Franco, B.; Graña Pereira, M.I.

SUMMARY This National Plan JACUMAR is coordinated from The production of the European lobster has been Galicia and the following Autonomous Communities carried out according to the foreseen objectives that take part: Andalusia, Catalonia, Balearic and Canary proposed 3.000 juveniles of restocking size (total Islands. length: 5-6 cm). Three larval cultures were conducted, the number of produced specimens were of more The main objective is to determine the basis on than 5.000. Approximately half of them were relea- which to design the restocking plans of marine spe- sed in the sea and the remaining ones are in grow- cies, through the study and definition of the zone, out phase up to release size in the raft of IGAFA. habitats and most adequate season of the year for the release, better marker strategy and re-catch, As regards to the turbot, the production was limi- adaptation of the specimens before the release and ted to the obtaining of the spawning, as the parent to detect, assess and mitigate the effect that we stock that we fitted out for a year did not reach the could define as involuntary restocking or escapes gonadal maturation in captivity. Only one female associated to the aquaculture. allowed us to carry out a larval culture, with eggs of doubtful quality that showed survival and pigmenta- For the compliance of these objectives, each tion results that were not too interesting. Autonomous Community carries out a subproject with those species more relevant for their interest, 2. Identification of the parent stock of the turbot so that the project has a global vision that intends to and genealogical traceability obtain results in the future marine restocking of fish and crustaceans. During this first year of project, this task was not performed because, as commented in the previous In the Autonomous Community of Galicia, the section, the parent stock did not reach the matura- following tasks are performed: tion in captivity; therefore it was decided to carry out this objective during the following years, when we 1. Obtaining of the juveniles for the release will be able to have a real stock of effective parent stocks. During the year 2008, larval cultures have been per- formed of two selected species, the turbot (Psetta maxi- The principal objective of this task is to assess the ma) and the European lobster (Homarus gammarus). usefulness of the genetic marker for the restocking

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fish follow-up. In this sense, non-invasive sampling 3. Assessment of the marker systems. methods are applied that avoid the sacrifice of parent stock and facilitate the re-catch samplings of released The type of used marks in a restocking activity is a progenies (fish market, fishing vessels, etc.). very important factor for the projects final success, as we will obtain most of the results from the recovery From the individual multilocus genotypes, genetic and identification of the re-caught specimens. diversity analyses will be performed in the stock of parent stocks using appropriate computing packages In the case of the European lobster, the magnetic and will be assessed comparatively as regards to the micro-labels seem to be the most effective marking references available in the wild natural stockings of system, due to the difficulty that this organisms offer this specie. to other type of marks, due to the shedding process.

With genetic criteria, a first selection, organization There are different marks in the turbot that will be and design of the crossings (maximum diversity, mini- borne out during the project discourse. Plastic labels mum relationship, considering the available diversity named “T-bar” have been used to present, of diffe- sources in nature) will be performed. Computing pro- rent sizes and colors, without observing any differen- grams will also be used in order to identify the ce in the loss or re-catch percentage. The tattoo mark parent-filial relationships as well as brotherly groups has also been used, by means of the Alcian Blue pig- (complete brothers and half brothers), as well as esti- ment injection in the ventral part of the fish, without mations of the relationship among all the pairs of assessing the results up to now. individuals of the considered stocks. In 2009, we will carry out experiences with other marking systems, the elastomers and the burn mark.

44 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Optimization of the octopus (Octopus vulgaris) grow- Guerrero Valero, S. out. Compound feed development (Galicia). Project coordinated by the AC of Murcia

START: January, 2007 INVESTIGADORES : FINISH: December, 2009 USC: Pascual López C.; Quinteiro, J.; Rey Méndez, M.; Rodríguez, C.J.; Seixas, P. FINANCIAL ASSISTANCE: National Plan for development of Marine Cultures RESEARCH SUPPORT: JACUMAR UC: Ocaña Falcón, S.

INTERN: CIMA: Ancosmede Garduño, C.

SUMMARY The rock octopus (Octopus vulgaris) is a species that that would be destroyed during the elaboration of has a great potential in the marine aquaculture, due fish meal. The ensilage takes advantage, under to its biological characteristics and high commercial controlled pH conditions and temperature, the value. However, the industrial development of the enzymatic activity of proteases and lipases of the octopus is limited at present due to the impossibility fish itself, facilitating the protein denaturalization, of obtaining hatchery juveniles for the grow-out limiting the unwished survival and microbiological phase, as well as due to the absence of an adequate activity. These ensilages and fermented fish pro- artificial fodder. ducts facilitate the digestion and absorption of nutrients and are set out as an alternative and fea- The first problem that we face in order to design sible technology for the development of new diets a fodder for the octopus is the adequate “presenta- for the octopus. An adequate structure should be tion” to be accepted and consumed completely. The given to the paste obtained with the ensilages in granulated fodders, extruded and half-wet are not order to be accepted by the octopus; therefore valid for the octopus as it breaks them up before hydrocolloids will be used as well the addition of consuming them; sometimes the octopus cuts the attracting substances. food up, swallows it, but it also makes an external pre-digestion sucking the juice. On the other hand, The primers developed with fresh paste of the raw the food is not always consumed immediately; the- materials present a lower humidity rate as regards to refore it has to be stable during long periods in the the raw materials, explained by the contribution of water. An alternative is to use the “wastes” or infra- the hydrocolloids; however, the batches where at least assessed resources of the industries that transform 50% of iris is contributed, its protein rate is quite simi- the fishing products, wastes that, sometimes, do not lar to the raw material, as the lipids. The primers have a good presentation, but however present an where the iris contribution is lower than 50% or null, adequate nutritive and hygienic-sanitary characte- the protein rate is the lowest, keeping the lipid one ristic. Thus, taking advantage of these wastes, still and of carbohydrates, but remarkably presents a hig- with a high nutritional value, several wet diets can her rate of ashes (contributed mainly by the crab be elaborated through the known ensilage techni- shell). que. This technique favors the action of the acido- philic enzymes of the wastes, releasing the nutrients Stability test of the fodder in water: The tests have in its easiest structure as fat acids and amino acids. been positive in most of the designed batches and On the other hand, it allows the presence of certain ensilages. With the rest, the result was negative as it thermolabile nutrients (omega 3 fatty acids, lysine) pulverizes with the water contact.

CONSELLERÍA DO MAR Annual Report CIMA 2008 45 SCIENTIFIC ACTIVITY

Figure.- Elaboration of artificial primers from fish paste and ensilage.

Acceptability test: The acceptance was only visual in mens, as they show a higher growth rate and no evi- two ensilages, in which they were caught and rejected dence has been observed of major aggressiveness immediately, however, this has been sub-optimal in among them (survival similar than the mixed group). part of the batches that they capture, they try to ingest and finally break up. Based on said results, the Genetic characterization of the wild stockings: It grow-out experiences have been performed with has been carried out from the evaluation of the pri- three primers obtaining a survival of 100% in both. mers sequences of the microsatellites and performing the comparison among the samples of octopus eggs Growth test: With the primers made up of iris and and the respective analyzed parents. The amplifica- mussel at 50%, only a slight growth is obtained in the tion conditions of the microsatellite sequences have female batch. Comparing natural diet and primers, been tried varying the parameters of the PCR, as the difference in mean weight reduction between the regards to the original conditions described until primers could be related with the increase in percen- achieving clean and repetitive amplification patterns tage as regards to the crab of 30% to 50% favoring a of each of the alleles. Up to now adjusted conditions better use and growth, however, to present the used are available for the analysis in two of the primers sets primers, in spite of the fact that they have been accep- (µOct/3F, µOct/3R and µOv12-F / µOv12-R). In the rest ted by the octopus, did not result to have enough of the sets, the conditions that ensure its efficacy for a nutritional value nor the adequate texture for an opti- majority of individuals to be genotyped could not be mal growth of the octopus. determined. In these last ones, the difficulties that appeared are the absence of amplification and unspe- Grow-out trials: Though it has been proved that the cific amplifications. The presence of unspecific strips males grow more than the females, the separation of has been confirmed previously by the isolated MS genders in the current condition of the octopus grow- from the µOct library. This fact is more critical with the out activity does not suggest a clear advantage, as this marking with fluorochromes and the resolution in an would entail a major work effort when sexing them. automatic sequencer. In this way, the acceptable In the future, and as long as the octopus life cycle gets amplifications dissolved in agarose or acrylamide gels closed in a profitable manner, it would be interesting with stain of ethidium bromide, are not treatable in to assess the possibility of obtaining only male speci- an automatic genotype.

46 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Prevention and protection study techniques of the De Coo Martín, A. principal predator species of the natural shoals bivalve mollusc resources and culture areas at the Galician RESEARCHERS: coast and estuaries. CIMA: García Fernández, A.; Santos Piñeiro, I. Guild of Vilaxoán: Alcalde Creo, A. START: 2007 Guild of Carril: Conde Varela, M.L. FINISH: 2009 Guild of Ribeira: García Galdo, J. Guild of Illa: Poza Chaves, G. FINANCIAL ASSISTANCE: Guild of Noia: Solís Pais, L.Mª. Consellería de Pesca e Asuntos Marítimos Aquarium Galicia: Crespo Lorenzo, J.A.; Fernández Xunta de Galicia Otero, D. PGIDIT-CIMA 07/03 RESEARCH SUPPORT: Fariña Iglesias, J.

SUMMARY The program counts with the collaboration of the present as well as the availability of a specific food, it guilds of Riveira, Illa da Arousa and OPP20, Vilaxoán, O results quite striking the capacity of distinguishing Carril and Noia and the ACQUARIUM of GALICIA. (without breaking the shells nor ingest the bivalve spe- During the year 2008, the following advances have cimens) among species. Likewise, but not in a first been recorded. phase, the predatory capacity of the triggerfish or pig- fish (Balistes carlinensis) or gilthead seabream (Sparus Knowledge of the diet guidelines of the predatory aurat) started to be documented. Likewise, the preda- species on bivalves: In tests performed at the tor practices have been proved in several types of per- Aquarium of Galicia, in O Grove, it could be proved the forator dogwhelks or none (Nucela lapillus, Ocenebra hunger of the Common Eagle Ray (Myliobatis aquila) erinaceus, Natica alderi, Murez brandaris and truncu- because of the consumption of bivalves; their capacity lus, Nassarius (Hinia) reticulates, Cyclope neritea. This to choose the type of species to be consumed and the last invasive species and not of Galicia, of crabs cavities and remains of shells that they leave in the (Carcinus maenas, Liacarcinus corrugatus, Atelecyclus substrate during the feeding, cavities and remains that rotundatus) and stars (Asteria rubens, Marthasterias match with the ones found during the inspections of glacialis). Both the species of crabs and stars present a beaches and intertidal zones, frequented by common high predation activity, and given the abundance of eagle ray of Illa e Enseada of O Grove, in the several Nassarious and Cyclope and its aggressiveness level (in departures to the sea carried out by the team. In the spite of not being perforator species) it can be conclu- Acquarium of Galicia, tests have been performed with ded, also, that these two species constitute a serious sand boxes where only one commercial bivalve species problem in the commercial bivalve stockings. has been deposited (grooved carpet shell clam, pullet carpet shell clam, short necked clam and cockle), pro- Quantitative importance of the stocking of bival- ving the capacity of distinguishing the different species ve predators. Samplings have been started in a by the common eagle ray couple of the Aquarium as natural environment in the natural beds of the Noia well as choosing and feeding themselves preferably Ria, Riveira and culture parks of O Carril, estimating with pullet carpet shell clam and cockles. Though this a high presence of species. The mean number of pre- preference by these species should not be generalized dator individuals per surface unit on which they bas- for now, being necessary to prove it with several expe- kets (table) can act. It has to be pointed out that the riences at the aquarium and natural environment and major density corresponds to Nassarius reticulatus, this might depend on the diet need that the predators followed by the hermit crabs and other crabs like

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Carcius maenas and Liocarcinus corrugatus and in natural bed (500.000 m2), or vast (5.000.000 m2), it minor presence, stars and sleepy crab (Atelecyclus can be observed the high quantities of the several rotundatus), with 0.002. Set out these possible species of predators that might be acting in the mean densities on a mean private park (1.000 m2) productive zones on a daily basis. or great dimensions (10.000 m2) or on a medium

Table. Number of mean individuals per surface unit in the samplings with baskets in Noia and Riveira, sup- posing 2.400 m2 of influence and a 25% of capture capacity.

The displacements and variability of the predator species could also be observed all along the seasonal cycle (Figure).

% in spring (s) and summer (s)

Figure. Spatial distribution in percentage of the predator species in the several zones of the natural (1) Bed, (2) Edge and (3) Channles in spring (s) and summer (s).

Interaction of protection techniques and preven- (intertidal beach) with seed of pullet carpet shell tion with the predator species. Tests of horizonal clam, short necked clam and grooved carpet shell meshes and acquarium baskets have been performed clam. with crabs and stars and in a natural environment

48 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

RESEARCH ACTION STAFF

TITLE: COORDINATOR: Integral microbiological control in molluscs hatcheries. Montes Pérez, J.

START: 2007 CHIEF RESEARCHER: FINISH: 2009 Martínez Patiño, D. (Subproxecto 2) FINANCIAL ASSISTANCE: Consellería de Pesca e Asuntos Marítimos RESEARCHERS: Xunta de Galicia Subproject 2 (CIMA): Da Costa González, F.; Ojea PGIDIT-CIMA07/01 Martínez, J.; Nóvoa Vázquez, S. PGIDIT-CIMA04/4 Subproject 1 (USC): Barja Pérez, J.L.; Prado Plana, S.

RESEARCH SUPPORT: Bouzamayor Yánez, M.V.; Cotarelo Jardón, M.J.; Páez Pérez, F.J.; Ruíz Misioné, M. Meléndez Ramos, M.I.; Penas Pampín, E.

INTERN: Balsa Freire, S.; Romanos Mondragón, L.

SUMMARY The main objective has been the establishment of samples taken was weekly. For the sowing it has a control protocol of the condition of the Molluscs been used, as in the rest of the project sections, the Culture Plant (CIMA of Ribadeo) from the microbio- Marine Agar means for the count of the heterotro- logy point of view. Samples of water have been phe marine bacteria and TCBS, selective mean for analyzed as well as of phytoplankton and different vibrions. The results indicated that the quality of the species of molluscs worked at the hatchery: grooved water used in the Cultures Plant is excellent, from carpet shell clam, pullet carpet shell clam and short the microbiology point of view. Only specific pro- necked clam; solinedae as the sword-razor shell and blems appeared that could be detected and solved the European razor clam and other species like the thanks to the control. It has to be added that the Donax clam. functioning of the ultraviolet radiation in determi- ned sections does not seem so effective as it has The design of the control protocol for the water been expected, therefore its use will submitted to circuit has been build considering that it should revision. allow the detection of: a) anomalies in the bacterial species, b) wrong functioning of some sterilization The phytoplankton provided to the parent stocks system, c) entrance of a too high bacterial load and and cultures as a prevention mean of a possible ori- d) presence of potential pathogens. Fifteen (15) gin of bacterial problems was controlled. The mixtu- points of sampling have been controlled distributed re of food has been taken as sampling points, as in by the 5 principal lines of the circuit. With these this way estimation could be achieved of the bacte- points, the final results are collected corresponding rial load that was contributed to the cultures. Again, to all the treatments (filtration, ultraviolet and war- the microbiology quality was very good, though it ming and piping stretches. The frequency of the has to be pointed out that the samples of one of the

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food circuits showed low growths, but too frequent The follow-up of the larval cultures was perfor- in the TCBS mean, which makes it advisable to revi- med on a weekly basis and the samples are collected se this point in order to minimize the risk that it before the water change, both the own culture implies for the cultures the presence of vibrion type water and the larvae. The development and survival bacteria, known molluscs pathogens. of the cultures of a same spawning with or without antibiotic has been compared. These data are going The parent stocks used to obtain spawnings have to be analyzed in a comprehensive manner with the been analyzed in order to know about their entran- results obtained of the bacteriological sows. In a ce to the Cultures Plant. These initial controls provi- first approach to the obtained data, it can be obser- de the necessary information to consider the possi- ved that the functioning of the antibiotic is inconsis- bility of carrying out a “cleaning” before the condi- tent; giving good results in some cases, but in other tioning, keeping them in water at low temperature cases the result is indifferent or even negative for or in case that this is not applicable, to take other the cultures. It is necessary to study the associated measures as to discard the first spawnings. The bacterial species that have been isolated, in order to intention is to fight in this way against a possible determine if the onset of resistant strains is respon- vertical transmission of bacteria present in the sible of this lack of effectiveness in some cases. In a adults and inoffensive for them, which are poten- precise manner, when mortalities have been registe- tially larval pathogens. red in any culture, samples have been taken obtai- ning a collection of isolated species. These species Controls have also been determined during the should undergo pathogenicity trials in order to stay time of the parent stocks in the hatchery, in assess its possible implication in the massive morta- order to know how the temperature and feeding lity events at the hatchery. conditions affected the quality, from the microbio- logy point of view, and in order to determine in this The application of the Denaturing Gradient Gel way the risks that might be entailed for the future Electrophoresis (DGGE) technique has been tuned obtained spawnings, as regards to the associated up for the knowledge of the microbiota associated bacterial species. to the larval cultures.

50 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Study of operability of the turbot (Psetta maxima) res- CIMA: Mariño Balsa, J.C. tocking and the European Lobster (Homarus gamma- rus) at the Coast of Galicia. RESEARCHERS: IGAFA: Pérez Benavente, G.; Rodríguez Villanueva, START: 2008 J.L. FINISH: 2010 RESEARCH SUPPORT: FINANCIAL ASSISTANCE: Cid González, E.; Fernández Franco, B.; Graña Dirección Xeral de Innovación e Desenvolvemento Pereira, M.I. Pesqueiro Consellería de Pesca e Asuntos Marítimos Xunta de Galicia

SUMMARY After the first experiences of the turbot and 5. To carry out sampling periodic campaigns at the European lobster release at the Galician coast, this sea and at the fish market, in order to keep a new Investigation Action tries to be a continuation of follow-up of the released specimen. the previous one, in the sense of keeping the follow- up of the released specimens and including the kno- 6. To start producing the first sea bream juveniles wledge obtained in the new restocking. for their release to the natural environment.

We could list the specific objectives that comprise 7. To prove the sector response as regards to the res- the following three years as follows: tocking plans.

1. To continue with the follow-up of turbot speci- The achievement of these objectives should be mens released in previous years and start the made on an annual basis, so the production, release of follow-up of the European lobster released in the the specimen to the environment and the follow-up year 2007. This follow-up is fundamental in order will take place during the three years of the project. to know the survival, growth and distribution of the restocked organisms. During 2008, we received the first notices about the recapture of fishes released during 2007, by the sailors 2. Production of turbot juveniles from the wild of O Grove and Cambados. Some of the specimens parent stocks, which was specifically constituted that have been recovered were in the sea over a year for the restocking program, since the last two and its growth was similar than that of the wild fishes. years. The production of European lobster juveni- les according to the techniques developed under In this first year, 8.000 alevins of turbot were relea- the AQUAREG “Lobster restocking” project. sed in the beaches of Mexilloeira and O Correiro, of the O Grove town, which were added to other 10.000 3. To carry out releases to the natural environment released in the same zone in the previous years and to of the produced and marked at the aquaculture 15.000 released between 2005 and 2006 at the Death plant. The releases will of 10.000 turbot juveniles Coast. per year and 5.000 of European lobster per year. The production of turbot alevins from the wild 4. To evaluate the different marking systems to parent stocks was within the initial objectives, for the allow us choosing what is most adequate for the release of this new Investigation Action; however survival of the fish and for its later recognition. during, this year it was not possible because the

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gathered specimens from the natural environment These zones have been selected previously with the during the year 2007, have not still reached good collaboration of the Fismare Company (a spin-off of gonadal maturations in captivity to allow us perfor- the University A Coruña), which was working in the ming the larval cultures. In this way, the released fish area during the last years. came from commercial fish farms of our community. Contacts were kept with Pescanova and Stolt Sea Farm To the releases specimens of the two species, a in order to buy specimens from different families, with follow-up was performed through samplings. In the a mean weight ranging between 20 and 60 grams, so case of the turbot, these were monthly captures as to favor as possible the diversity of the specimens to carried out with poles by restocking technicians, and be restocked. The fishes from the fish farms were in the case of the European lobster, the follow-up was taken to IGAFA where they have been labeled and preferably conducted by the fishermen and control taken care of until the release. The labels that have boats of the Marine Reserve of Mariñazos, that sub- been used where of the “T-anchor tags” type that has mitted all the European lobsters that have been fished a “t” based shape, attaching the turbot body. These under a detector at the protected marine area and tags have a number printed in it corresponding to surroundings. each individual and the telephone number of IGAFA, for the fishermen to be able to get in touch with the The results obtained during this first project year, plan responsible parties after capture. Besides this tag, resulted encouraging as regards to the number of tur- the turbots were marked with a blue color tattoo, in bot specimens that have been recovered from the the ventral part, in order to assess the loss percentage release that took place in the month of May. of the conventional labels. Considering the fishes fished during the sampling campaigns and the notices received by the fishermen, As regards to the European lobster, 5.000 juveniles the recapture percentage of the release in spring was were released of a mean total length of 6 centimeters in the region of 2% of the released alevins. at the Protected Marine Area of Miñarzos at Lira and its surroundings. In the case of the European lobster, due to the lower growth rate of this species and the difficulty of By means of the larval culture techniques and grow- the marking system, we did not obtain any recapture out in rafts, developed during the Aquareg Lobster result up to now. On the next year, some of the relea- Restocking project during the year 2008, 7.000 sed specimens in the year 2007 might reach the com- European lobster juveniles were produced from which mercial size, and therefore the possibility of being fis- 50% have not yet reached the restocking size and will hed and marked increases: being able to reach the be released in the year 2009. Each specimen, that first results of this species release. takes the time of 5 and 6 months to reach this size, is marked with an identification magnetic micro-tag of 1 millimeter of length, that stays in the animal during its whole life, in spite of the changes, and that might be detected through a metal detector.

The releases were carried out all along the year, in May, September and November, by means of profes- sional divers and the advice of the restocking techni- cians, in order to choose the best release habitats.

52 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Start-up and validation of a grow-out system of Fernández Álvarez, A. clam seed at the pontoon of the dock of O Vicedo (Lugo). RESEARCHERS: CIMA: Guerra Díaz, A. START: June, 2007 CETMAR: de Santiago Meijide, J.A. FINISH: June, 2009 RESEARCH SUPPORT: FINANCIAL ASSISTANCE: CPAM: Martín Salas, F. Consellería de Pesca e Asuntos Marítimos CETMAR: Álvarez Padín, A.; Couso Dacosta, M.S.; Xunta de Galicia Gómez Valladares, V. PGIDIT-CIMA 07/05

SUMMARY During 2008, the grow-out of the clams’ seed kept until reaching the target size that corresponds continued, at the facilities located in one of the to the seed retained in the sieve of 7 mm (T7), what pontoons of the O Vicedo port, at the mini-hat- suggests a size of approximately 10 mm, measured chery surroundings from where the seed comes according to the anterior-posterior axis. Size consi- from. dered as adequate to start the grow-out phase. The time that the seed remains in the grow-out facilities The seed used in the grow-out is of three clam is of approximately five months, though it depends, species, the grooved carpet shell clam (Ruditapes among other factors, on the grow-out starting, decussatus, L., 1758), pullet carpet shell clam initial load (g/cm2), initial size, the performance or (Venerupis pullastra, Montagu, 1803) and the short not of the splits, sieving, etc. necked clam (Ruditapes phillippinarum, Adams & Reeve, 1850). The grow-out system consists of a When the seed batches reach the target size, they series of containers, where the claim seed is stabled are delivered to the guilds for their sowing in the provided by forced flow generated by a pump (1.5 beaches. All along the year, a total of 2.124.329 clam CV), forcing the circulation of water through the seeds were distributed, from which 1.319.691 were seeds, pulling along the faeces and pseudo-faeces, short necked clam, 760.838 of pullet carpet shell keeping the seed clean. Moreover, the continuous clam and 43.800 of grooved carpet shell clam. flow avoids the fixation of epibionts at the bottom and on the walls of the container, reducing the cle- In a parallel manner, a grow-out experience was aning and maintenance works versus other carried out with seeds of the three species of the systems. above-mentioned clams, from mid-May (May 15th 2008) until early September (September 3rd 2008). At the beginning of January of the year 2008, the expansion of the grow-out facility started functio- It started with three batches of farm clams, each ning, as the initial facility turned out to be ineffi- batch in a container provided with forced flow, with cient for the seed grow-out coming from the mini- initial sizes of 4.36 ± 0.39 mm for the grooved carpet hatchery; the expansion consisted in the placement shell clam, 4.08 ± 0.56 mm for the pullet carpet shell of a pump in a similar power as the existing one, clam and 3.06 ± 0.31 mm for the short necked clam. allowing to duplicate the number of containers used The initial density, the same for the three batches is during the seed grow-out. of 0.66 g/cm2.

The seed retained in a light mesh sieve of 3 mm The mortality that has been registered in each of (T3) is moved to the grow-out facilities where it is the samplings and for each batch of clams can be

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considered null. Losses have been registered in each tence during the last month has not been verified in of the batches, mostly at the beginning of the expe- the batch of the short necked clam (t = 0.085; p- rience, when the seed is smaller. value = 0.932 and GL = 85).

Splits have not been done all through the expe- The grow-out experience will come to an end rience in none of the three batches of clams, suppo- when the 45% of the grooved carpet shell clam seed sing loads at the end of the experience of 6.04, 6.14 reaches the target size. In this period, the short nec- and 4.66 g/cm2 for the grooved carpet shell clam, ked clam does not reach the target size. short necked clam and pullet carpet shell clam, res- pectively, with final outputs in the region of 60%. It can be concluded that the system of the contai- ners with forced flow is efficient for the seed grow- In order to prove the growth existence, the statis- out with size of 3-4 mm up to 10 mm, during a tical Student´s T has been applied to the mean sizes period ranging between four and five months, with of two consecutive samplings of each of the clam high densities and very low mortalities. batches. There has been a growth from the very beginning up to the end of the experience in the The installation of the grow-out system in the batches of the grooved carpet shell clam and pullet pontoon suggests an easy access from land. carpet shell clam. On the contrary, the growth exis-

54 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Follow-up and validation of mollusc mini-hatcheries. Guerra Díaz, A. In addition: Optimization of the fixation of larvae in the mini-hatcheries. RESEARCHERS: CIMA: Martínez Patiño, D.; Nóvoa Vázquez, S. START: January, 2006 IGAFA: Andrés Rivas, M.C.; Lastres Couto, M. FINISH: December, 2008 CETMAR: de Santiago Meijide, J.A.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: Consellería de Pesca e Asuntos Marítimos CIMA: Búa Santamaría, I.; Fernández Besada, M.; Xunta de Galicia Miranda Bamio, M. PGIDIT-CIMA06/02 En O Vicedo (CETMAR): Alvarez Padín, A.; Couso PGIDIT-CIMA04/4 Dacosta, M.; Gómez Valladares, V. En Camariñas (COFRADÍA): Graña Pérez, L.; Lampón Lampón, V.; Pombal Juncal, M.; Vidal Pouso, O.

SUMMARY The objective of the Investigation Action leads to needing a strict conditioning that cannot be gua- three lines: a) support and tutorship for the installa- ranteed in the facilities of the modular hatcheries; tion of a mini-hatchery associated to the IGAFA at also the variability of response to the culture of the Illa de Arousa, Pontevedra, in which three techni- seed in all the phases, difficult the determination of cians in aquaculture production, with a scholarship stable production protocols. in mentee company regime, train themselves in the management of the installation that later will be It has been focused on the continuous micro transferred to other areas; b) coordination of the algae production systems, with automatic produc- performance that is developed through CETMAR tion systems, harvesting and distribution of the and that considers the productive development of phytoplankton to the different culture units. The the mini-hatcheries of Camariñas (A Coruña) and O previous treatment of the sea water that reaches Viceo (Lugo) and c) coordination and implementa- the culture reactors and the adjusted harvesting tion, at the most adequate zones and conditions, procedures (considering the lightness, environ- and in coordination with the technical agents of ment, species, etc.) are elements that are associa- CPAM, of the sowing of the clam seed that are ted in order to achieve a high and stable micro obtained in the mini-hatcheries. algae production. In the final memory of “scholar training in mentee company regime of IGAFA”, One main objective of the project was addressed the performed tests are detailed. in stating the procedures and work routines in the installations of the mini-hatcheries, determining The seed of 2 mm size (or less) was marked as work methods that might allow obtaining a stable target, and the most adequate to obtain from the production of clam seeds during the periods of mini-hatcheries, and pass to the exterior grow-out natural spawning of the species. Mainly, the inten- until it reaches the size of 12 mm (T7), from which sive production of the following species of seeds it can be cultured outside without high risks of has been promoted: pullet carpet shell clam mortality. Associated and as a complement of (Venerupis pullastra), short necked clam (Ruditapes these facilities, trials are being conducted with dif- philipinarum) and less of the grooved carpet shell ferent grow-out systems, that cover the size 2-12 clam (Ruditapes decussatus). The spawning of these mm, with efficiency, easy management and low last species, grooved carpet shell clam, have been operative costs. unstable even in the periods of natural spawning,

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Table. Relation of distributed seed (> 12 mm) by Rias and Guilds in the year 2008.

Therefore, another objective of the mini-hatche- the month of December, with seed loss reaming ries located at Camariñas and O Vicedo was the star- completely useless and without the possibility of ting of the grow-out system, for the seed in the 2 having access to the system installed in the pontoon. mm sieve (T2) o lower. The forced inverted flow This, adding the similar events in the previous year, system by “air-lift”, tried at the pontoon of the questions the future use of this type of seed grow- sport wharf of Camariñas gave good results, what out activity. entailed the installation of another similar system, at the O Vicedo port. Initially, the size of the grown- In the table, the seed production is summarized out seed at Camariñas was of sieve T2 (> 3 mm), up to November, which has been grown-out in the then, trials have been conducted with a system of systems indicated and distributed to the associative trays in column, also in inverted forced flow, that entities of the shellfish sector and, based in the cri- allows the grow-out of the clam retained in the terions of the technicians of DXRM. Also associated sieve T1, 2. This allows shortening the stay time of to these deliveries and in collaboration with the the seed in the interior of the farm, making cheaper beach technical teams of the DXRM a follow-up of the production costs and facilitating the manage- harvesting and incidences is being performed in ment of the installation. The installation of O Vicedo order to optimize and validate the results of the (Lugo) has been affected by strong storms during seed production.

56 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Grow-out and culture of Queen Scallops (Aequipecten Pérez Acosta, C. opercularis) and King Scallop (Pecten maximus). RESEARCHERS: START: 2008 Tarea 1: Cerviño Eiroa, A. FINISH: 2010 Tarea 2: Sánchez Mata, A.; Molares Vila, J. IEO: Román Cabello, G. (agreement coordinator) ENTIDADES FINANCIADORAS: Dirección Xeral de Innovación e Desenvolvemento RESEARCH SUPPORT: Pesqueiro Iglesias Gonzales, P. (IEO) Consellería de Pesca e Asuntos Marítimos Louro Pico, A. (IEO) Xunta de Galicia PGIDIT-CIMA 08/04 INTERN: Bea Bernabé, B. Associated to the Agreement of collaboration bet- ween CPAM and IEO of A Coruña.

SUMMARY The development of scallops’ culture is proposed and meat content) according to the cohort and cultu- at the Rias Bajas, from the seed captured in collec- re place. The points of culture in suspension will be: tors. This project is part of a collaboration agree- Cangas, Aldán (Ría de Vigo) Raxó (Ría de Pontevedra), ment with the IEO, and is made up of two parts. The O Grove and at Cambados (Ría de Arousa). first one: capitation, grow-out and culture of the queen scallops (Aequipecten opercularis) and king • Grow-out of the queen scallop in suspension: scallop (Pecten maximus), that will be in charged by growth control, survival and meat content the IEO, consists of the application of the capitation according to the culture place. techniques for the obtaining of the seed. The second part: Grow-out and culture of the queen sca- • Only at Cambados, sow of queen scallop seed llop (Aequipecten opercularis) and king scallop of 40 mm. Production estimation by the result (Pecten maximus) in charge by the CIMA, comprise of the commercial fishing. the development of culture techniques with the seed obtained and is divided in two tasks: • Diversification of the culture techniques in suspension by the application of new grow- Task 1: that includes the culture in suspension of out systems, in opposition to the traditional the queen scallop until the commercial size, in which system of oyster baskets (trays, lantern-nets). the growth, survival and meat contents will be Comparison of results. determined, in function of the cohort, origin and culture point, and b) In relation to the king scallop. Estimation of the structure of the scallops’ natural stockings in the Task 2: that will comprise the culture of the king sow zone. scallop on bottom, after a period of grow-out in sus- pension up to 30 mm; then this seed will be marked, • Sow of king scallop seed of a bigger size than sow and fishing will be carried out for the control of 30 mm. at the bottom, in a selected area of its growth and survival. the IV zone of Cambados.

Objectives • Growth control, relation size-weight and sur- a) In relation to the queen scallop. Response of the vival of the sowed king scallop seed. seed to the grow-out in suspension (growth, survival

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• Direct evaluation of the natural stocking out at different densities, in order to determine the during a cycle of two years. <1.5 mm seed management and another of grow- out under standard conditions. This last one showed Results. As regards to the capitation: collectors good growths with an increase of side of 134% in were placed in the five chosen points and, though four culture months (Initial height 18 mm, now 42 all the samples have not been analyzed yet. The mm). This fact and the mortality absence suggest maximum obtained fixations took place at Aldán that this zone is especially appropriate for the cultu- and Cangas. In this last point, though 5000 units/bag re of this species. The experience carried out at O have been registered in some line of collectors, they Grove seems to indicate lower growths than at lack of infrastructure for the time being and the Aldán. works could not be done. The obtained production from the collectors in this first year was in the region In relation to the king scallop culture, from the of 415.000 seeds at Aldán, 250.000 at Cabío, 82.500 tens of thousands of king scallop units that was at Meloxo and 25.000 at Raxó, constituting encoura- expected to be obtained after the anchoring of ging results. In general, the period of maximum fixa- several thousands of collectors at 6 points of the Rias tions corresponds to the collectors anchored in May Baixas, specifically 3 at Arousa, 2 at Pontevedra and and the beginning of June, except at Raxó that took 1 at Vigo, a capitation of one seed or none by the place in April. A gradual increase is observed as collector has been obtained, with a result of a range regards to the number of seeds present in the collec- of size between 7 and 10 mm. This low fixation rate tors that correspond with new fixations that take might be due to the anchoring zones do not present place as successive waves all through the time, rea- the adequate conditions for the fixation of this spe- ching the register of up to 800 units per bag at the cies (mud bottoms, great depth). This problem pre- moment of removal. In relation to the predators, sents backgrounds. It has been observed in all Raxó and Meloso are the points where more abun- Europe, that the culture of the king scallops in sus- dance can be found, both of stars (Asterias rubens), pension presents several problems; therefore the as hedgehog (Psammechinus vulgaris), overall in the tendency is towards its bottom culture from 30 mm. collectors anchored in June and Liocrcinus sp., was Moreover, at Galicia, the presence of the ASP abundant at all the points, except at Cabío, appea- (Amnesic Shellfish Poisoning) toxin makes it difficult ring mainly in collectors anchored at mid June and and hinders its sale. In a near future, samplings will at the beginning of July. be carried out on the natural stockings in order to quantify the juveniles stock of king scallop available As regards to the culture of the queen scallop in at the rías, given that the seed grow-out phase of suspension, experiences were carried out in two this species will not be possible to be performed for locations only: Aldán and O Grove. In the first zone, such a low volume of seeds. two experiences are being developed, one of grow-

58 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

3.1.2 AREA OF PATHOLOGY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Offsprings production of the corresponding seeds Villalba García, A. to F3 Generation, in a program of genetic selection to produce a stock of flat oyster Ostrea edulis tole- RESEARCHER: rant to the bonamiosis and with a high rate of sur- Fuentes González, J.M. vival. RESEARCH SUPPORT: START: January, 2008 Gregorio Chenlo, M.V.; Meléndez Ramos, M.I.; FINISH: December, 2008 Pazos Pazos , J.C.; Penas Pampín, E.

FINANCIAL ASSISTANCE: DXIDP. CPAM. Xunta de Galicia PGIDIT-CIMA08/01

SUMMARY The most important problem that affects the With this background that confirms the benefits grow-out process of the European flat oyster in the of the selection program, it is a priority for our Galician coast is the high mortality, to a great team to transfer experience, technology and biolo- extent due to diseases caused by the protozoan gical material to the industrial sector, for it to Bonamia ostreae. As a solution to this problem the make good use of this program success. However, development of a selection program of an oyster considering the perspective that months would plan lineage has been considered with a high sur- elapse before the approval of an investigation pro- vival rate until completing the grow-out, based on posal that might involve the oyster sector compa- the tolerance of the diseases, in particular the nies and the achievement of financing through an bonamiosis. In a study developed in the period adequate public call, in October 2007, it has been 2001-2003, previous to the selection program star- set out the development of a short investigation ting, several cohorts have been evaluated (genera- action to allow the production of the seed corres- tion F1) obtained from different geographic ori- ponding to a new generation (F3) of the selection gins of European flat oyster and it was proved that program before losing the potential parent stocks there is a relevant variability as regards to the sus- chosen from the generation F2. The objective was ceptibility to the diseases and the survival rate in the production of several cohorts of oyster seeds the stockings of the European flat oyster and that using as parent stocks oysters chosen from each of the exposure of a natural stocking to a extended the cohorts that proved to be more beneficial disease through the years, might have determined during the evaluation process of the previous a natural selection process of tolerance to the dise- generation (F2) in the selection program, as well as ase. These results served as a foundation for the a group of oysters collected randomly from natural starting of the selection program from cohorts that beds at Galicia that would serve as reference. The showed a beneficial performance in the previous produced seed will be used in a later project of phase. Thus, several selected cohorts took place technology transference to the oyster industrial (generation F2) whose performance, during the sector. grow-out process (2005-2007), was compared with non-selected cohorts used as reference, proving In order to ensure that the required mixing in that the best cohorts in terms of growth and survi- order to achieve the new generation (F3) of the val were the selected cohorts. selection program had success, it should have been

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very important to condition the parent stocks massive mortality and in the few cases in which the during the winter of 2007-2008, which is the time pediveliger larva phase was achieved, the fixation of the year empirically shows to be as the one that percentage was worthless. The microbiological and guarantees a higher larval variability at the hat- histopathological analyses that have been perfor- chery. However, the bureaucratic procedures that med did not allow distinguishing the original cause the Administration imposes, delayed the hiring of of the scarce larvae feasibility. After reaching the personnel until May 2008, therefore the opportu- convincement of the uselessness to keep on collec- nity of conditioning the parent stocks in winter ting spawning and culturing larvae in such time of was lost. The parent stocks of four cohorts selected the year and in order to minimize the loss of from the F2 and from two stockings of reference parent stocks, with irreparable consequences in the (collected from a bank of the inner of the case of the oysters of F2, it has been decided to Pontevedra Ria and another from Ortigueira Ria) transfer the parent stocks to the natural environ- were transferred to the CIMA hatchery in mid-May ment, distributing them in baskets that were hang 2008, when the oysters are mature in the natural from a raft at the Arousa Ria, in order to wait for means, therefore a specific conditioning was not the next favourable time for the conditioning. In necessary to induce the gonadal maturity. The the middle of October 2008, the parent stocks were parent stocks remained at the hatchery until the transferred again to the hatchery and a conditio- beginning of July and released several spawning ning process started. Once 3 months elapsed, on from the moment of their distribution in rafts. January 16th 2009 the first spawning was registe- However, all the spawning, both those derived red. The larvae of the two first spawning that have from the selected cohorts and those from the oys- been obtained in this new period showed an opti- ters collected directly from a natural bed, were mum aspect and behaviour after the release and integrated by a high percentage of larvae of during the first culture days. doubtful feasibility from the very release moment, as they presented morphological abnormalities in When this summary has been edited, January the veil and erratic behaviour (constant displace- 22nd 2009, the hope is kept that the larvae viability ment in short ratio circle). Even so, 30 larvae cultu- in this year period is favourable and a sufficient res were started with the cohorts that showed the amount of seeds is obtained from the planned best initial aspect. All the larvae cultures suffered crossbreeding.

60 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Gene Identification of resistance/susceptibility as Abollo Rodríguez, E. potential indicators of disease in the flat oyster (Ostrea edulis) and in the Pacific (Crassostrea gigas). RESEARCHERS: Cao Hermida, A.; Villalba García, A. START: August, 2005 FINISH: July, 2008 RESEARCH SUPPORT: Ramilo Álvarez, A. FINANCIAL ASSISTANCE: a) DXID. Consellería de Innovación e Industria INTERNS: Xunta de Galicia Comesaña Lestayo, P.; Martín Gómez, L. PGIDIT05RMA50101PR b) DXIDP. CPAM. Xunta de Galicia PGIDIT-CIMA06/01

SUMMARY In Spain, the main natural beds of the European adapted to the Galician marine ecosystem and the flat oyster, Ostrea edulis, can be found at the O. edulis as host. The two types of bonamiosis, Galician rias. These beds have been very productive both the one caused by the B.osteae as the one in the 30s and 40s, but suffered and exhaustive caused by the B. exitiosa are considered lethal for exploitation that gave place to almost its total the infected oysters, reaching death when the disappearance at the end of the 60s. Due to the maximum infection levels of the tissues are achie- shortage of oysters, a massive importation of oys- ved; there are no control or eradication methods ters from France took place with the aim of kee- known. At present, the culture of the Pacific oyster, ping the culture activity in some way. Due to these Crassostrea gigas, suggested an alternative for the importations, two parasites have been introduced survival of the sector in many European countries. in Galicia: Marteilia refringens, that hardly affec- This species is characterized for having an accelera- ted the production of the European flat oyster in ted growth rhythm, culture easiness, low mortality Galicia, and Bonamia ostreae that caused a devas- and resistance to the bonamiosis. Considering this tating effect. From April 1979, the massive mortali- differential resistance characteristic versus the ties attributed to the B. ostreae caused the collap- infection by Bonamia, we focus in order to develop se of the flat oyster at Europe; consequently diffe- a functional genomics project in which we tackle rent strategies were developed to counteract the the study on the defense system of the European effect of the disease, with uneven results, though flat oyster and the Pacific oyster versus the bona- in no case did they reach a great success. At pre- miosis. sent, most part of the production of the European flat oyster at Galicia come from the cultures in rafts The experimentation started with the inocula- and a small part from the capture from natural tion of the Bonamia cells to European flat oyster beds. Recently, our investigation team informed and Pacific oyster individuals. The extraction of about the presence of a new species of Bonamia, B. hemolymph and the separation of the hemocytes exitiosa infecting the European flat oyster, O. edu- started after a post-inoculation period; being the lis in the Galician waters, extending its geographic effector cells of the immune system in the molluscs, and host range. The demographic values registered being responsible of the inflammation processes, for the infection showed that in the studied zone repair, encapsulation and phagocytosis. From a 57% of the analyzed oysters was infected by the B. stocking pool of hemocytes we proceeded with the exitiosa indicating that this pathogen is perfectly extraction of RNA, cDNA synthesis and later the

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achievement of suppression subtractive hybridiza- gories using the GO TREE program, allowing us to tion (SSH) gene library. A SSH is a very resolutive choose those genes of immunologic interest that technique that allows the comparison of two had to be cloned. In order to determine the com- mRNA stockings, and the cloning of those genes plete codifying sequence of each gene, we used that express themselves in one stocking but not in the SMART RACE cDNA Amplification Kit techni- the other. Each of the suppression subtractive que. Finally, for each of the cloned genes, its hybridation gene libraries have been sequenced expression was studied through the quantitative and said sequences have been analyzed using the CRP using oysters free from infection as well as BLAST (Basic Local Alignment Search Tool). The with different levels of infection intensity. genes have been classified under functional cate-

62 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Neoplasic disease in marine bivalves of commercial Carballal Durán, M.J. interest. RESEARCHERS: START: December, 2006 CIMA: Abollo Rodríguez, E.; Cao Hermida, A.; Díaz FINISH: December, 2009 Costa, S.; Villalba García, A. USC: López Romalde, J. FINANCIAL ASSISTANCE: DXIDI. Consellería de Innovación e Industria RESEARCH SUPPORT: Xunta de Galicia Meléndez Ramos, I.; Penas Pampín, E. PGIDIT06PXIB501132PR INTERN: Riveiro Arjomil, F. Rodríguez Galván, B.

SUMMARY The disseminated neoplasia is a disease that con- examination of the histological cuts stated that 4% sists of the proliferation of undifferentiated and of the oysters suffered from neoplasia in March, pleomorphic cells that invade the tissues of several being 34 mm the minimum size in which this dise- organs and that result in the substitution of the ase has been detected. The maximum prevalences healthy cells by the tumoral ones, entailing the were of 8% and were detected in April and July. death of the affected individuals. This disease From June onwards, the protists Bonamia ostreae affects some marine bivalves of the coasts of the and Bonamia exitiosa have been observed with a United States and Europe, including the Galician maximum prevalence of 12%. Rickettsias, ciliates coast. The disseminated neoplasis that affect the and some copepods have been observed; all of European flat oyster, Ostrea edulis and the cockle, them with a scarce pathological relevance. The Cerastoderma edule of the Galician rias are studied accumulated mortality was of 35% from the star- in this project. In both species the neoplasias reach ting of the experiment until September 2008. epidemic levels and have been associated to inten- sive mortalities. The project consists of two investi- With the intention of evaluating the possible gation lines: the disease etiology study and the transmission of the disseminated neoplasia of the characterization of the associated genetic altera- European flat oyster, Ostrea edulis, to the Pacific tions, and the study of the effects that cause these oyster, Crassostrea gigas, monthly samples were diseases in the bivalve stockings. Our previous stu- collected of Pacific oysters during the year, to dies have already tackled the second investigation which histological cuts of 5 µm and dyed with line in the case of the neoplasia that affects the hematoxiline and eosine and were examined with cockle stockings, therefore only the first investiga- the optic microscope. These Pacific oysters grew in tion line is dealt as regards to this species. the same raft as the European flat oysters. No case of neoplasia was detected in the oyster C. gigas, Disseminated neoplasia in Ostrea edulis indicating a lack of transmission of this disease. In January 2008, European flat oysters of autoch- thonous origin of 20-30 mm of length have been Those oysters diagnosed, by means of microsco- placed in small baskets in a raft placed at pic techniques, with an advanced neoplasia have Cambados where the disease in the oysters had been selected to tackle the oncogene cloning been previously diagnosed. Monthly samples had objective. After the extraction and quantification been taken from 25 individuals that had been pro- of the total RNA, the cDNA was undertaken cessed through usual histological techniques. The through retrotranscription. The cloning of the

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oncogenes was performed using the technique functions in the carcinogenesis of the vertebrates. known as homology cloning. The obtained CRP The Hsp70 and Hsp90 proteins are related with p53 in products were bond following with the transfor- the control of the cell cycle and the proliferative res- mation of the competent bacteria Escherichia coli. ponse. The p53 protein has also been detected in the The positive clones have been differentiated in the neoplastic cells of the bivalve molluscs as Mytilus edu- middle of the growth and the inserts have been lis, Mytilus trossulus and Mya arenaria but not in the amplified by the CRP and sequenced in specialized cockle Cerastoderma edule. Therefore, western blot- services. The obtained sequences are being analy- ting analyses were performed in order to examine zed using the BLAST utilities, which allow carrying the expression of the Hsp70, Hsp90 and p53 proteins out a comparison with the GenBank database. muted in the cells and plasma of the cockle hemolymph affected by the disseminated neoplasia Disseminated neoplasia in the cockle with different intensity levels. The muted p53 protein Cerastoderma edule in cockles with moderate to severe neoplasia has been detected, not detecting it in those without neo- The alteration and the absence of the gene p53 plasia or with slight neoplasia. The Hsp70 and Hsp 90 functionality is one of the most usual defects in the proteins have been detected in neoplastic cells and human cancers, taking place in more than the 50% of not in plasma, so the intensity of these proteins incre- them. Therefore, the p53 gene is classified as a sup- ased with the increase of the neoplasia intensity. A pressor tumour gene as it produces apoptosis and the relevant positive association between the levels of destruction of the cancerous cells. The p53 proteins the Hsp70, Hsp90 and the muted p53 protein has and the Hsps (heat shock proteins) have important been detected.

64 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Perkinosis in the Spanish Coast: characterization of the Villalba García, A. taxonomic variants of the parasite, of its live cycle, and its immunological answer to the host. RESEARCHERS: CIMA: Abollo Rodríguez, E.; Cao Hermida, A.; START: October, 2006 Carballal Durán, M.J. FINISH: September, 2009 USC: Calaza Cabanas, M.; Castro Alberto, J.; Gómez Pardo, B.; Vilas Peteiro, R. FINANCIAL ASSISTANCE: IFAPA: Navas Triano, J.I. Ministerio de Ciencia e Innovación Gobierno Balear: Grau Jofre, A.; Valencia Cruz, J.M. AGL2006-11809/ACU DXIDP. CPAM. Xunta de Galicia RESEARCH SUPPORT: PGIDIT-CIMA07/02 Gregorio Chenlo, M.V.; Meléndez Ramos, M.I.; Penas Pampín, E.; Ramilo Álvarez, A.; Vicente Fernández, R.

INTERNS: Fernández Boo, S.; Ramos Caamaño, J.M.; Vicente Fernández, R.

SUMMARY Parasites from the Perkinsus kind are present in where the identification has not yet been perfor- the five continents and in many cases they cause a med: P. olseni has been detected in Ruditapes philip- high mortality of molluscs. The economic importan- pinarum and R. decussates of the Ebro delta, in R. ce made it necessary to adopt measures at an inter- decussates, Cerastoderma edule and Venerupis national level to avoid its expansion and mitigate its aurea of Sardinia (Italy), in R. philippinarum of effects. In the Spanish coast, the following species Britain (France) and in warty Venus clam Venus have been identified: P. olseni and P. mediterraneus verrucosa of Minorca; P. mediterraneus has been that affect several species of molluscs with commer- detected in V. verrucosa (sometimes in mixed infec- cial interest. One of the objectives of the project is tions with P. olseni) of Minorca. to determine if there are other unknown species or different genetic lineages within the species at the Production of clonal cultures of Perkinsus spp. in Spanish coast, with pathogenicity differences, that vitro. A variable number (10-20) of clonal cultures in would require difference surveillance and control vitro of Perkinsus olseni have been determined at level. Another objective is to clarify the transmission the CIMA, derived from each of the 5 grooved car- routes of the disease in the environment, which is pet shell clams R. decusstus of the Arousa Ria, 4 gro- important to implement fight strategies. The third oved carpet shell clams of the Pontevedra Ria, 8 gro- objective is to identify and characterize the expres- oved carpet shell clams of Carreras Ria at Huelva, 8 sed genes and the proteins whose synthesis is indu- short necked clam R. philippinarum of the Ebro ced in the immune response of the clam versus the delta, as well as the clonal cultures of the species perkinosis. To present, the following activities have Perkinsus mediterraneus derived from two been developed: European flat oysters Ostrea edulis of the Mahon inlet (Balearic Islands). All these clonal cultures are Specific identification of perkinosis agents in dif- being used in the comparison of different variables. ferent geographic areas. At the CIMA, using the Besides, there are clonal cultures of Perkinsus olseni PCR-RFLP technique, the species of Perkinsus have derived from 3 clams R. philippinarum of Japan and been identified that infect molluscs in some zones clams Austrovenous stutchburyi of New Zealand.

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Genetic differentiation analysis and population ment secreted by the cultured cells. Gels separating structure of Perkinsus spp. The team of the Genetic the intercellular proteins of clonal cultures samples Department of the USC arranged 13 micro-satellite derived from 5 different clams of each zone have markers for the Perkinsus olseni genome, which are been performed. The comparison among the gels is being used in the comparison of the parasites of this pending in order to detect qualitative and quantita- species obtained from different locations. The adop- tive differences. ted experimental design supposes the analysis of 5 clonal cultures derived from each of the 5 different Evaluation of the perkinosis transmission possibi- clams collected in each zone study. The comparison lity from alive infected clams to healthy clams. At has not been completed yet, but a scarce variability the CIFPA “Agua de Pino”, experiments have been of the parasite at the Spanish coast has been deter- conducted in which mixed clams couples have been mined, with homozygosis in the most part of the loci kept (one infected and the other not) in order to micro-satellites. Some different alleles have been determine the relative risk (RR) and the relative detected in the parasites of Japan, while the parasi- opportunity (RO) of infection by cohabitation with tes of New Zealand show different remarkable dif- an alive infected clam with one dead infected clam. ferences as regards to the Spanish and Japanese After staying 74 days in glasses of 100 mL with open ones. The detection of heterozygosis in some loci flow, the probability of getting the infection was suggests that the trophocytes of the parasite are 10/30 in cohabitation with dead clam and 3/61 with diploids. a live clam. Consequently, the disease transmission is possible without the infected clam dying. The RR of Variability analysis of the protein expression in infection with a dead clam was 6.2 times higher vitro cultures of Perkinsus spp. The protocol for the than with a live clam. The RO was 8.4 higher. A separation of proteins by means of bidimensional second experiment proved that the transmission electrophoresis has been arranged at the CIMA, probability from live clams increases when the flow both for those that are present in the in vitro cultu- is half-closed. re cells and those present in the culture environ-

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RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Tumour alterations that affect the golden carpet Shell, Carballal Durán, M.J. the flat oyster, and the cockle in Galicia. Characterization of the disease and effects on the RESEARCHERS: populations. CIMA: Abollo Rodríguez, E.; Cao Hermida, A.; Díaz Costa, S.; Villalba García, A.; Iglesias Estepa, D. START: December, 2008 FINISH: December, 2010 RESEARCH SUPPORT: Meléndez Ramos, M.I.; Penas Pampín, E. FINANCIAL ASSISTANCE: Consellería de Pesca e Asuntos Marítimos INTERNS: Xunta de Galicia Riveiro Arjomil, F.; Rodríguez Galván, B. PGIDIT-CIMA07/06

SUMMARY The objectives of this project are focused on the were surrounded with an iron frame, and placed on study of the neoplasic alterations that epidemic the bottom. Monthly, the bottom trays have been levels have reached in commercial bivalves of lifted and were placed on the ship where the num- Galicia, that are the ones that affect the cockle ber of dead and alive ones has been estimated. The Cerastoderma edule, flat oyster Ostrea edulis and a clams were not used again in order to estimate the new tumour impairment, diagnosed in the golden mortality of the following month. carpet shell Venerupis aurea. In December 2006, we started a project about neoplasia of the flat oyster The analysis of the histological cuts under optic and the cockle financed by the Innovation and microscope proved that compact masses of abnor- Industry Ministry. Recently, we have observed the mal cells were observed in the affected clams in mul- presence of a tumour in the golden carpet shell with tiple focuses of the connective tissues of the gills, a high prevalence and intensity. The study of the organs of the visceral mass and foot. The prolifera- effects that this disseminated neoplasia induces on tion of these neoplasic cells in the conjunctive tissue the clam population will be tackled in this project, of the interlaminar septum of the gills led to their and its extension in the natural beds of Galicia. dilatation and the narrowing of the water channels. Moreover, a comparative study of the morphologic The neoplasic cells were big in comparison with the and functional characteristics of the neoplasias that hemocytes that flow in the connective tissue, and affect these bivalves, and the possible transmission had a big and vesiculous nucleus with a clear nucle- of the disease among them will be assessed. olus. It was usual to observe cells in mytosis in these masses of tumour cells. The ultra structural study Neoplasia in the golden shell clam Venerupis aurea. showed that the neoplasic cells of the gill and of the Monthly samples have been taken from 30 golden hemolymph had a big and pleomorphic nucleus shell clams of the subtidal bed of Bohido (Ria de with accumulations of chromatin and frequently Arousa) using a dredge driven from a ship. The with a nucleolar. Abundant mytochondrias were clams were taken to the laboratory where the histo- observed in the cytoplasm and the Golgi apparatus pathology exams have been performed. Some clams was observed in some cells. Viruses have not been have been processed for transmission electronic observed. microscopy. The mortality assessment was perfor- med on a monthly basis, and for its 75 clams from The epidemiological study indicated that after the the bed have been collected that were distributed in analysis of 18 monthly samples, the neoplasia preva- 5 plastic boxes (60x35x20). The boxes were filled lence ranged from 0% to 57%. The seasonal varia- with sediment sand; they were covered with a mesh, tion of the prevalence was not adjusted to a seaso-

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nal model. The four intensities in which the neo- parasites observed in the clams have not been plasia was classified have been observed monthly. relevant due to the low prevalence or scarce The prevalence of serious cases varied from 0% to pathogenic power. 13.8%. The monthly mortality percentage varied from 1.9% to 26.3%, with a mean value of 12.3%. Disseminated neoplasia in oysters Ostrea edulis, The monthly mortality showed a temporal varia- cockles Cerastoderma edule and golden shell clam tion related to the prevalence variation, especially Venerupis aurea in the first half of the study, therefore increases or In order to improve the diagnosis techniques, falls of the prevalence were associated with the early markers of this disease are searched in order same tendency of the mortality on the following to detect the initial phases. With this intention, month. This correspondence was not so clear in immune-essays in the neoplasic cells of the cockle, the second half of the study, maybe because the flat oyster and golden shell clam of Galicia have values of both variables were higher and showed been performed with the 1E10 monoclonal anti- less monthly variation. Anyway, the seriousness of body that recognizes the neoplasic cells of the the injuries of the very affected clams and the soft-shelled clam, Mya arenaria. The results of correspondence between the increases of the pre- these studies showed that no reaction was produ- valence parallel to the mortality suggest that the ced with the neoplasic molluscs of Galicia, indica- mortality is due to the neoplasic disease. The other ting a high specificity of this antibody.

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3.1.3. AREA OF COASTAL OCEANOGRAPHIC PROCESSES

PROJECT STAFF

TITLE: CHIEF RESEARCHER: Miltids Culture: expansion and sustainability. Blanco Pérez, J.C. Subproject: Evaluation of the impact of the methods and levels used for the toxin control in mussels. RESEARCHERS: INTECMAR: Arévalo, F.; Correa, J. START: 2007 Consello Regulador o Mexillón de Galicia: Alcaide, FINISH: 2009 A.; André, G.; Longa, A.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: National Plan for development of Marine Cultures CIMA: Mariño, C. JACUMAR INTECMAR: Muñíz, S.

SUMMARY This National Plan, in which the Autonomous results, the same happened with the Jellett test that, Communities of Catalonia, Galicia, Andalusia, Balearic with the G. catenatum toxins profiles, seems to work and Canary Islands, and the Autonomous City of Ceuta correctly, but it needs additional tests with other take part, is coordinated by the first of the mentioned toxins profiles in order to evaluate its reliability communities and is addressed to the expansion possibi- correctly. lities evaluation of the mitilids culture in Spain, both through the study of new areas and/or culture methods Three techniques have been proved for the quanti- (open sea), as well as of new species (Perna perna), toge- fication of the type DSP toxicity: the mouse bioassay, ther with a study about the adequacy of some of the HPLC with detection by means of mass spectrometry new quantification techniques of bio-toxins for its (HPLC-MS) and phosphatase inhibition test of ZEU- implementation in the health control systems. In the Inmunotec. In relation to the mouse bioassay, both case of Galicia, specifically, the proposal is to study com- techniques produced both “false negatives” and paratively the technical and economical efficacy of some “false positives”, though with the HPLC--MS techni- of the new quantification systems in the detection of que less false negatives were found in the samples in admissible levels of toxins and in the processing of large which the mouse bioassay estimated higher toxicities. work volumes of the control systems based in these Both alternative techniques to the bioassay gave techniques. Moreover, the intention is to study the inci- approximately the same number of opposing results dence in commercialization closures, and therefore eco- with the bioassay, but the samples in which the discor- nomical, of the admissible level of toxins concentration dances took place were not the same ones. The quan- or fixed toxicity for each technique or group of techni- tifications obtained through HPLC--MS and phospata- ques. ses are not lineally related. The response of the phos- phatases is asymptotic, showing the asymptote appa- Several techniques have been compared for the type rently below what is suggested by the test specifica- PSP toxicity determination; most of them using samples tions, but showing a clear curvature even below this of an event originated by the Gymnodinium catenatum. level. Anyhow, the phosphatases tend to sub-estimate The toxicity quantification from the concentrations of the values obtained through HPLC-MS. the different toxins matched well with the measure- ments by means of the mouse bioassay but only when The times requested for each technique and its costs the toxins have been analyzed through HPLC with post- started to be estimated, as well as the maximum pos- column oxidation. The post-column oxidation caused sible work flow. Moreover, the closure effects are also remarkably different results as regards to the mouse being quantified by means of a recompilation of data bioassay, overestimating the highest concentrations. In of the closure seasons and the mussel commercializa- all the cases in which the mouse bioassay gave positive tion in different forms.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Reduction of the type PSP and DSP toxins incorpora- Blanco Pérez, J.C. tion in the rafts of mussels. RESEARCHERS: START: 2006 CIMA: Pérez Acosta, C. FINISH: 2008 OPMEGA: Franco, M.; Mondragón, E.; Somoza, A.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: OPMEGA, in the framework of a corporate project CIMA: Mariño, C. co-finances by the DXID OPMEGA: Martín, H. PGIDIT06RMA012E

SUMMARY This project, financed by OPMEGA with the help that suffered a remarkable loss of the condition of DXI+D, and in which CIMA acted as Investigator rate. Both the grouping of the ropes and the use Team, had the aim to look for methods to atte- of a mesh led to the incorporation of toxins in an nuate the effects of the toxic events through the amount of half than those of the control (in an reduction of the toxins intake. experiment during one month), being, therefore, effective for the shortening of the closures by Several experiments were conducted with the toxins and constituting promising methods for objective of evaluating the efficiency for the the reduction of the impacts of the toxic events. reduction of the toxins accumulation of several However, from the practical point of view, at pre- procedures in order to reduce the water flow in sent the use of any of the techniques is complica- the mussel ropes. The depth ropes have been ted because, in order to do it in an optimal way, it changed; they were surrounded by a mesh or is necessary to have a capacity to predict how the were grouped in groups of 4. The depth change toxins concentration will develop in the water, so of the ropes has a difficult predictable effect and as to decide when the flow reduction processes varied in the different performed experiments. start and when they have to stop, as from this The part of the ropes that remained close to the depends the important reduction of the condition bottom accumulated fewer toxins than the others rate.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Comparison of methodologies for the determination Blanco Pérez, J.C. of paralyzing toxins in bivalves related to the Paralytic Shellfish Poisoning (PSP). Uses in Spanish RESEARCHERS: aquaculture. INTECMAR: F. Arévalo, J. Correa, A. Vidal Subproject: Comparison of methodologies for the CSIC: J. Franco, P. Riobó, B. Paz determination of paralyzing toxins in bivalves related Sanidad Exterior Vigo: E. Cacho to the Paralytic Shellfish Poisoning (PSP). Uses in Galician aquaculture. RESEARCH SUPPORT: CIMA: Mariño, C. START: 2007 FINISH: 2010

FINANCIAL ASSISTANCE: National Plan for development of Marine Cultures JACUMAR

SUMMARY This National Plan, in which the Autonomous methodologies. The objective is the proposal of an Communities of Galicia and Andalusia take part, is optimal surveillance model of the paralyzing toxins coordinated by the first of the mentioned communi- that will ensure the diet safety, strengthening the ties and is focused in the comparison of available confidence of the consumer and should ensure the methodologies for the determination of paralyzing viability of the productive sector at long-term. toxins present in the bivalves and related to the paralyzing intoxication by shellfish consumption In this project, which is in its first year of effecti- (PSP) and its application to the aquaculture in Spain. ve development, the materials of the toxic events Comparative studies will be performed among registered were collected in Galicia that result methodologies and an applicability assessment of necessary for the evaluation of the different techni- the methods as daily tools for the determination of ques to be used. Some cultures of PSP toxins dino- paralyzing toxins within the production zones flagelated producers are being developed, also for management. The laboratories responsible of the its use in the evaluation experiments of the techni- surveillance programs of the shellfish production ques that will be developed in 2009. The main con- zones of Catalonia, Galicia and Andalusia take part, tribution to present is the development of a HPLC together with other laboratories specialized in the technique and of UPLX with post-column oxidation determination of paralyzing toxins. These laborato- system and fluorimetric detection, that allows the ries will ensure the samples obtaining, the determi- quantification of the PSP toxins that contribute nation of the paralyzing toxins by means of the dif- mostly (usually more than 90%) to the toxicity of ferent methodologies and an assessment of the the bivalves, in a fast manner and in only chromato- applicability within the different surveillance graphic injection.

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RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Investigation action about toxicological episodes of Blanco Pérez, J.C. phytoplankton: in bivalve molluscs found in Galicia. (EPITOX Action). RESEARCHERS: Sub Project 6- Ligaments of the DSP toxins in the cells of CIMA: C. P. Acosta the mollusc, acceleration of the elimination of the INTECMAR: F. Arévalo, J. Correa toxins and the ASP through non digestible substances and analytic aid towards the epidemiology and genetic RESEARCH SUPPORT: sub projects. CIMA: Mariño, C.

START: 2008 INTERNS: FINISH: 2011 A. Escudeiro, A. Mauríz, G. Álvarez

FINANCIAL ASSISTANCE: Consellería de Innovación e Industria Strategic action, agreement with EPITOX

SUMMARY Several relevant results have been obtained in this mostly in the cytosol, which suggests that the lack project, included in a strategic investigation action of a carrier for these substances, more than its lin- about the toxic events. kage to bio-molecules or determined cellular struc- tures, might be the cause of the great retention In relation to the DSP toxicity in the mussel, it has capacity of these compounds in the digestive gland been found that the okadaic acid, the main toxin of of the king scallop. this type, is in a very high percentage in the cytosol and associated to a determined bio-molecule type, Moreover, it was necessary to develop purifica- whose its precise identity is being studied at present. tion and fast analysis techniques of the two types of toxins. In the case of the ASP toxins in the king scallop, the situation is very different, finding the free toxins

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Toxic episodes of phytoplankton origin in the bivalve Fuentes González, J.M. mollusc of Galicia. Sub project 8: Production of families of the mussels in RESEARCHER: hatcheries for genetic studies. CIMA: Villalba García, A.

START: 2008 RESEARCH SUPPORT: FINISH: 2011 CIMA: Gregorio Chenlo, M.V.

FINANCIAL ASSISTANCE: Consellería de Innovación e Industria. Dirección Xeral de Investigación, Desenvolvemento e Innovación Strategic action, agreement with EPITOX Xunta de Galicia

SUMMARY The mussel culture in Galicia is undoubtedly the the toxins or obtaining varieties of mussels with most important activity of the Spanish aquacultu- less captation capacity and/or retention of the re and one of the most important in the European toxins. The existence of genetic differences in the aquaculture. From the biological point of view, capacity of the bivalves to ingest toxic phytoplank- the culture enjoys of good health; it has not been ton opens the possibility for the achievement of detected yet neither serious pathological pro- varieties with less captation and/or retention of blems nor worrying reductions in the growth of toxins by means of the application of genetic the cultured mussels; not even, as it happens in improvement techniques. However, before the other European countries, shortage of the wild application of these techniques, it is necessary to seed, the main raw material for the development know certain characteristics or genetic variables of of the culture. However, a serious problem of envi- the populations that are going to be used in the ronmental nature is threatening this and other improvement programs such as hereditability of bivalve cultures in Galicia in a serious and recu- the features to be improved, genetic components rrent manner during the last decades: the appea- of the variation, correlation among features, etc. rance of toxic events of phytoplankton origin. For the achievement of these variables and for the Given that the proliferations of the toxic phyto- later implementation of the genetic improvement plankton that causes the toxicity events are natu- programs it is essential to produce different mus- ral processes, the achievement of a definitive solu- sels families for which it is also necessary to be tion at short-medium term seems improbable. The able to control the crossbreeding by means of the minimization of the impacts of the toxic events application of the culture techniques in captivity. can be performed through two main routes. The These techniques have been developed and used first route consists in not allowing the develop- successfully during the last four decades for the ment of toxic phytoplankton populations, which experimental and commercial production of seve- seems to be not feasible given the impossibility of ral species of bivalve molluscs worldwide. Recently, acting on water masses that move in large not our investigation team, together with partners of confined spaces. The second route consists in redu- other European investigation centers, arranged cing the toxic phytoplankton intake by the the mussel culture techniques in captivity, stres- molluscs. At the same time, this can be done in sing mainly in the management and conditioning two manners: developing treatments that accele- of the parent stocks, in the induction of the spaw- rate the depuration or difficult the assimilation of ning, in the larvae culture and the grow-out of the

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produced seed (See Memory CIMA 2007). The main correlations of these features with others of com- objective of the sub-project is the production in mercial interest as growth rate, feasibility, perfor- captivity (farm and seedbed) of several families of mance and meat, etc. Considering that the investi- mussels and the grow-out of these families under gation project “Toxic events of phytoplankton ori- culture conditions in rafts. Having several families gin in the bivalve molluscs of Galicia” in which this of mussels (parents genetically known and evalua- sub-project is included, it has been recently been ted) faced to the toxic events (during the grow- approved (September 2008), we find ourselves at out phase), estimations of hereditability of the this moment starting the conditioning of the indi- features of interest can be performed (intoxication viduals that are going to be used as parent stocks capacity and/or retention) as well as disposing of for the obtaining of the families.

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3.1.4. MARINE RESOURCES AREA

PROJECT STAFF

TITLE: CHIEF RESEARCHER: IIncorporation of immunodetection tecniques in the Fuentes González, J.M. monitorization of the mussel larvae in Galician coastal waters. I. Adaptation of the sampling protocols and RESEARCHERS: larva determination. UV: González-Fernández, A; Pérez Estévez, D.

START: August, 2005 RESEARCH SUPPORT: FINISH: August, 2008 Gregorio Chenlo, M.V.

ENTIDAD FINANCIADORA Consellería de Innovación e Industria Xunta de Galicia GIDIT. Sectorial Program of Marine Resources

SUMMARY The mussel culture, Mytilus galloprovincialis, in main problem that was set out was the difficulty of Galicia started at the mid of the last century with identifying and separating the mussel larvae from the installation of the first rafts at the Arousa Ria. other species larvae of bivalve molluscs coexisting in Since then to present date, this sector grew remar- the plankton in a reliable and at the same time fast kably turning into the most relevant of the Spanish manner. The resolution of this problem was tackled in aquaculture. This culture consists of the grow-out in a coordinated manner by the investigation teams of rafts of the obtained juvenile individuals, both from the Immunology Area of the University of Vigo and the intertidal populations of this species, and from the Culture Bioecology of the Mussel Culture of the post-larvae captured by means of collector ropes CIMA, in the research project PGIDT00MAR5101PR: hang from the rafts themselves. Notwithstanding, immunological and biochemical identification of the the recent studies have proved the advantages of mussel larvae Mytilus galloprovincialis larvae (2000- the collector ropes use, both capture studies are still 2003). As result of this study, two monoclonal antibo- being used at present. However, the collector ropes dies of mouse have been obtained named M36.5 and are used more and more by the mussel industry and M22.8 (registration no. OEPM P200300629), that its facilitated use, with new legislation, by the recognizes the mussel larvae specifically. Once obtai- Spanish Fishing Administration. Since 1999, with the ned, these antibodies should now be incorporated to aim of advising both the mussel sector in its strategy the monitoring of the mussel larvae, which is still of the collector ropes placement and the Spanish being done weekly in the Galician coast waters. For Fishing Administration itself in its regulatory policy such purpose, this research project has been designed (Decree 406/1996), the INTECMAR (former CCMM) is and is made up of four principal objectives: still issuing weekly reports about the abundance of the different larvae phases of the mussel in the 1. To determine which the most adequate conserva- water of the Galician Rias. At present, these reports tion is method of the plankton samples for the are based on data obtained by the Bioecology and use of the monoclonal antibodies. Mussel Culture Team of CIMA under the PGIDT- CIMA-04/1 research action: evaluation of the larva 2. To determine which is the most adequate prepa- abundance and recruitment of the seed of the ration procedure of the samples in the laboratory mussel Mytilus galloprovincialis in the Galician in order to perform the larvae incubation with coast. Since the beginning of these studies, the monoclonal antibodies.

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3. To incorporate the larvae incubation phase with the project. The results of these experiments have the antibodies, in the monitoring program. shown that there are no relevant differences in the number of mussel larvae for both methods. 4. To perform the determination of the larvae However, and as the most important conclusion of abundance of the mussel by means of epifluo- this project, it suggests the special use of the new rescence microscopy and to compare the results larva epifluorescence immunodetection method in obtained with the current determination the determination program of the mussel larvae method, which is the clear field microscopy. abundance in the Galician Rias, as it is faster than the traditional method; based in the observation of The objectives 1, 2 and 3 have been handled the larva morphology under clear field microscopy, during 2005 and 2006 (see Memory CIMA 2006); in and, overall, because it can be implemented by the 2007 and 2008 we have focused in the performance technical personnel without previous knowledge of several experiments related to the objective 4 of about the larva morphology of the bivalve molluscs.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Study of the detoxification and grow-out of the King Sánchez Mata, A. scallop Pecten maximus (Linnaeus, 1758) in the Vigo Ria. RESEARCHERS: CIMA: Molares Vila, J. START: November, 2008 Pereira Productos del Mar S.A.; Sánchez Mata, A.G. FINISH: November, 2010 RESEARCH SUPPORT: FINANCIAL ASSISTANCE: Pereira Productos del Mar S.A.; Varela Sóñora, M.T. Consellería de Innovación e Industria PGIDIT. Programa Sectorial de Recursos Mariños Xunta de Galicia Code: 08MMA010E

SUMMARY The commercial extraction of the King scallop tides (detoxification, growth, survival and meat (Pecten maximus L.) in Galicia, one of the most content rate) according to the origin and cap- sought-after marine resources, is limited by the ture date. accumulation of the ASP toxin produced by the microalgae. The ASP toxicity in P. Maximus consti- 2. Study on the detoxification process in the natu- tutes an important problem in Galicia, since 1997 ral environment, following of the auto depura- when it was detected for the first time (Arévalo et tion by means of monthly analyses of the ASP al., 1997) due to the extended closures that affect content. the fishing industry causing serious economic los- ses. 3.King scallop culture: growth control, survival and meat content according to the place and The main objective in this study is to move matu- depth of the culture, pproduction estimation. re specimens of natural beds of Pectum maximus L. affected by biotoxins, to zones free of them, with In the experimental farms (a floating platform the aim of producing the auto depuration in the located in the grid number 18 and a culture line in culture facilities in the less possible time and maxi- the grids 30 and 31), the monthly follow-up of the mizing the commercialization possibilities of the growth, the gonad condition index and the analy- species. ses of the biomasses in ash free dry weight (PSLC). Likewise, periodical registers of the environmental This experience takes place in the Vigo Ria. King variables of the water and bottom sediment are scallops from the natural beds of Cies (Zone I) and taken: salinity (%), temperature (°C), Eh (mV), pH, Cangas (Zone II), where the largest incidence of red oxygen concentration (mS/cm). In a parallel way, tides of the Vigo Ria take place, are moved to the the monthly follow-up of the auto depuration pro- culture farms located in the inner zone of this Ria, cess is carried out by means of analysis of the ASP at the San Simon Inlet (polygon Redondela B), content. where the phytoplankton species producers of this biotoxin do not find the adequate oceanographic Results conditions to develop. Previous to the immersion in the culture systems, the initial size of each specimen has been registe- Specific objectives red. A month later, the first measurement was 1. Analysis of the king scallop response with high taken for the growth monthly follow-up, from a concentrations of ASP as regards to the move randomized sample of 132 individuals in the long- to the internal zones of the ria free from red line (73 individuals of the size class 1 and 2 and 59

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individuals of the classes 3 and 4) and of 23 indivi- In the first follow-up month of the culture at the duals in the platform (size class 5). farms, a mortality of 2.70% has been registered in the long-line (13 individuals from a total of 481 speci- To present, no relevant growth increase has been mens) and of 6.05% in the culture platform (13 indi- found, considering that the study has only a month of viduals from a total of 215 specimens). development.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: King Scallop Phase 1: Study of the stock populations of Sánchez Mata, A. King Scallop (Pecten maximus, L., 1758) in Ares, Arousa and Vigo Rias. RESEARCHERS: CIMA: Molares Vila, J. START: August, 2005 Pereira Productos del Mar S.A.; Sánchez Mata, A.G. FINISH: August, 2008 RESEARCH SUPPORT: FINANCIAL ASSISTANCE: Pereira Productos del Mar S.A.; Varela Sóñora, M.T. Program of Marine Resources DXID. Consellería de Innovación e Industria Xunta de Galicia PGIDIT 05RMA023E

SUMMARY The populations of Pecten maximus L. (1758) show (saturation %), salinity (psu cc) and conductivity a distribution linked to the batimetry, the substrate (mS/cm) of the beds of P. maximus L. take expected type and the level of exposure to the waves. These values, being variables all along the different beds three factors have an influence in the same way in of each rias, finding themselves within the charac- the structure of the populations of the three rias. The teristic values range for the king scallop growth in maximum densities are in the level of depth determi- the banks of these rias. ned by the isobaths from 5 to 50 m, overall, in the cliff zones, where the King scallop rest as they are zones The biochemical analyses of the contents in that favor the growth. In relation to the exposure to lipids, proteins and carbohydrates, carried out in the waves and the currents, maximum abundances the muscle and gonad of the king scallop, suggest have been found in the inner and protected zones of a higher nutritive value of the king scallops that each ria. Thus, for the Rias of Ares-Betanzos and Vigo are consumed with muscle and gonad than those the highest concentrations of King scallops were from which only the muscle is consumed. found in the beds of the North bank of both of them and in an interior third party. However, in the Arousa The zones in which the king scallops presented a Ria, the most productive bed is in the South bank, lower concentration of biotoxins were the protec- close to Cambados. The obtained results indicate that ted ones, zone I and II of the Rias of Arouse and the stock of the scallop beds in the Rias of Ares- Vigo. In the Ares-Betanzos Ria, however, only a Betanzos, Arousa and Vigo would allow the extrac- value has been registered, in the Ares bed. The tion of the resource as regards to the stability of the direct observation of alive and dead individuals populations’ structure, considering the high values in during the samplings of the Vigo Ria, showed a the biotoxin concentration (µg domoic acid/g meat) high mortality of the king scallop in the North found during the development of this study. bank and in all the external zone, following a decreasing gradient towards the South bank and As a general guideline, the beds with higher food the inner zone of the ria. This might be due to the availability in suspension (phytoplankton) are at the contribution of the oceanic waters with southwest inner and medium zones of the rias, matching with wind influences that are typically produced in the zones with high density of the species. The environ- Galician Rias, leading the red tide to these loca- mental variables measured in the water and bottom tions. sediment (pH, Eh (mV), Ta (°C), oxygen concentration

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: King Scallop Phase 2: Evaluation of the stock popula- Sánchez Mata, A. tions of King Scallop in Pontevedra, Muros and O Ferrol Rias and study of the dynamic of populations in RESEARCHERS: Vigo and Ares Rias. CIMA: Molares Vila, J. Empresa Pereira Productos del Mar S.A.: Sánchez START: November, 2006 Mata, A.G. FINISH: November, 2010 RESEARCH SUPPORT: FINANCIAL ASSISTANCE: Varela Sóñora, M.T. Program of Marine Resources DXID. Consellería de Innovación e Industria Xunta de Galicia PGIDIT 06RMA021E

SUMMARY A similar distribution pattern has been found for species settles. The register of the environmental the total and commercial stocks of the Pecten maxi- variables, water measurement and bottom sediment mus L. in the Rias of Pontevedra, Muros-Noia and (pH, Eh (mV), Ta (°C), oxygen concentration (satura- Ferrol. A heterogeneous distribution has been obtai- tion %), salinity (psu cc) and conductivity (mS/cm) of ned, conditioned to the higher or lower exposure to the P. maximus L. showed characteristic values of the the winds and marine currents. Thus, the highest different beds of each ria, finding themselves within densities and biomasses of the King scallop, both for the expected values range for the king scallop the total stock and for the commercial one, corres- growth. ponded to the inner zones and to the most protec- ted zones of the wind in each ria : in the north bank The inner zones of the estuary of the Rias of Ferrol for the three rias: O Ferrol Ria, Muros and Noia Ria and Pontevedra, in the zones II, Bazán bank, for the and Pontevedra Ria. The results indicate that the first and I, bank of the Aldán Ria for the second one, stock of the king scallop beds in the rias of Ferrol, showed high contents of biotoxins in the analyses Muros-Noia and Pontevedra allow the extraction of performed in whole body and once a year. The the resource as they present a stable population’s necessary time for the depuration of the biotoxins is structure (density and biomass). slow in all the rias, constituting an important factor to be considered in the re-sowing processes or in the The three rias were characterized for presenting aquaculture activities, as the frequent closures of heterogeneous sediment bottoms and cliffs with the king scallop fishing regulated by law, they have pronounced slopes where the kings scallop beds are a relevant negative impact on the economic feasibi- located, constituting the suitable habitat where this lity.

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RESEARCH ACTION STAFF

TITLE: TECHNICAL MANAGER: Geographic Information System Oriented towards the Molares Vila, J. Management of Marine Resources (SIGREMAR). RESEARCH SUPPORT: START: January, 2008 CIMA: Giráldez Rivero, R. FINISH: December, 2008 INTECMAR: Rodríguez Ruibal, L.

FINANCIAL ASSISTANCE: INTECMAR CPAM Xunta de Galicia

SUMMARY The use of geographic information systems in the Unfortunately, the work conditions of many tech- management of the handcrafted fish farming is nical assistances and the temporality of their con- every day more frequent in the coastal waters all tracts did not favor the generalized use of the over the world. The main advantage of these systems SIGREMAR application, and with the passage of is its facility to integrate information and be able to time there are a few guilds that still design their analyze it on a geographic basis. To apply this tech- plans with this system. Moreover, in the course of nology to the shellfish in Galicia has been the main the years, the massive use of INTERNET left obsole- objective of the SIGREMAR project. At the beginning te the information transfer system among the tech- of the project, the information tools were develo- nical assistances and the data bases centralized at ped, a data base created in Access, managed by the CIMA. means of a written application of Visual Basic. The information system is designed to be able to mana- In spite of not having reached the objective of ge the shellfishery resources through an adaptable implementing an information system of general use co-management model based on the communities. in all the Galician fishing entities for the design and With this management model, the shellfish commu- management of the shellfish exploitation plans, nities, with the help of technical assistance, have the other results have been obtained that justify and opportunity to adapt the exploitation plans to the compensate the effort invested in this project: changes of the resources and market conditions. • The SIGREMAR application served as basis for The exploitation plans are designed in the shellfish the development of the monitoring system of entities. Usually, the person who carries out this task the recovery plans of the shellfish beds of is the technical assistance, that besides is in charge of “Lombos do Ulla” and “O Bohído”. performing the follow-up of the development of the plans. These technicians, frequently underestimated, • The SIGREMAR application will be the informa- have been the main piece in the progress of the tion system used by the Assessment Unit and shellfish management in Galicia that has been regis- Scientific Follow-up of the Protected Fishing tered during the last decade. In order to make the Marine Areas declared by the Galician Fishing best of its potential, all the technical assistances of Administration. the community have been involved in the SIGREMAR project, carrying out seminar and workshops with • The elaboration of several protocols of sam- the aim of showing the functioning of the applica- pling, fruit of the interaction between the tion and to interchange knowledge about the sam- technical assistances and the investigators pling methods and information analysis. involved in the project.

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• The contribution to the formation of the tech- that other services of the Galician Fishing nical assistances communities that, though Administration asked us for help. Thus, we got intangible, is a vital subject for the good func- into step by step in the complex world of the tioning of the fishing management. shellfish management. After a decade of work, the SIGREMAR project comes to an end, but the • The elaboration of several layers with geo-refe- knowledge achieved during its development will renced information that have been shared with allow us to face new challenges related to the the geographic information system of INTEC- management of the marine resources. MAR. This system feeds the “Eventualities Plan due to Contamination in the Galician In all the projects it happens that without the Seascape”. These layers have been used in collaboration of certain key persons, it would be investigation projects related to the coastal difficult or impossible to carry them out. However, effluents as CONTINMAR and EROCIPS in SIGREMAR to show the list with the names of (Emergency Response to coastal Oil Chemical the principal collaborators is an impossible task and Inert Pollution from Shipping). due to the high number of participants and colla- borators, both from inside the Ministry of This project that started in 1999 with the only Fisheries and Maritime Affairs, as well as from the intention of helping to improve the management fishing entities. goose barnacle grew in complexity to the extent

82 CONSELLERÍA DO MAR Memoria CIMA 2008 SCIENTIFIC ACTIVITY

RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Action Plan for the recovery of the shellfish bed Molares Vila, J. “Lombos do Ulla” and “O Bohído” (Arousa Ria). RESEARCHERS: START: January, 2008 CIMA: Sánchez Mata, A.; Carballal Durán, M.J.; FINISH: December, 2010 Villalba García, A. INTECMAR: Iglesias Estepa, D. FINANCIAL ASSISTANCE: CETMAR: Parada, J.M. DXIDP and DXRM CPAM RESEARCH SUPPORT: Xunta de Galicia CIMA: Giráldez Rivero, R. CETMAR: Carreira Vázquez, P.; Darriba Santiago, C.; Martínez Verde, G.; Rodal Mallo, M. INTECMAR: Rodríguez Ruibal, L.

SUMMARY In Lombos do Ulla, the results of the shellfish cam- we consider that the bed is still quite below its poten- paign 2007-2008 have been the best ones from the tial production. The accumulation of a great quantity beginning of the recovery plan of the bed in 2002. A of algae on the bottom, mainly the ulva algae, might great part of the success was due to the excellent be the principal cause of the poor recruitments of recruitment of the cockle in 2006, and the high captu- bivalves with commercial interest registered during res of the grooved carpet shell clam and the short nec- the last three years. The maintenance of the extrac- ked clam. On the other hand the pullet carpet shell tion prohibition of the mature dosinia limits the acti- clam continues to be much reduced. vity of the shellfish industry to the less deep zone of the bed, hindering that the laboring destroys part of In O Bohido, the results of this campaign have the accumulated algae and reducing considerably the been a bit better than the previous ones; however, profitability of the shellfish campaign.

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In all the resources a seasonal growth pattern has named “granulomatosis” present mainly in the adult been observed. In the cockle and the grooved carpet specimens can be the cause of the mortality increase. shell clam, short neck clam and in the pullet carpet Peaks of high mortality during summer took place shell clam the growth was the maximum in spring during the last three years in the grooved carpet shell and minimum or null in winter, while in the golden clam, which were associated to periods of high tem- carpet shell was the maximum in spring and autumn peratures. The short necked clam did not suffer any and minimum in summer and winter. mortality event from the beginning of the follow-up. In the pullet carpet shell clam, events were observed The natural mortality rate was quite different in of high mortality associated to strong reductions in each of the studied species. In the cockle it was kept the water salinity. In the golden shell clam, a modera- in low levels until November 2007; from that moment te mortality was observed during most of the year, the mortality increased gradually. A pathology being lower in autumn and winter.

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SCIENTIFIC ACTIVITY

B.- PROJECTS WITH PARTICIPATION OF RESEARCHERS AND CIMA STAFF

PROJECT STAFF

TITLE: CHIEF RESEARCHER: IDENTILARVAS. Genetic characterization of species of CSIC: González Sotelo, C. (team 1 and coordinator). bivalves’ mollusc and PCR in real time design systems USC: Rey Méndez, M. (team 2). for its application in the detection and identification of larvae in our planktons. RESEARCHERS: CIMA: Guerrero Valero, S. START: 2007 CSIC: Pérez Martín, R.; Chapela Garrido, Mª J.; Blanco FINISH: 2010 Comesaña, M.; Sánchez Díaz, A.C; Suárez Otero, Mª I. USC: Besteiro Rodríguez, C.; Quinteiro Vázquez, J. FINANCIAL ASSISTANCE: DXDI. CPAM RESEARCH SUPPORT: Xunta de Galicia UC: Ocaña Falcón, S. Code: IN825E INTERN: CIMA: Ancosmede Garduño, C.

SUMMARY The study of the zooplankton community has a any phase of the biological cycle for said species, vital importance for the understanding of the will determine its detection in a faster, automatic dynamic and structure of the populations and also and reliable manner, being applicable to the study of the correct management of the wild cultured of the populations dynamic, monitoring of the marine resources. The methodological difficulties reproductive cycle, detection of foreign species for the study of the ecology of the first phases of and diet traceability. The foreseen results include the biological cycle difficult these studies, mainly the genetic characterization of the considered spe- hindering the discrimination of the larvae belon- cies and the design and implementation of the ging to different types of species. However, the detection and identification systems through PCR advances of the genomics and the availability of at real time, the isolated DNA from plankton sam- technologies and methodologies that are very effi- ples and belonging to said species. cient for the DNA analysis, allow reconsidering the study of the plankton samples from a molecular • The identification of the species to which a point of view. Therefore, the design is proposed, as bivalve larva belongs to obtained through a well as the development and implementation of zooplankton sample. systems of detection, identification and quantifica- tion of plankton larvae samples through PCR at • The formulation of larvae quantification real time. With this aim, the genetics characteriza- systems through PCR at real time. tion of three groups of species of special interest is formulated: mussels, clams and oysters, and based • The monitoring at geographic and temporal on this information, the design of primers and pro- scale of the productive cycle of the cultures bes for the specific considered species. The selec- and natural populations of the considered tion of these marine resources was based that they bivalve species. support cultures in the Galician Rias, in the impos- sibility of distinguishing the veliger larvae from • The invasive nature of several molluscs’ species these species and in the existence of quite a lot of is of particular interest in several areas like de information about genetics. The development of Galician Rias where there are activities poten- identification systems through PCR at real time, at tially related with the introduction and inva-

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sion of allochthonous species, the aquaculture ment and elaboration of the commercialized pro- navigation. Therefore, an adequate measure duct, including fresh, frozen and canned products. for the prevention of the introduction and its follow-up is the detection of these species in The project, that has three stages, is performed their larval phases in environmental samples of according to the scheduled flow chart, with a first ballast tank water. stage of obtaining of biological material, both of adults obtained from natural population and cultu- The estimation of the genetic diversity values in re like larvae, proceeding with the extraction of plankton samples will allow having a valuable DNA and the analysis of the information available measure about the present diversity in natural to present with the aim of detecting sequences populations directly comparable with the existing with the capacity to house PCR systems diagnostic one in cultures. The taking into consideration of at real time. The second stage is focused in the the largest genetic diversity in the cultured species genetic characterization of the considered species, can generate a higher feasibility of the culture by means of the direct sequence of different frag- during environmental changes or events of patho- ments, being at present with the mitochondrial logies. sequences of the cytochrome b and 16S rRNA of all the species collected during the first phase. To pre- The genetic characterization of the larvae’s of sent, these two phases have been practically commercial interest species in the plankton can be carried out. A third phase remains that consists of considered as the first step in a traceability system the analysis of the systems of detection, identifica- within the food sector, allowing an adequate iden- tion and larval quantification, through PCR at real tification of the populations, as well as the detec- time designed through the candidate sequences tion of the genotypes / haplotypes of interest for mentioned in the second section, assessing the effi- the identification of the geographic origin of a ciency, specificity, sensitiveness, adjustment of the product. Moreover, after the development and chemical and thermodynamically conditions of the validation of the detection systems through PCR at PCR and adjustment of the multiple reactions, to real time for larval phases, these systems are conclude with the technique validation experi- directly applicable at any phase of the develop- ments.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Optimization of industrial production techniques of A’OSTREIRA, S.L.: Veiga Blanco, E. the flat oyster. (Ostrea edulis). RESEARCHERS: START: 2008 CIMA: Cerviño Eiroa, A.; Montes Pérez, J.; Meléndez FINISH: 2011 Ramos, I.; Penas Pampín, E. USC: Barja Pérez, J.L.; Núñez García, S.; Prado Plana, FINANCIAL ASSISTANCE: S. DXIDI Plan Galego de Investigación, Desenvolvemento e INTERN: Innovación Tecnolóxica Balsa Freire, S. Consellería de Innovación e Industria Xunta de Galicia Code: 08MMA007E

SUMMARY The CIMA takes part as collaborator, jointly with the parent stocks that will be used for the diffe- the Microbiology Department of the University of rent spawning. Santiago in a project presented by the company A Ostreira S.L., to the Sector Summon of the Marine During the annuity of 2008, it has been collabora- Environment Galician Plan. ted in the elaboration of the project for its presen- tation to the Summon and once approved (October The Investigators of the CIMA will develop the 2008); several meetings were held with the partici- part of the project dedicated to the grow-out in pating teams in order to put this into practice. rafts placed in the Cambados area D (Ria de Arousa) of the seed of the flat oyster produced at It started with the selection of the parent stocks the Barizo hatchery. They will also take part in the of different origins for the starting of the spaw- control, from the pathological point of view, of ning phases in the hatchery.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Action investigation about toxic episodes of phyto- Arévalo Bartolomé, F. plankton origin in the mollusc bivalves of Galicia. (Epitox Action). RESEARCHERS: Subproject 1. Epidemiological detection aspects of CIMA: J. Blanco. intoxications effect of cooking in toxicity and the size INTECMAR: J. Correa, A. Vidal. of the portion of bivalves. RESEARCH SUPPORT: START: 2008 CIMA: C. Mariño. FINISH: 2011 INTECMAR: S. Muñíz.

FINANCIAL ASSISTANCE: Strategic action, agreement EPITOX Consellería de Innovación e Industria Xunta de Galicia

SUMMARY The intention of this subproject is to contribute to principal combinations mollusc/toxin of the improve the assessment of the permissible toxin Autonomous Community. It has been proved that levels by means of the evaluation of the toxic con- the cooking does not affect in the same manner tent of the bivalves that might produce intoxica- the concentration or the content of the toxins of tions (in case one is detected), the evaluation of the the ASP type in the clam and in the cockle. In this typical portion in Galicia and the changes produced last species, toxins is lost and, in a less extent, its by the cooking in the toxicity of the main bivalves of concentration, with the vapor cooking, while in Galicia. the clam, the content is practically kept and the concentration increases, due to the reduction of The activities developed within this subproject the body weight by the dehydration. Other experi- are focused mainly in the evaluation of the effects ments have been performed but the data are still of the usual cooking procedures in Galicia in the being analyzed.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Action investigation about toxic episodes of phyto- Moroño Mariño, A. plankton origin in the mollusc bivalves of Galicia. (Epitox Action). RESEARCHERS: Subproject 3. - Improvement of the control system of CIMA: J. Blanco. bivalves affected by biotoxins through the develop- INTECMAR: P. Montero; J. Mouriño; J.J. Pérez ment and implementation of dynamic models. Queiro; M.D. Doval González; Y. Pazos Martínez; J. Correa; F. Arévalo. START: 2008 FINISH: 2011

FINANCIAL ASSISTANCE: Strategic action, agreement EPITOX Consellería de Innovación e Industria Xunta de Galicia

SUMMARY This subproject intends to improve the efficacy of the control activities. The developed activities within the control system, implementing in the INTECMAR the subproject to present have been focused in the system, and the mathematic models of accumulation election of the models to be used and the modifica- and elimination of the toxins developed in the tion of some of them in its original programming. CIMA, once modified to be integrated in the infor- The adaptation of the chosen models will start in mation systems used and feed the data provided by 2009.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Analysis and depuration of lipophilic toxins, bitter Uribe, E. domoic acid that allows the exportation of Chilean wedge clams (Mesodesma donacium) and clam (Mulinia RESEARCHERS: edulis) to European markets from Coquimbo Bay. CIMA: J. Blanco. UCN: Ávalos, P.; Aburto, J.; Espinoza, P.; Hiche, G. START: 2008 FINISH: 2010 RESEARCH SUPPORT: CIMA: Mariño Cadarso, M.C. FINANCIAL ASSISTANCE: FONDEF (CHILE) and Collaboration Agreement with INTERN: the Xunta de Galicia and the Universidad Católica Álvarez, G. de Coquimbo. UCN (Chile)

SUMMARY Coquimbo bay (Chile) has one of the most impor- amnesic, lipophilic toxins and the bitterness in the tant beds of the Chilean wedge clams, with an annual bivalves. Moreover, it will solve the microbiological extraction potentiality of 2000 tons (management problems by means of a small depurator station. The area) and a bed that has not yet been exploited of results will allow keeping a continuous extraction of clams where 421 fishermen work. these resources, favoring the families of the Handcraft Fishermen Association and Divers at the During the last years, these natural beds have been Coquimbo Bay achieving stable incomes and impro- frequently closed for the extraction, especially for the ving the life quality. export due to the presence of toxic or harmful micro- algae and microbiological problems, which has nega- From the Galicia and the EC point of view, this pro- tively affected the economy of the fishermen and the ject will improve the health guarantees of some of companies that elaborate sea products. This project the Chilean products that are frequently consumed in develops investigation that allows disposing of speci- the countries of the Community. fic detoxification treatment to face the presence of

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Exploration and follow up of harmful microalgae for Uribe, E. the activity of culture of pectinides (II, III and IV Region). RESEARCHERS: START: 2008 CIMA: Blanco Pérez, J.C. FINISH: 2009 UCN: Rosales, S.; Díaz, R.; Espinoza, P.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: FIP (CHILE) and collaboration agreement between CIMA: Mariño Cadarso, M.C. the Xunta de Galicia and the Universidad Católica de Coquimbo (Chile). INTERN: Álvarez, G.

SUMMARY The data gathered through systematic surveillance place in spring and summer or at the beginning of samplings (monitoring) of the Sanitary Program for autumn, exactly during the high season of the sca- Bivalve Molluscs (PSMB) for the United States and/or llops harvesting. One coincidence that causes great the European Union since the year 2000 to present damages to the activity, whenever that both are the date, performed in those coastal bays of the north only dates, in which the scallop (frozen or fresh-refri- zone, in which, the oyster and scallop farming activity gerated) is extracted to be shipped to the export des- indicate that the phytoplankton is dominated during tination market. most part of the year, by diatoms, standing out for spring-summer, the species Leptocylindrus danicus, From the six sheltered bays, of the areas of Pseudonitzschia australis Detonula pumila, Antofagasta, Atacama and Coquimbo in which the Skeletonema costatum and Thalassiosira aestivalus, north scallop is cultured, the Tongoy Bay, is the one among others, with abundances that exceed the that presented most of the events of paralyzing poi- million of cells/liter. In turn, the dinoflagelated, scar- soning, 13 events (sampling date) from April 2000 to cely represented during almost all the seasons of the March 2007; all sub toxic events whose contents have year , increase their abundance, late at summer and not exceeded the 40 µg/100 in which the causal agent at the beginning of autumn (February and March), has not been detected. matching with the highest sea temperatures, being the Protoperidinum brochii, P. pellucidum, P, oceani- On the other hand, the ASP is the second poison cum, Dinophysis acuminate, Prorocentrum gracilis, P. with more occurrences and the only one that someti- micans and Displopsalis lenticula, the most recurrent mes has reached the toxic levels, having determined species. more than 5 events, at the (Inglesa) English Bay and 8 events at the bay of Tongoy; all of them in summer The sporadic appearance of some of the phyto- and levels lower than 12 µg/g for this last location. plankters that produce bio toxins, such as, those that However, the diatoms Pseudo-nitzschia australis has cause the diarrheic poison and amnesic poison of the been registered since the summer of the year 2000 bivalves, has been ratified with the detection of the (before this date its classification was P. seriata) appl- three marine bio toxins, Paralyzing Shellfish ying plans of contingence with intensive samplings, Poison(PSP), Diarrheic Shellfish Poison of the and that have included analysis of finished product Shellfishes (DSP) and Amnesic Shellfish Poisoning since the winter of that same year, in spite that it was (ASP), in events, mainly, sub toxics, in living specimens not until the year 2003 that it could be associated for of scallops of the north, cultured in the centers loca- the first time, the specific dominance of P. australis ted at the Bays of Antofagasta, Atacama and with the appearance of the ASP. The samplings per- Coquimbo areas. Most of these events have taken formed during the years 2004 and 2005 rendered

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contradictory information between a higher abun- toxic species of phytoplankton and the presence of dance of cells of this species and the occurrence of the the relevant toxins at present, according to the inter- ASP. However, during 2006 and 2007, it has indeed national scientific community, in several species of showed a higher correlation versus its specific domi- bivalves of commercial interest. nance, in autumn-winter and the late detection of the ASP (outdated). From the point of view of the European Community countries that import bivalves from Chile, In spite of all the efforts performed up to present, the knowledge of the toxins present in the produc- the knowledge of the possible incidence of the diffe- tion areas might contribute to a better control and rent poisons is still remarkably incomplete, therefore, therefore to a higher safety of the imported foods. this project tackles a detailed study of the presence of

94 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

PROJECT STAFF

TITLE: CHIEF RESEARCHER: SEMAUMEX: Development of an automatic splitting Maroto Leal, J. (CETMAR) system of the mussel. RESEARCHERS: START: July, 2006 Fuentes González, J.M. In charge of the project in FINISH: June, 2009 CIMA: Domínguez Pérez, L.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: PGIDIT. Programa Sectorial de Recursos Mariños Fernández Abuín, I.; Gregorio Chenlo, M.V. Consellería de Innovación e Industria Xunta de Galicia

SUMMARY The mussel Galician aquaculture, in spite of being phase is performed at present manually or semi fruit of a contrasted experience that dates back to automatically; the irregularity in the control of the several decades ago, is subject in an endemic form parameters that govern the current machinery to a series of circumstances that makes fear for its (speed of the belts, number of mussels in the raft, stability. Unlike the fish farming, this is not a com- etc.) entails a lack of precision that passes unnoticed pletely controlled resource. This, the closure of the and whose consequence is the irregular density of areas and the consequent extraction due to the pre- the individuals all through the whole available sence of toxins, they conclude in an industrial acti- space. The joint intention of the CTAG and the CET- vity with continuous collapses and in a shortage of MAR in this investigation project is to introduce the markets than other competitors settled in other robotic control systems in the current system to countries make the best to position their product ensure that during the moment of the rope attach- and try to displace the Galician mussel in part. To ment, the harvesting has been performed under these interferences that destabilize the sector, other constant conditions, which would derive probably in interferences of socio-economical order are added a substantial improvement of the mollusc size. The as it is the large automatization of the business net- first year and a half of the project has been used in work (3200 rafts belonging to 1800 producers of the development of the rope attachment machine business size and dimension highly uneven). The prototype by the CTAG and to carry out the first fight by the Galician agents involved to this sector field tests in a raft located at the Pontevedra Ria goes through many facets: commercial policies, com- that the companies Hermanos Ruano have put at mercialization of the mussel in value added sizes the disposal of the project. These field tests have (MAP), improvement in the detection systems, been supervised both by engineers of the CTAG and analysis of toxins, etc. However, there are basic technicians and biologists of the CETMAR and CIMA. aspects related to the own phases of the culture, Once the new rope attachment prototype machine that in spite of all the existing knowledge and expe- has been delivered by the CTAG to the CETMAR rience, they still admit sensitive improvements that (October 3rd 2008), the CIMA started a preliminary might cause an increase of the tonnage produced by experiment on October 13th 2008 with the object of raft or in the homogeneity of the species as aspect comparing the provisions of the new prototype with that might increase the sales prices. The rope attach- the traditional rope attachment machinery used by ment process of the mussel is essential for the futu- the company Hermanos Ruando and built by Talleres re results of exploitation when determining the Aguin S.L. This first experiment had also as objective density of the specimens along the ropes and, con- to assess the effect of the density of the rope attach- sequently, the growth indexes and the homogeneity ment on the productive mussels’ variables (size, of the specimens. This last aspect is highly valued by weight, biomass and final density). Therefore, 40 the producers and by the destination markets. This ropes have been prepared of culture of 12 meters of

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length with divided mussel of approximately 5-6 cm from first sampling have been analyzed and consi- of size. Ten ropes have been prepared with the tra- dering that these results did not adjust themselves ditional machinery at a density of 500 mussels per to the ones expected from the previous experimen- meter and the other thirty ropes by the new pro- tal design, it has been decided to start a new expe- totype machinery at three different densities: 10 riment at the beginning of the year 2009. This ropes at a STANDARD density, similar to the one strong discrepancy among the results obtained and used by the traditional rope attachment machinery, the expected ones was attributed to a deficient cali- 10 ropes at a LOW density of approximately 300 bration of the new experimental prototype and also mussels per meter and 10 ropes at a HIGH density, of to the accidental mixture of the mussel batches of approximately 700 mussels per meter. Once the data different size in the experimental ropes.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Analysis of evidences of the impacts due to the clima- METEOGALICIA: Pérez Muñuzuri, V. te change in Galicia RESEARCHERS: START: January, 2007 CIMA: Blanco, J.; Molares, J.; Fernández, A. FINISH: June, 2008 CETMAR: Fernández, M.L.; Parada, J.M.; Navarro, E. CPAM: Bañón, R. FINANCIAL ASSISTANCE: IIM, CSIC: Álvarez Salgado, X.A.; Fernández Reiriz, Consellería de Medio Ambiente e Desenvolve- M.J. mento Sostible IEO: Bode, A.; Varela, M.; Ruíz, M.; Porteiro, C.; Xunta de Galicia Santos, B. UV: Francés, G. Other institutions.

SUMMARY The information available at Galicia about the parasite infestation. In this species, the effect of the abundance of the shellfish resources is not sufficient temperature on the parasite infestation can be com- to perform a strict study about the evidences of the pensated by the effect of an increase of the high rain- climate change. This study tried to ease this defi- fall events on the infestation itself. In the wedge ciency with information about the sale of the princi- clam, these variables do not explain the pattern of pal species with commercial interest: the cockle, gro- the observed sales; therefore it is convenient to think oved carpet shell clam, pullet carpet shell clam, about other sort of factors. In the case of the goose wedge clam and the goose barnacle. The obtained barnacle, the evolution of the Atlantic Multidecadal results allowed deducing the effects of the meteoro- Oscillation (AMO) and the progressive relaxation of logy about the populations of the analyzed species, the coastal upwelling’s might be the causes of the assuming that the evolution of the sales reflect the continuous increase of the Galician coastal produc- abundance of their populations. tion during the last 14 years and the expansion of the “elongated” morphotype without commercial inte- A negative influence could be observed in the coc- rest, along the Galician coast. kle as regards to the sales of this resource due to the high rainfall events. The floods produced by the The results obtained suggest that, other factors intense rains cause massive mortalities in the main that have not been analyzed due to the lack of data shellfish banks, which are located at the estuary of might be influencing remarkably in the abundance the largest rivers. This effect has a remarkable local of these resources. This is the case of the hydrodyna- nature. Similar patterns were found in the sale of the mism, the incidence of parasites and/or predators or pullet carpet shell clam and wedge clam and, to a less the influence of the coastal uses on the habitat of extent, in the grooved carpet shell clam. The results these species. It was left on record the need to point out that the production of these three resour- determine continuous records of physic-chemical ces might be related with some common factor that variables of the marine environment and the popu- shows an apparent cyclic behavior. In the pullet car- lation parameters of the main shellfish resources pet shell clam, an increase of the temperatures might with the objective to dispose of historical serried explain the reduction in the sales both for its influen- that might allow to suggest variations in the pro- ce on the physiology of the species and favoring the duction, associated to the climate change.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Study and characterization of intracellular prokaryote López Romalde, J. (USC) Rickettsia type and other potentially pathogenous oxi- dative bacterias for the clam. RESEARCHERS: CIMA: López Gómez, C. START: December, 2006 USC: Magariños Ferro, B. FINISH: December, 2009 RESEARCH SUPPORT: FINANCIAL ASSISTANCE: CIMA: Meléndez Ramos, I.; Penas Pampín, E. Plan Nacional de I+D Nacional CETMAR: González Rodríguez, A. Ministerio de Educación and Ciencia AGL2006-13208-C02-01 INTERNS: De Castro Puente, M.; Otero Fraga, M.J.

SUMMARY The general objective of this project is to know tology standard techniques were used, using as the pathologies of the species of the grooved carpet routine stain the Harris hematoxylin – eosine, and shell clam (Ruditapes decussates) and short necked Pinkerton stain for the study of the rickettsias. The clam (Tapes philippinarum), being the main objecti- culture technique of tioglicolate was also used for ves the identification and characterization of poten- the diagnosis of Perkinsus. In the bacteriological tially pathogenous oxidative bacteria for the clams study, a total of 539 isolated have been obtained and the development of methods for the detection from which, and up to present, 340 have been pre- and isolation of intracellular prokaryote of the liminary characterized using classic bacteriology Rickettsia type, with the aim of determining its tests in tube and plaque, as miniaturized identifi- importance as clam pathogenous. cation systems. Moreover, as complementary iden- tification method, the partial sequence of the In previous works we have studied the pathology gene 16S rRNA of the isolated, has been perfor- of populations of grooved carpet shell clam and med using universal primers for the mentioned short necked clam in Galicia, detecting the presence gene. of different parasites through an histopathology study, in which the presence of rikettsia type orga- On the other hand, it started with the study of the nisms were pointed out both in the digestive gland detection of the type Rickettsia intracellular micro- and in the gill and a study of the fermentative bac- organisms by means of molecular methods. A total teria community was carried out associated to these of 44 samples have been processed from Camariñas bivalves, finding a great predominance in the Vibrio (grooved carpet shell clam and short necked clam), type. However, the studies focused on oxidative bac- Redondela (grooved carpet shell clam) and Carril teria are scarce, in spite of the fact that some species (short necked clam) during 2007 and 2008, both of Pseudomonas have been described as pathoge- from fresh individuals and from paraffinized sam- nous for the molluscs. ples used for the histopathology studies. After the extraction of the bacterial DNA, its amplification Material and Methods. During the year 2008, took place using universal primers for the gene 16S the samplings of the population of grooved carpet rRNA, as well as specific genes of type addressed to shell clam continued (Redondela and Camariñas) the genes, ompA, ompB and ompR (proteins of and short necked clam (Carril and Camariñas). The external membrane), gltA (genes encoding citrate samples were collected in intertidal areas on a synthase) and groEl (heat shock protein). bimonthly basis. For the histopathology study, his

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Results fermentative isolated (64.4%) and oxidative (35.6) kept more or less a constant through the year, Histopathology study. The samples of 2007 and except for the locality of the Arousa Ria where the 2008 have all been processed and its observation percentage of oxidative bacteria was reduced to a under the microscope has practically been ended. 6% in summer. The presence of the following symbionts have been detected: 1) intracellular colonies of the rickettsia The phenotype characterization did not result type in the gill and digestive epithelium; 2) bacterial very useful for the identification of the isolated, the- bags in the gill water tubes (approximately 100 µm) refore a partial sequence of the gene 16S rRNA was included inside a fiber cover derived from the con- performed. The predominant types were junctive tissue of the base from the gill epithelium Pseudoalteromonas (55.6%), Alteromonas (14.8%) (also possible rikettsian type organisms); 3) and Shewanella (7.4%), though only a minority of Plasmodes of the Haplosporidium type in the diges- the isolated ones could be assigned to determined tive epithelium; 4) Steinhausia spp. microsporide, in taxons. This indicates the existence of possible new oocytes; 5) Perkinsus (atlanticus) olseni present in bacteria species among the clams isolated. different organs; 6) Non-identified gregarines in intestinal epithelium and oocytes of the Nematopsis It is interesting to point out that abnormal morta- spp. Gregarine in gill; 7) Free ciliates in the surface lities have not been observed during the study and channels of the gills water; 8) turbellarian period, except for the appearance in Camariñas Paravortex type in digestive lumen and Urastoma during February. The predominant isolated have type in gill and paleal cavity; 9) Sporocysts and meta- been identified as the Pseudoalteromonas spp., cercaria of trematodes in several organs; 10) though Vibrio tapetis has also been detected. The Copepods in gill. From these organisms, it can be virulence factors study has started in order to deter- considered potentially pathogenous, due to the mine the pathogenic potential of these isolated. intensities observed or due to the existence of bibliographic references of its pathogenicity: 1) As regards to the detection of Rickettsian type Perkinsus olseni; 2) The rickettsia type organisms; 3) intracellular micro-organisms through molecular the Haplosporidium type organisms and 4) the tre- methods, though the PCR with universal primers for matodes. Infiltrations were also detected as well as the gene 16S rRNA gave positive results in all the the presence of granulomas always associated to the cases; with the rest of the genes, ompA, ompB and presence of Perkinsus, except for some infiltration ompR (proteins of external membrane), gltA (genes case that is not associated to the presence of any encoding citrate synthase) and groEl (heat shock pathogen. protein), a positive result was obtained from a gill sample for the amplification of the gene ompA. At Bacteriology Study. The bacteriology study pro- present, studies are being performed in order to ved an increase of bacteria number during the determine the causes of the amplification absence months of summer, though the proportion between in the other cases.

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RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: potential pathogens that affect the grooved carpet USC: López Romalde, J. Shell clam culture (Ruditapes decussatus) in the Pontevedra Ria. RESEARCHERS: CIMA: López Gómez, C. START: October, 2004 USC: Rodríguez Díaz, R. FINISH: October, 2010 RESEARCH SUPPORT: FINANCIAL ASSISTANCE: CETMAR: González Rodríguez, A. DXIDP. CPAM CIMA: Meléndez Ramos, I.; Penas Pampín, E. Xunta de Galicia PGIDIT-CIMA 07/04 INTERN: De Castro Puente, M.

SUMMARY The principal objectives of this project are on one vibrios (TCBS). Besides the isolation and biochemical hand the detection of potential pathogens for characterization of the microbiot associated to the populations of grooved carpet shell clam in culture, culture of the grooved carpet shell clam, the presen- and on the other hand its relation with several envi- ce of the Vibrio tapetis clam pathogen has been ronmental parameters. The design of diagnosis analyzed by means of PCR. methods and the characterization of the potential pathogens, together with the knowledge of variabi- The data of the environmental factors are provi- lity of the environmental parameters, shall help in ded by the INTECMAR. the knowledge of the cause or causes of massive death events that took place during the last years, Results. In the histopathology study the presence knowledge that will be useful to determine possible of the following symbiont organisms could be obser- preventive measures in order to avoid de problem. ved: 1) intracellular colonies of the rickettsian type in the gill and digestive epithelium; 2) bacterial bags Material and Methods. During 2008, bimonthly in the gill water tubes (approximately 100 µm) inclu- samples have been done of the grooved carpet shell ded inside a fiber cover derived from the conjuncti- clam in three areas of the Pontevedra Ria (Ameixal, ve tissue of the base from the gill epithelium (also Aguillóns and Placeres); samples of the different possible rikettsian type organisms); 3) Plasmodes of areas are used for a histopathology and bacteriolo- the Haplosporidium type in the digestive epithelium gic study. (higher intensities were observed than those detec- ted to present in the clam in other areas of Galicia, For the histopathology study, samples of 20 indivi- but esporulation could not be observed); 5) duals have been processed using the histology stan- Perkinsus (atlanticus) olseni present in different dard protocol (routine stain, Harris hematoxylin - organs (the highest densities were detected in eosin); for the diagnosis of Perkinsus (atlanticus, Placeres); 6) Non-identified gregarines in intestinal olseni) the culture technique in tioglicolate has also epithelium; 7) Free ciliates in gill; 8) turbellarian been used. Paravortex type in digestive lumen and Urastoma type in gill and paleal cavity; 9) Metacercaria of tre- In the bacteriology study, harvests of serial dilu- matodes mainly in foot; 10) Copepods in gill. From tions of inter-valve liquid is being performed and of all these organisms, it can be considered potentially the homogenate of hepatopancreas, gill, gonad and pathogenous, due to the intensities observed or due stratum in a general environment for marine bacte- to the existence of bibliographic references of its ria (Marine Agar) and in another selective one for pathogenicity: 1) Perkinsus olseni; 2) The rickettsia

100 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

type organisms; 3) the Haplosporidium type orga- The results of a sample that has been analyzed in nisms and 4) the trematodes. A case of disseminated April after a mortality event, showed similar bacteria neoplasia has been detected in Placeres affecting counts than those obtained in the routine samples, different organs and of high density. The infiltra- being not indicative of a bacterial infection. Thus, for tions and the presence of granulomas observed the total aerobe bacteria, 2.83 x 103 and 1.37 x 104 mostly are associated to the presence of Perkinsus; CFU/mL and between 4.72 x 102 and 3.02 x 108 CFU/mL except for some infiltration case that is not associa- for vibrios could be observed, depending of the har- ted to the presence of any pathogen. vested organ. Six bacteria predominating in the har- vest of the different types of organs have been isola- In the bacteriology study, similar bacteria count ted and have been biochemically and genetically cha- could be obtained in different areas. In the area of racterized through the sequence analysis of the gene Ameixal, the aerobe marine bacteria count was bet- 16S rDNA. The identification of these isolated mat- ween 5 x 102 and 108 CFU/ml and between 2 x 102 ches with the species Pseudomonas putida, Vibrio and 108 CFU/ml vibrios. In the area of Aguillóns, the gigants, V. lentus, V. tasmaniensis and Vibrio sp. obtained counts were between 2.55 x 103 and 108 CFU/ml and 3.10 x 102 and 108 CFU/ml for total mari- There are no virulence trials described for the first ne bacteria and vibrios, respectively, Similar results four species, and only one of them, V. gigantis, has were found in Placeres with 1.65 x 103 and 108 been isolated from sick animals, oysters in these CFU/ml in total marine bacteria and 2 x 102 x108 case. The last isolated one corresponds with a non- CFU/ml for vibrios. described species that still belongs to the Vibrio type, has been isolated both in hepatopancreas and The seasonal distribution was homogeneous in in stratum. The virulence analyses have proved that the three study areas, producing an increase in the it is a non-pathogen bacteria for larvae and adult number of bacteria from September both in total clam both grooved carpet shell clam and short-nec- aerobe bacteria and in vibrios. ked clam.

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RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Design and application of the recirculation systems of Rodríguez Villanueva, J.L. (IGAFA) the larval and grow-out phase of the marine fish and continental aquaculture. RESEARCHERS: CIMA: López Gómez, C. START: November, 2008 IFAFA: Fernández Souto, B. FINISH: October, 2010 IGAFA: Figueiro Casas, R.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: DXIDP. CPAM CIMA: Soto, Nerea. Xunta de Galicia

SUMMARY The investigation action has been approved in optimum growth and achieve a quite high September 2008 and the work developed until the quantity, meaning that the water temperature end of the year consisted in the purchase of the has to be increased in several degrees with a necessary equipment for the assembly of the clo- high energy cost. sed circuits of the larval phase and grow-out of marine fish. 3.Design and application of recirculation systems in the grow-out of fishes of fresh- The objectives stated at the Action and that will water in order to prove the advantages of be developed during 2009 and 2010 are as follows: these systems in the growth of these species.

1.Design of a recirculation system to apply in 4.Prove the functioning of the closed circuit the larval culture of marine fishes in which the systems through the control of the parameters optimum temperature is of 14-17°C and in that have more influence in the efficacy of the which, in order to keep said temperature, the recirculation circuits. water has to be cooled with a very high energy cost. This system will also have a conti- 5.Microbiology analysis in different points of nuous living alimentation system incorporated each of the recirculation systems for the qua- (rotifer and brine shrimp) in order to feed the lity control of the water and evaluate the effi- larvae during the 24 hours a day. cacy of the applied sterilization systems.

2.Application of recirculation systems for the 6.Evaluation of the growth and survival of the grow-out of marine fishes. In this culture cultured species in the recirculation systems phase, most of the marine fish species need a and comparison of the results with the tradi- temperature of 18-20°C in order to have an tional systems.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Analysis and study of culture factors and conditions of Peleteiro Alonso, J.B. (IEO-Vigo) industrial production of the Senegalese sole (Solea Project coordinated by the AC of Andalucía senegalensis). Subproject: Improvement of the parameters in the RESEARCHERS: reproduction of the Senegalese sole (Solea senegalensis). IEO-Vigo: Olmedo Herrero, M.; Cal, R.

START: January, 2006 RESEARCH SUPPORT: FINISH: December, 2008 CIMA: Cores González, M.J.; Pazos Sieira, G. IEO-Vigo: Álvarez-Blázquez Fernández, B.; Gómez FINANCIAL ASSISTANCE: Ceruelo, C.; Lago, M.J. National Plan for development of Marine Cultures JACUMAR Agreement between CPAM and the IEO-Vigo

SUMMARY Five Autonomous Communities take part in the • To prove the role of the bacteria in the disease National Plan: Andalusia, Valencia, Catalonia, and to count with a tool to prove the control or Cantabria and Galicia. For the Autonomous prevention mechanisms. Activity B-2: Infection Community of Galicia, the CIMA and IEO, Vigo takes experiments controlled with bacterial strains part in this project. present in the healthy and sick soles.

The specific objectives of the project for 2008 in Activity A-3: During the last years (2004-2008) an the Autonomous Community of Galicia are: autochthonous parent stock of the Senegalese sole has been constituted in the Autonomous • To prove the feeding effects in the amount of Community of Vigo, made up of 87 specimens of 1.3 the spawning and its quality. Activity A-3: Diet kg of mean weight approximately and a relation of the parent stockings of the sole. male/female of 1/1 (Stock T-2). This stock was rene- wed during 2008 in the region of 10% and was fed • To determine the possible differences in the since its acclimatizing with semi-wet pellets made of sperm and oocyte quality of the sole, in func- a mixture of mussel, squid, blue fish and white fish, tion to its origin and the culture conditions. with the aim of obtaining spawning of quality that might allow the culture of larvae of this specie and • Activity A-5: Characterization of the Senegalese the quality control of the gametes (Activity A-5). sole gametes. On the other hand, considering that the speci- • To develop and apply hormonal treatments for mens F-1 used all through the year 2007 in hormo- the stimulation of the spermiation, ovulation nal induction experiences, a large part has been and spawning of the sole. Activity A-6: sacrificed for the obtaining of samples or died due Hormonal stimulation of the sole reproduction. to the manipulation, a new stock of autochthonous specimens has been constituted deriving from our • To use light cycles and artificial temperature for own parent stocks, with the aim of preparing and the induction, synchronization and displace- consolidating it as stock for future experiments with ment of the sole spawning period. Activity A-7: parent stock of second generation. This stock was Environmental handling of the sole reproduc- fed with a commercial dry fodder specific for sole tion. parent stock of the Skretting house (Stock T-1).

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Activity A-6: The activities carried out in this sec- Within this activity, a series of larvae cultures have tion of hormonal stimulation, have been performed been done, according to the traditional protocol of with LHRAa (Releasing hormone luteinizing hormo- larvae culture for this species, with the aim of obtai- ne) and were done with the aim of obtaining game- ning biological material for the development of vac- tes from the specimens F-1. For this, batches of dif- cines (immune system) with the University of ferent origins have been done and have been har- Santiago, for the determination of the biochemical monized or not, according to the origin. composition of the larvae with the CIMA, for stress studies with the Aquaculture Institute of USC and for Two experiments have been performed. One of the obtaining of individuals to keep the progeny artificial fecundation, for which the females F-1 corresponding to the year 2008 and the genetic have been induced with doses of 25 ugr/kg of analysis of the offspring. LRHRa. The obtained eggs due to abdominal pressu- re have been fecundated with sperm providing from As regards to the Activity B-2, samples have been the wild males and males F-1, obtaining fecundated sent to the IRTA center for their bacteriological analy- eggs in both cases. The larvae have been cultivated sis. following the traditional protocol, up to the three months. Other activities Besides the mentioned activities, the parental test With this experience, it has been proved that both have been carried out that we perform with the spe- the oocytes and the sperm from the F-1 parent stock cimens born from the autochthonous stock that we are feasible. have available. In this sense, our intention is to obtain information about the stock, as regards to the fre- On the other hand, artificial fecundations have quency and intervention of the individuals (males and been carried out (gametes obtained by abdominal females) in the reproduction process, that is to say: pressure) with oocytes from the F-1 females, indu- ced with LHRHa and with sperm of wild males. In • Which specimens have spawning this case, no good results were obtained. • How frequently • Fidelity of the couple Activity A-7: As in the last year (2007), spawning have been obtained from the two stocks, however The study of the relationship level among the stock those from F-2 (wild parent stocks) have been feasible parents and to know which of them and to what while those of stock T-1 (F-1) have not been feasible. extent have taken part in the reproductive process, Both stocks have been submitted to dim light control shall allow optimizing the stock feeding spaces. during the day and a thermo-period control in ramp signal that started during the month of March and Samples of eggs and larvae have been taken for was kept until November, obtaining a response after the determination of fat acids. These samples are 2 months approximately. The feasible spawning was being analyzed at the CIMA and the results are eva- kept until November, when the thermo-period was luated by the investigators of the respective autono- cancelled in order to offer a recovery period for the mous communities. spawning of the next year (2009). They obtained 14.6x106 total eggs from which 9.6x106 have not been All the results of the experiments performed along feasible and 5.2x106 have been feasible, and have this year, as well as those that might be pending from been used for the culture of 1.4x106 larvae, from previous exercises, are being treated and evaluated, which 430.000 have been cultured approximately. for the preparation of the project final report.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Pigment characterization and light regulation in the Garrido Valencia, J.L. (IIM,CSIC) dynoflagellates. RESEARCHERS: START: 2006 CIMA: Zapata Gago, M. FINISH: 2010 IIM-CSIC: Martínez Lorenzo, M.L.

FINANCIAL ASSISTANCE: RESEARCH SUPPORT: Xunta de Galicia CIMA: Campaña Ferro, E. PGIDIT06PXIB602143PR

SUMMARY A study about the pigment composition of the llates of the Kryptoperidinium and Durinskia type dynoflagellates has been performed, using high with chlorophyles observed in pinnate diatoms. Type resolution chromatographic techniques. Among the 5: dynoflagellates with crptophyceae chloroplasts 60 analyzed species (110 strains), representatives of present in some species of the Dinophysis. Type 6: the groups generated from hydro and liposoluble dynoflagellates with chlorophyll b, Lepidodinium toxins are included. The species with chloroplasts chlorophorum type. that contain peridinin as majority carotenoid repre- sent the 85% of the studied species. The remaining Unlike of the commonly determined, some dyno- 15% correspond to the obtained dynoflagellates flagellates with peridinin might present chlorophyll with chloroplasts, by tertiary endosymbiosis whose c1 besides the chlorophyll c2 (Peridinium and origin varies from haptophytes (Karenia, Gamnierdiscus). Karlodinium and Takayama) to diatoms (Durinskia and Kryptoperidinium), to dynoflagellates that As result of this study, new carotenoids have been obtained the chloroplasts from a chlorophyceae detected whose characterization through mass spec- (Lepidodinium) through a possible secondary endos- trometry is under publication phase. ymbiosis. The variation of the pigment composition and the The autotrophe dynoflagellates represent a wide photosynthetic parameters as regards to the light, pigment diversity that we group in 6 pigment types. have been studied in representatives of the diffe- Type 1: dynoflagellates with peridinin. Type 2: dyno- rent pigment types through HPLC and fluorescence flagellates of the Karenia type with a derivative pro- in vivo. The initial experiments allowed obtaining file of fucoxanthin with any precedent among the variation guidelines in the curves of the electric haptophytes. Type 3: dynoflagellates of the transportation (ETR) as regards to the light (PAR) Karlodinium and Takayama type. Type 4: dynoflage- through a PAM fluorimeter.

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PROJECT STAFF

TITLE: CHIEF RESEARCHER: Characterization of the quality of the breaded fish. IEO-Vigo: Peleteiro Alonso J. B. Sub Project: Characterization of the quality of the Project coordinated by the AC of Canary Islands breaded fish. RESEARCHERS: START: January, 2008 IEO-Vigo: Olmedo Herrero, M.; Álvarez-Blázquez FINISH: December, 2011 Fernández, B. CIIMAR: Pinheiro Valente, L.Mª. FINANCIAL ASSISTANCE: National Plan for development of Marine Cultures RESEARCH SUPPORT: Junta Nacional Asesora de Cultivos Marinos (JACU- CIMA: Cores González, M.J.; Pazos Sieira, G. MAR) IEO-Vigo: Gómez Ceruelo, C., Lago M.J. y Pérez Rial Agreement between CPAM and the IEO-Vigo E.

SUMMARY In this project, the following Autonomous • Qualitative stage: in this phase, the most impor- Communities take part: Canary Islands, Andalusia, tant aspects will be obtained for the fish consu- Principality of Asturias, Balearic Islands, Cantabria, mers at the moment of choosing and consuming Catalonia, Galicia and Murcia, with a total of 43 the product as well as their beliefs and opinions investigators and the performance of the project about the aspects related with its consumption, Activity 1 has been foreseen in two stages, a quali- both for the one deriving from the extractive fis- tative and a quantitative one. hing and for the breeding one. New discussion groups will be done, one for each participating The activities for the first year of the project in the Autonomous Community plus the Community of Autonomous Community of Galicia have been per- Madrid. In each discussion group pictures and/or formed as it has been foreseen. products will be included to enrich the discus- sion. Activity 1: To evaluate the perception that the consumers have in the fish quality in general and The groups will be made up of 8-10 persons who the breeding fish in particular. will be selected from the socio-demographic and con- sumption criteria discussed and agreed a priori. Such The diet behavior of the consumers is extremely meetings will be recorded in video and audio for the complex and difficult to predict. Normally, three later subjective and objective analysis through statis- great aspects condition it: the sensorial properties, tical analysis techniques of the textual data. the marketing (product, price, distribution and communication) and the individual psychological This first part of the activity, the qualitative stage, characteristics (beliefs, attitude, hopes, etc.). One was performed at the CIFP Compostela (Integral of the objectives of this project in a first stage is to Center of Professional Formation) at Santiago de evaluate one of these aspects, the perception of Compostela. For such purpose a discussion groups of the fish quality in general and the breeding fish in 10 persons was created. The data provided by the dis- particular that the consumers have. For such pur- cussion group, collected in video and audio tapes, will pose an extensive study shall be performed in the be prepared by the personnel of the IRTA along the eight involved Autonomous Communities to which year. the Madrid Community shall be added as reference of the inner market. This activity will be divided in • Quantitative stage: from the ideas and beliefs two stages: obtained during the previous stage, a question- naire will be designed to allow assessing quan-

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titatively the information obtained during the For the performance of this quantitative stage of first stage. Thus, nine studies about the consu- the Activity 1, a coordination meeting was held in mers will be done; one for the Autonomous Madrid for all the Autonomous Communities might Community involved in the project, and the prove in situ, how the survey should be done. For Madrid one, on a sample of 100 consumers cho- the recruitment of consumers, a company of sen according to a filter questionnaire, basically Catalonia has been hired, VIVA Investigación, that considering their socio-demographic and fish was in charge of the management and necessary consumption characteristics. Given the impor- infrastructure to carry out the survey in each tance of the correct sampling of the population Autonomous Community, as the hotel hiring, the to be studied, the capitation of the consumers payment to the consumers and their recruitment. will be subcontracted to a specialized company. The consumers´ attitude versus the fish will be The first survey was done in Madrid to be able to assessed through the questionnaire, as regards help the rest of the members of the project. In the to the extractive and breeding fish, using a sim- Autonomous Community of Galicia, the survey was plified version of the Planned Behavior Theory. done at the hotel of Vigo (Pontevedra); for such pur- Moreover, a Joint Analysis will be included with pose 105 persons have been summoned. Along the which the relative importance of 3-5 key aspects day in groups of 25/26 persons, in four calls, and will be assessed obtained from the first stage consisted of two parts. The first one was a joint (for example, the origin, fishing type, presenta- analysis in which the consumer had to identify the tion, etc.) as well as the combination of the dif- fish purchase preferences, and a second part with a ferent levels of each of them that provide a hig- survey in which the consumers had to answer ques- her usefulness for the consumer and a higher tions related to the family habits as regards to the purchasing probability. Finally, the study will be consumption of fish. The data obtained are evalua- completed with the determination of the risk ted at the IRTA Center (Autonomous Community of perception associated to the fish consumption Catalonia). (Anisakis, wastes, diet intoxications, etc.) in the consumer’s mind. On the other hand, for the performance of the activities two and three foreseen for next year, con- From all this information, several scientific and tacts have been established for the purchase of disclosure publications will be prepared with the necessary fish for the sensorial analysis with a panel principal obtained results, that might serve as basis of judges trained about fish from extractive and bre- for the definition of marketing strategies, both for aded fish and the for the biochemical analysis of the the product of the extractive fish as well as the bre- fish from the extractive and breaded fish that will be aded one, focused on a higher satisfaction of the sent to the Autonomous Communities of Murcia, consumer’s need according to the demands and Catalonia and the Canary Islands for the sensorial beliefs and, therefore, that might facilitate an incre- analysis and the Autonomous Communities of ase in the consumption of the food. Murcia, Catalonia, Canary Islands and Andalusia for the biochemical analysis.

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RESEARCH ACTION STAFF

TITLE: COORDINACIÓN TÉCNICA: Feasibility study of the culture of the Pacific Oyster or DXRM: Fernández Martínez, R.; Pérez Pazó, X. Giant Oyster (Crassostrea gigas) in the Galician rias. COORDINACIÓN ADMINISTRATIVA: START: 2006 CETMAR: Fernández Cañamero, M.L. FINISH: 2009 RESEARCHERS: FINANCIAL ASSISTANCE: CIMA: Guerra Díaz, A.; Montes Pérez, J.; Iglesias DXRM and DXIDP Estepa, D. CPAM INTECMAR: Rodríguez González, L. Xunta de Galicia CETMAR: Gómez Gesteira, J.L.

RESEARCH SUPPORT: Meléndez Ramos, M.I.; Penas Pampín, E.; Balsa Freire, S.; González Casal, G.

SUMMARY Crassostrea gigas usually named as Pacific oyster, • Identification of the wild populations of this spe- Japanese oyster or giant oyster is the oyster species cies established in different rias located at the with the larger worldwide production. Its introduc- north of Galicia (Ria do Baqueiro, Ortigueira, tion in different European countries was brought Viveiro and Ribadeo) and the Beaches of about due to its optimal growth and reduced mor- Anguieira (Foz) and the one of Lake (Burela). talities. Likewise, in Galicia, its experimental culture is carried out as from more than 20 years in rafts, From the seeds fixed in the collectors placed in starting from the seed native from France. July 2007 in O Baqueiro Ria and detached in February 2008, an over-raised culture is being per- The feasibility study of this species is promoted by formed in the beach of Moledos (Vicedo), whose the DXRM and it has been stated as an extension of oysters reached a commercial weight during the project “Impulse of the Pacific oyster culture November of that year. (Crassostrea gigas) in different Galician rias”. Its principal objectives are. In March 2008, more than 3.500 units of “naissan” oyster from France have been received at the facili- • Evaluation of its reproductive success in Galicia ties of CIMA. These oysters were separated in three from the quantification of the recruitment and batches of the same weight that were destined to the influence of environmental parameters on three different cultures: over-raised culture also in this regard. the beach of Moledos (April), over-raised culture in the beach of Corón, close to CIMA (April) and a cul- • Performance of a comparative study among ture in a raft at the Grove A park in (May). wild oysters and culture oysters in relation to the gametogenic development and the possible During the whole 2008, the environmental para- pathologies that might affect them. meters were evaluated in parallel by means of the collocation of a CTD in one of the culture tables of • Evaluations of the feasibility of a comprehensi- each area and a temperature recorder in the case of ve culture of Pacific oyster in the intertidal area the raft. The high values of this parameter that have of O Barqueiro Ria. been recorded during the summer months have been related to the existence of a productive period.

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A mortality follow-up of the over-raised cultured gigas in Galicia. Since July 2007 and on a monthly oysters is performed in each study area bimonthly, basis, a histological follow-up is being performed of using three previously marked plastic bags. This wild oysters collected from two locations of the O same scheme was followed in the same way with the Baqueiro Ria. The microscopical analysis of the his- collected oysters and those from France. In the case tological preparations showed that the natural of the oysters located in the raft, three oyster bas- populations established in this ria present an kets were kept for this objective. annual gonadal cycle characterized for presenting a gonadal resting period along the autumn, a star- In August 2008, the samplings of O Barqueiro Ria ting of a gametogenic development during winter, coast ended; most of the routes were walked, using followed by a progression during spring and rea- the help of a watercraft in the inner zones as well as ching the maximum gonadal maturity in June and in those in which the cliffs did not allow the access July. The spawning takes place massively between to areas where populations of Crassostrea gigas the months of July and August. Said pattern was were. Each 30-50 m approximately, a photo was observed during two consecutive years (2007 and taken in digital format of a surface of 1 m2 and the 2008). The histological examination of the tissues coordinates of each photographed point were regis- revealed the presence of symbiont organisms of the tered with the help of a GPS. Then, through the prokaryote, protozoan and metazoan group. Thus, enlargement of the images, the oysters were coun- basophiles inclusions have been observed, probably ted of each of the squares. A total of 212 sampling corresponding to organisms of prokaryote nature in points have been registered, whose density data will the epithelium of the intestine with very low preva- be used in the performance of cartography of the lence’s and intensities of minimum infection. The ria incorporating them to a geographic information ciliates of the Trichodina kind close to the gills and system. border of the stratum were usually observed among the protozoans. Copepods in the intestinal A comparative study is being done between the lumen were detected in the metazoan group with wild oysters and the culture oysters as regards to the very low prevalence’s. No case was detected that gametogenic development cycle and the possible might cause damage to the tissues of the analyzed parasites and pathologies that might affect the C. oysters.

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Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

C.- PROJECTS AND RESEARCH LINES PROMOTED AND/OR FINANCED THROUGH THE DXIDP

RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Genetic markers for the identification of the “Mexillón Consello Regulador DOP Mexillón de Galicia: Longa de Galicia”. Portabales, M.A.

START: 2007 RESEARCHERS: FINISH: 2009 Consello Regulador DOP Mexillón de Galicia: Lee, R.S. FINANCIAL ASSISTANCE: Departameto de xenética-UDC: Méndez Felpeto, J.; DXIDP; CPAM. Xunta de Galicia Fernández Tajes, J.

SUMMARY The Regulatory Council of the Origin Mytilus spp. the ones that group the species of the Denomination MEXILLON DE GALICIA has among its highest commercial interest. With the aim of protec- mission to defend the brand “Mexillón de Galicia” ting the consumers and ensuring their right to infor- ensuring that the mussel that appear under this mation, there are ground rules related to the labeling denomination belongs to the species Mytilus of the food, to which the Protected Origin Galloprovincialis, it originates from the traditional Denomination Mexillón de Galicia has to be added to and natural beds of recollection of the Galician coast the case of the mussel. or from the spat collectors of the rafts, which are pro- duced according to the traditional raft method in the All this creates the need to count with univocal Galician rias. identification techniques of the different commercia- lized species under the mussel term in order to avoid Several factors as the progressive increase in the the fraud as regards to the labeling rules and prevent food international commerce, the globalization of that other mussels benefit from the image and pres- the offer and demand and the increase in the number tige that the Mexillón de Galicia has. Likewise, the of drawn out species that do not have a consolidated Regulatory Council needs to count with adequate commercial denomination, create the need of coun- analytical techniques for the authentication and veri- ting with univocal identification techniques of the fication of the Galician mussel at any stage of produc- several commercialized species to avoid the fraud as tion, processing and marketing to guarantee the regards to the labeling rules. compliance of the regulations associated with the protected origin denomination. For the specific case of the mussel, 250 and 300 thousand tons of this shellfish are annually produced Under these determining factors, the aim of this in Galicia. In Galicia, the myticulture in rafts is an acti- project is to develop analytical techniques to allow us vity with more than 60 years of history and has achie- to identify and differentiate the Galician mussel, in ved such a development that it is placed as one of the order to take action against illegitimate practices on most important social and economical activities in the the origin of the raw material. municipalities where they are present. In spite of that, our markets receive an increasing volume of In this third year, we follow a sample collection pro- foreign mussels in different presentations. gram in the main production areas and at present the Regulatory Council has a collection of individuals of The mussel term gathers an important number of the main species of the cultured mussel (Perna viridis, species of the Mytilidae Family with a wide geogra- P. canaliculus, Mytilus galloprovincialis, M. edulis, M. phic distribution, being the Perna spp. species and the chilensis, M. californianus, M. coruscus, M. trossulus,

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Choromytilus sp. and Aulacomya spp.) that counts (intronic regions, PLII, Glu-5, Calmodulin, Actin, with more than 2.400 specimens from the most anonymous regions of DNA), RAPIDs, library of small important production areas (China, Spain, France, segments of genomic DNA through the randomized Italy, Chile, New Zealand). fragmentation of the DNA with the restriction enzy- me Hind III and PCR-SSCP. At present we are finishing Besides, we have developed a fast method that the analysis of the results and we count with several allows the identification at an individual level of the candidates genomic regions that we have to confirm specimens of the several species included within the in a deeper study. Mytilus species: M. galloprovincialis, M. chilensis, M. edulis, M. trossulus, M. coruscus and M. californianus. To sum up, we count with an analytical tool that Given the interest of the achieved results, the protec- will allow us to increase the surveillance and the con- tion of these species is being managed as utility trol in the market of the correct labeling of the mus- models in the Spanish Patent and Trademark Office. sels present in the national markets regardless of the authentic product format and with the last objective As regards to the objective of finding specific mar- of protecting the mussel under the Origin kers for the Galician mussel, we have been intensively Denomination “Mussel of Galicia” preventing frauds working; we tried different variable genomic regions associated to the species replacement.

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PROJECT STAFF

TITLE: INVESTIGADOR RESPONSABLE: INDAQUA: Selection of indicators, determination of Gabeiras Verez, J.M. (CPAM). reference values, design of programs and protocols, Carballeira Ocaña, A. (Invest. Principal, USC) and measures for environmental studies in marine Project coordinated by the AC of Canary Islands aquaculture. RESEARCHERS: START: January, 2008 Grupo Ecotox-USC: Aboal Viñas, J; Fernández FINISH: December, 2010 Escribano, A.; Villares Pazos, A.; Real Rodríguez, C. C. del Mar (Univ. de Cádiz, UCA): Carballeira Braña, FINANCIAL ASSISTANCE: D.C. National Plan for development of Marine Cultures JACUMAR RESEARCH SUPPORT: DXRM (CPAM): Otero Llovo, J. UCA (Fac. C. del Mar): del Valls Casillas, A; Romanó de Orte, M. USC: Espinosa Gallego, J. (Inst. de Acuicultura); E. Fernández Pulpeiro; (Dpto. de Zoología) Rey Asensio, A. (Dpto. Ecología) UC: Cremades Ugarte, J. (Dpto. de Botánica)

BECARIOS DEL PROJECT: González Viana, I; Romero Parraga, J.; Ares Rial, J.A.; Fernández González, V.

SUMMARY The following Autonomous Communities take ning of reference levels and will feed a DPSIR part in this National Plan: Galicia, Canary Islands, cause-effect conceptual model (Driving Forces, Valencia, Murcia, Catalonia and Andalusia. Pressures, State, Impacts and Reponses) generated by the Marine Sciences and Applied Biology 1. Coordinated activities between the involved Department of the (University of Alicante). Autonomous Communities: The creation of a data- base was started with the aim of identifying the 2. Activities of the investigation team of the indicators that are being used in the Autonomous Autonomous Community of Galicia. While in the Communities in the evaluation of the quality and involved Autonomous Communities dominate the condition of the marine environment related to marine fish farming in cages, in the case of Galicia the aquaculture facilities. The Environmental it is preferably developed in land facilities. Impact Assessment (EIA) Study and the environ- Unfortunately, the bibliography about SP for this mental surveillance plans (ESP) have been revised type of facilities is quite scarce what makes us to and the information available about the Galician design new control methods and prove the efficacy facilities has been gathered. The intention is to in different scenarios of our coast. The project con- adjust the limit values from these data and estima- templates an integrated environmental surveillan- te the probabilities reference values of the varia- ce system based on three complementary central bles used in the SP. In a parallel way, a bibliogra- points: phic revision has been done orientated towards the SPs, the most relevant texts have been chosen • Chemical analysis of pollutants in the environ- and a database has been constructed. These tasks ment and in organisms in order to determine will facilitate the selection of indictors, the obtai- the influence area and the potential toxicity.

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• Performance of bio-trials and biomarkers 2.2. Toxic and eutrophication risk. The applica- study, using native and transplanted organisms tion of different bio-trials has been set out as well to determine the toxic and eutrophication risk. as the biomarker study in order to evaluate the toxic and eutrophization risk related to the fish • Study on the communities related to the farms farms. to determine the impairment risk in the ecolo- gical integrity. Toxic risk. The intention is to apply bio-trials bat- tery in order to evaluate the ecotoxicity of the bio- 2.1. Influence area and potential toxicity. The cides authorized in the marine aquaculture, of first objective was to set out a method able to dumpings and average receptor. The battery is separate the maximum potential area of affection. made up of four tests: the bacterial bioluminescen- Theuse of markers has been considered in the envi- ce; micro-algae, embryos of sea urchins and amphi- ronment and organisms but the isotopic relation pods. The objective is to fix the ecological safety ð15N determined in macro-algae was the best des- levels of the biocides. criber of the extension and intensity of the farm influx integrating the effect of the hydrodynamic Given the sensitivity for this type of biocides, conditions in the dispersion of the dumpings with first, it has been started with the miniaturized trial the bio-availability of N. taking into account: 1/ of the micro-algae for two species (P. tricornatum that the selected macro-algaes as priority biomoni- and I. galbana); and the evaluation of the biocides tors of the Galician Environmental Specimen Bank and of dumpings from six farms have been ended. (BEAG)1 are two fucus types (F. vesiculosus and F. The study of mixtures and the estimation of the ceranoides) and 2/ that no study was found in the toxicology parameters together with the rest of bibliography about the evaluation of the influence the tests will be done in 2009. area through ð15N in this species, so it has been set out: This central point is complemented with the exposure of biomarkers study and the effects on • To study the spatial variability of ð15N in collec- native and transplanted organisms from two sce- ted algae in the Galician coast following the narios (Lira and Xove) and located at an environ- Coastal Network of BEAG (48 localities). mental gradient mode. M. galloprovincialis has been recollected for the histopathology and mole- • To qualify the contamination level of the coast cular biomarkers analysis and transplants of the considering the ð15N in algae from the determi- juvenile pullet carpet shell clam (survival test) and nation of the probabilistic regional reference adult (biomarkers kinetic). levels. Eutrophication risk. Two types of biotrials of field • To be able to perform detailed studies, someti- fertility have been designed for the evaluation of the mes, it is necessary to use other algae species eutrophication: with one micro-alga (Ulva spp.) and due to the absence of the biomonitors sta- with the community of the native phytoplankton in tions. In order to normalize the results the ð15N the two indicated scenarios. The end points of the among pattern species and among them and phytoplankton test were: chlorophyll fluorescence other algae (F. espiralis, F. serratus, C. tomento- and pigment composition. The growth and the ð15N sum, Ulva spp., etc.). have been added to the Ulva tests as describer of exposure. Approximately, it has been observed an • The influence area of the six farms has been inhibitor effect in the zone close to the focus (<100 studied through ð15N and the bio-accumulation m) followed by a growth activation strip (100-300 m) of trace pollutants (i.e. Cu, Sn, Pb, etc.). recovering the normality from 400 m.

• The study of the relation ð15N- morphology has The adjusting of the miniaturized biological been started in Fucus for its use in retrospecti- assay with microalgae to evaluate the potential of ve studies. fertility in the dumpings.

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2.3. Study of the integrity of the ecology. Given tures able to profit from the wastes poured by the the high hydro dynamism of the coast where our farm fishing plants. For this purpose two bio-trials marine farms are placed, it has been set out to have been designed, one with the pullet carpet apply experimental techniques (native phytoplank- shell clam and the other with L. saccharina, in the ton community and colonization trials) in order to two selected scenarios. At present, the follow-up evaluate the ecological integrity in the area of of the culture are being performed (vigor, bio- influence as well as classic bentonic bio-indication accumulation of pollutants and nutrients, produc- in the two previous scenarios. The best results have tion and epiphytism). been provided by the colonizing community of artificial substrates placed in gradients as regards To sum up, for the interest of Galicia, the main to the dumping focus in two times. 2 months (per- objective of this project consists of the establis- formed) and 4 months. The end points of this bio- hment of the scientific bases on which to add pro- trial are: biomass, pigment concentration and spe- tocols and Environmental Surveillance plans with cific composition/abundance of the colonizing the purpose of facilitating to the Administration community; the ð15N has been used as describer. the environmental management of the marine farm fishing on land. 2.4 Other complementary studies. It has been set out to study the feasibility of the polytrophic cul-

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RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Sanitary Management in Aquaculture (GESAC). Areoso Casal, E. (CPAM). 1. Adaptation to the new ground rules. 2. Characterization and standardization of condi- RESEARCHERS: tions of animal health in marine aquaculture: crea- CIMA: Iglesias Estepa, D.; Villalba García, A. tion of epidemiologic maps and strategies of elabo- DXRM: Hidalgo Cortijo, A.; Alonso Juste, M.J.; Marí ration for the design of a net for epidemiologic sur- Puget, J.; García González, M.A.; Fernández Llamas, veillance. D.; García Iglesias, B. CMR: Muñoz Barbero, A. START: 2007 INTECMAR: Darriba Couñago, S. FINISH: 2009

FINANCIAL ASSISTANCE: National Plan for development of Marine Cultures JACUMAR

SUMMARY This National Plan includes two subprojects coordi- and the update of the guidelines of responsibi- nated respectively from the Autonomous Community lities and follow-up programs, in collaboration of Catalonia and Murcia in which the Autonomous with the General Sub-Direction of Animal Communities of Galicia, Andalusia and Canary Islands Health of the Ministry of the Environment and also take part. Rural and Marine Affairs (open document to be updated). Subproject 1. Its objective is to facilitate the collabo- ration among agents and administrations, to allow • Compilation of available information about obtaining information related to the marine aquacul- prevention measures, control and eradication of ture diseases in order to create epidemiological data- relevant disease as defined in a list correspon- bases that might allow operating with scientific crite- ding to the first year of work. ria in the decision taking as regards to the protection of the animal health of the exploited species in the • Identification of the most adequate diagnosis marine aquaculture. methods for each disease of the list and plan- ning of inter diagnosis laboratories exercises All this with the aim of promoting the consensus in with the National Laboratories as regards to the the procedures or strategies of the national and auto- disease of the molluscs of Vigo and fish of nomous programs of the new legal framework com- Algete. pliance, the Royal Decree 1614/2008 dated October 3rd, related to the zoo-sanitary requirements and the • Design of surveillance programs for each deter- aquaculture products, as well as the prevention and mined disease/species, proposing strategies to the control of certain diseases of the aquatic animals, guarantee the traceability. that transposes our national legal rules the Resolution 2006/088/EC, of the Council of the same title. • To identify the needs of formation as the fami- liarization with the diagnosis methods and The performance lines of this second year have work techniques in epidemiology as well as the been: concepts related to the risk evaluation.

• To continue with the analysis of the new legal • Preparation of technical document/guideline framework for each Autonomous Community that contains the list of relevant selected disea-

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ses, the list of susceptible species for each of besides doing sowings in culture environments in them, the programs of proposed surveillance order to detect the presence of pathogen bacteria’s. and the control and prevention known measu- res. A first sampling has been scheduled to take place between mid May and the end of June and the second Subproject 2. Its objective is the preparation of sampling since mid November until the end of 2008. acting strategies for the design of an Epidemiology The samplings have been done with the help of the Surveillance Network for the diseases of interest in aquaculture companies that have cages in the sea, and the fish marine aquaculture by the competent as they have always done in the taking of samples of authorities, where specimens from farms and speci- fish of their exploitations, also in the samples of fish mens from the wild environment are controlled. that prowl around them. And also with the collabora- tion of fish suppliers in four important fish markets of We performed two pathologic samplings in 2008, Galicia in order to obtain the two groups of extractive both in all the aquaculture exploitations of Galicia fish from which we did the sampling, the same species and the wild fish in order to detect virologies (infec- that are cultured in Galicia and the species that are tious anemia, hemorrhagic septicemia, hematopoie- considered possible lookouts or monitors for these tic necrosis, pancreatic necrosis and betanodavirus), pathologies in the wild environment.

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RESEARCH ACTION STAFF

TITLE: CHIEF RESEARCHER: Adjusting an intensive culture system of octopus para- Sánchez Conde, F.J. larvea Octopus Vulgaris and importance of the bioche- mical composition in the larval diet. CIMA ACTION COORDINATOR: Guerra Díaz, A. START: 2008 FINISH: 2011 AGREEMENT COORDINATOR: Iglesias Estévez, J. FINANCIAL ASSISTANCE: DXIDP RESEARCHERS: CPAM IEO: Sánchez Conde,F.J.; Iglesias Estévez, J.; Otero Xunta de Galicia Pinzás, J. Specific Agreement between CPAM and the IEO CIMA: Guerra Díaz, A. (coordinación). Vigo RESEARCH SUPPORT: IEO: Lago Rouco, M.J.; Fuentes Moledo CIMA: Pazos Sieira, G.

SUMMARY The general objective of the specific agreement red that once they have hatched it was grown with the CPAM and the IEO is to start an intensive in tanks of 100 and 500 liters. culture technique of octopus paralarvae Octopus vulgaris, to offer good growth and survival results, 3.Culture of octopus paralarvae: The intention determining the importance of the lipids and pro- was to standardize the culture in volumes of teins as nutritional requirements of the species. 100 and 500 liters with the aim of reproducing The octopus culture experiments are performed in the different treatments as in 1000 liters the the CO of Vigo of IEO and the biochemical analysis consumption of the adult Artemia is very high of preys and paralarvae in the CIMA of Corón and in many cases it can only be worked with through the Investigation Action related to the one tank, without reproduction; therefore it is Agreement. not possible to obtain the statistical confirma- tion of the used treatments. The tests perfor- The activities performed during 2008 have been med and the provisional results are listed related to the following aspects. below.

1.Parent Stocks: Acquisition of adult octopus 4.Sampling: Sampling taking (Artemia, zoo- specimens to constitute the parent stock of plankton, zoeas of spider crab, paralavae) and 2008 with the aim of obtaining spawning and lyophylization for a later biochemical analysis. paralarvae for the culture tests. Spawn spider crabs have also been acquired with the aim of 5.Biochemical analysis: Biochemical analyses of getting zoeas for the later processing (lyophy- proteins, lipids, types of lipids and fat acids are lization) and biochemical analysis. performed of the samples from the performed experiments. 2. Auxiliary cultures: Cultures of microalgae have been performed for its use in the grow-out Specifically, as regards to the culture of the octo- and enrichment of Artemia: Nannochloropsis pus paralarvae, a standardization attempt has spp, Isochrysis spp, Tetraselmis suecica and been performed to volumes lower than 1000 l. Rhodomonas spp. Artemia has also been cultu- Each of the performed tests are detailed below.

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Methodology of the culture was similar but the survival was lower in the tanks of The density of paralarvae was of 10 per liter. The 100 (12% versus 57%). In this case, higher values are food that has been used was adult Artemia (over 1.5 obtained after 21 days of lipids and proteins life mm of length) put on weight during 6 to 10 days (14.38 and 44.48% respectively) in the paralarvae with Isochrysis galbana cultured in the plant and cultured in tanks of 1000 L versus those cultured in enriched 24 hours with a concentrate of tanks of 100 L(11.82 and 40.55%). There are no rele- Nanochloropsis spp., with a concentration of 106 vant differences in the composition of fat acids in cells/mL. Said Artemia was provided to a concentra- the cultured paralarvae compared to the volumes tion that ranged between 0.5 and 1 in/mL. The quoted with EPA contents between 11-12 ng/ug dry “green water” technique was used in the culture weight, DHA in the region of 2 ng/ug dry weight tanks keeping a concentration of 106 cells/mL of and ARA between 2.5 and 3 ng/ug dry weights. This Nanochloropsis spp. last one, the culture could be continued up to the day 70. These results were confirmed in later expe- The culture was performed in a close circuit riences, obtaining always low results of weight and during the first days (5-7), which was opened on survival, therefore it was decided to redesign the alternate days during four hours using water at the culture technology in volumes of 100 L with the aim same temperature than the culture (22-23°C). of improving these results in the next project annuity. The registered parameters during the culture were the following ones: Artemia density, phyto- Three tests were conducted in cultures at 500 plankton density, temperature and dissolved oxy- liters: gen, dissolved gas pressure, nitrates and ammo- nium, dry weight of the paralarvae each 7 days, lar- 1. “Hamazaki Method”. Standardization in 500 val survival of 30 days of life. Samples were taken liters in two tanks with 10 paralarvae/L. Survival for the biochemical analysis at Day 0 and 30 of life. <10%.

Two tests with tanks of 100 liters with three 2. “Hamazaki Method”. Standardization in 500 replies and a “control” tank of 1000 liters were per- liters in two tanks with 10 paralarvae/L. formed. In the first test the weight on day 21 was Biochemical samples on day 0 and 30. Final sur- higher in the tank 1000 L (p<0.05) and the survival vival (day 30): 27.8% and 100%. was similar. The percentages of the lipids and pro- teins were similar in the paralarvae cultured at 100 3. “Hamazaki Method”. Standardization in 500 and 1000 liters being the lipids, especially the phos- liters with addition, two days a week, alive zoo- pholipids, slightly higher in the paralarvae cultured plankton and with 10 paralarvae/L. Biochemical in tanks of 100 L and the proteins in the tanks of samples on day 0 and 30. Final survival (day 30): 1000 L. In accordance with the lipid level, the hig- 20.6% and 14.4%: hest values in the content of the fat acids (Eicosapentaenoic acid) EPA were also obtained as The biochemical analyses are being performed of well as docosahexaenoic acid (DHA) and arachidonic the samples corresponding to these tests whose acid (ARA). In the second test, the weight on day 21 results will be obtained in 2009.

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CENTRO DE INVESTIGACIÓNS MARIÑAS

3.2. DOCTORAL THESIS AND ADVANCED STUDIES DEGREES (DEA) CARRIED OUT AT THE CIMA

TITLE: DOCTORAL THESIS RESEARCHER: Comparison of immunological parameters among popu- Comesaña Lestayo, M.P. lations of Ostrea edulis with different susceptibility to the bonamiosis and the resistant specie Crassostrea gigas. DIRECTOR: Villalba García, A.; Cao Hermida, M.A.; Casas Liste, PRESENTATION DATE: July, 2008 S.

SUMMARY The culture of the flat oyster, Ostrea edulis, in been: total and differential count of hemocytes Europe is bogged down due to the bonamiosis, an (CTH and CDH), phagocytosis and respiratory burst infection caused by the protozoan Bonamia ostreae. (production of superoxide anion [O2-], hydrogen The oyster sector continued its activity with the peroxide [H2O2] and nitric oxide [NO], inhibitors of introduction of the culture of the Crassostrea gigas several protease activities (papain, thermolisin, species, which are resistant to the infection and to trypsin and chimotrypsin), antibacterial activities present only one Irish oyster company alone and against Vibrio splendidus and Micrococcus spp., phe- investigation teams of France and Galicia have tried noloxidase, acid phosphatase and N-acetyl-ß-gluco- to select a variety of O. edulis tolerant to the bona- saminidase and finally heat shock proteins (HSP70). miosis. These parameters have been analyzed with colori- metric methods (and also electrophoresis in the case There are O. edulis populations in Galicia that are of the HSP70) in serum and/or hemocytes lysate in contact with the parasite since its first detection, supernatant (HLS) carrying out comparisons at three in Europe and in Ireland there are still populations levels. (1) among groups of O. edulis with different that have never been exposed while there are susceptibility to the bonamiosis, (2) between O. edu- others that have been subject to selection programs lis and C. gigas and (3) among subgroups of O. edu- after years of disease pressure, so they show a lower lis oysters infected by B. ostreae and subgroups susceptibility (mortality, infection prevalence and without detected infections. Moreover, focusing in intensity have been reduced). The molecular and the inter-specific differences, a study has been done cellular bases of this lower susceptibility are ignored in 2006 of the interaction between the parasite and and its discovery would help to find a variety tole- the hemocytes of O. edulis and C. gigas, with several rant and resistant to the infection in a faster and contact experiments of B. ostreae with hemocytes of more effective manner. While searching a molecular each species, analyzing the respiratory burst and the explanation to the intra and inter specific differen- production of the non-specific esterase activity by ces as regards to the susceptibility to the bonamio- means of flow cytometry. The species C. gigas was sis, a comparison has been made of some parame- characterized by a much more stable CTH and a hig- ters of the immune system among groups of O. edu- her proportion of basophile granulocytes than the lis of Ireland and Galicia, using the resistant species groups of O. edulis. The production of O2- was simi- C. gigas as reference. In November 2003, oysters lar between the species and lower in the hemocytes from a population that has never been exposed to of the infected oysters. The production of H2O2 was the bonamiosis, oysters less susceptible of Ireland variable depending on the group of O. edulis with and Galicia and a group of oysters of the resistant an inhibition in the stimulated hemocytes of the C. species C. gigas have been kept in a raft at the gigas. Generally, the HLS of the C. gigas showed a Arousa Ria (Galicia, Spain) affected by the bonamio- lower inhibition of the protease activity, phenoloxi- sis, taking samples of hemolymph in February and dase activity and N-acetyl-ß-glucosaminidase than May 2004 and April 2005. In November 2004, oysters the groups of O. edulis. The phagocytic and antibac- from the same group of Irish flat oyster and C. gigas terial activity and production of NO have been simi- have been put at Cork Bay (Ireland) carrying out a lar in all the oyster groups. Only the acid phosphata- sampling of hemolymph in June 2005. The immuno- se activity was higher in C. gigas compared to the logic parameters analyzed in the hemolymph have groups of O. edulis. All the infected subgroups of O.

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edulis showed lower activities than the correspon- unperturbed in the hemocytes of C. gigas. Some of ding subgroups without detected infections, except the defensive parameters that have been analyzed for the N-acetyl-ß-glucosaminidase activity. The heat could explain, at least in part, the differences of sus- shock proteins of 70 kDa were expressed effectively ceptibility to the bonamiosis between O. edulis and in C. gigas being a useful weapon to get over the C. gigas, while none of them seems to explain the invasion of pathogens with success. In the contact in differences of intra-specific susceptibility among vitro experiments between the parasite and the groups of O. edulis. The global view of the results hemocytes, only in the hemocytes of O. edulis and proved that the higher tolerance to B. ostreae in O. inhibition of the respiratory burst and non-specific edulis does not come together with a higher capa- esterase activity could be observed in the presence city of general defense, but it obeys to a more spe- of B. ostrae, while these parameters remained cific selection process versus this specific pathogen.

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TITLE: DOCTORAL THESIS RESEARCHER: Lipidic metabolism, fatty acids in the larvae culture of the Nóvoa Vázquez, S. pullet carpet shell clam, Venerupis pullastra (Montagu, 1803). Calidad Ovocyte quality , larva and nutritional with DIRECTOR: an approach to the use of lipid microencapsulation. Rodríguez Rodríguez, J.L.

PRESENTATION DATE: February, 2008

SUMMARY The nutritional quality of the bivalve molluscs, Likewise, given the importance that the nutritio- specifically the pullet carpet shell clam (Venerupis nal quality has in the good results of the larvae pullastra), makes that the demand for the con- developments of this species and the total depen- sumption in the human diet be high and in a con- dency that exists of the phytoplankton as main diet tinue increase. However, the evolution of its pro- source, it has been tried to investigate the possible duction by means of extractive fishing since 1997 use of Gelatin- acacia microcapsules (GAM) as nutri- to 2008 shows a reduction up to 48% (from tional supplement. For this, microcapsules have 2.129,473 to 1.113,187 kg). To guarantee and incre- been manufactured with different lipid components ase its production it is not enough with the stated to provide the different essential fat acids but legal controls, but it would be necessary that the microcapsules have been manufactured before that aquaculture to satisfy the market demands. The incorporated a marked fat acid (deuterated arachi- optimization of its culture for the obtaining of the donic acid) in order to demonstrate its non-toxicity, good quality seed is the main objective of this its intake, digestion and assimilation by the fed lar- work. vae.

In view of the above, it has been set out to analy- According to the results obtained in the different ze the larvae cultures of Venerius pullastra, carried studies of the analyzed cultures, it has been conclu- out at the Marine Culture Center of Ribadeo ded first and in relation to the larva development (CIMA) from spawning of parent stocks from the (from 22 days) that its daily growth is of 11.23 natural environment and thus be able to go into µm/day adjusting itself to a lineal regression straight depth as regards to the importance of the ovocyte line of y = mx+b (R2 = 0.986) type and they reach a quality of the different spawning and the nutritio- seed over 750 µm approximately after two months, nal needs of the cultures, performing a study on with a production of 26% from the fertilized egg. the evolution of the fat acids in polar lipids (struc- tural) and neutral lipids (reserve) in cultures with As regards to the contents in fat acids in the diffe- good and bad results. These studies have been per- rent phases of its larval development it has been formed in the different phases of the larvae cultu- concluded that the content in linoleic acid in the res (ovocytes, larvae D, larvae veliger, larvae pedi- lipids of reserve (principal fat acid of the series φ6) veliger and post-larvae). Considering the nutritio- of the ovocytes is directly related to the good results nal dependencies of these phases, from the ovocy- obtained in its culture. An optimum relation φ3/φ6 te recently fertilized to the formation of the larva is suggested in the neutral reserves of ovocytes of D, depending on the stored vitelline reserve in the the pullet carpet shell clam that might be comprised eggs. A mixotrophic stage from larvae D to five in an interval of 1.5 to 2.0 and this relation is being days larvae where there is a mixed diet (endoge- determined by the diet provided to the parent stock nous and exogenous) and, finally, a planktotrophic before the spawning. stage that comprises from the veliger life stage previous to the metamorphosis (larva pediveliger) When studying the results of the evolutions of the up to the previous metamorphosis stage (postlarva) fat acid contents in the different stages it could be and with a diet that turns to be completely exoge- determined that there is no clear mixotrophic nous. period in the larvae of the pullet carpet shell clam

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considering that the larvae D are growing (they phosis. Also the experiences performed with the increase the fact acid of the membrane) and store microcapsules proved that they can be used as a sup- reserves (they increase the neutral fat acids) during plement to the microalgae diet confirming the inta- the early veliger period, which is a characteristic of ke, digestion and assimilation, both in structural an assimilation of the exogenous diet. Moreover, we lipids and reserve. Likewise, it could be used as a could prove that the larvae of the pullet carpet shell working tool to incorporate oils or fats with diffe- clam give priority to its larval development in gro- rent profiles in fat acids and in this way facilitate the wing (lean larvae). lipid metabolism studies of fat acids in larvae of marine bivalves, being able to trace its incorpora- Finally, as regards to the quantity and nutritional tion, as its possible bio-transformation (elongation quality of the food, we conclude that it will have an and/or desaturation) by means of the own enzyma- impact on the growth rates and on the time in tic machinery of the organism. which the larva is ready to perform the metamor-

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TÍTULO DEA: RESEARCHER: Reproduction cycle, biochemistry and pathology of the Louzán Pérez, A. parent stock of the Donax trunculus (Linné, 1758), from a natural bed in Valdoviño (NOT in Galicia), for the obtai- DIRECTOR: ning of the seeds in a hatchery. Martínez Patiño, D.; Nóvoa Vázquez, S.

PRESENTATION DATE: July, 2008.

SUMMARY The natural beds of wedge shell in the Galician onwards, ending towards the month of August. It coast suffered a regression phenomenon. There are shows a maximum spawning in the month of July; fish markets where the wedge shell commercializa- from this month onwards the minimum rate value tion took place in Galicia, but at present they do not of the gonadal condition is given coinciding with any more due to the depletion of this resource. the post-spawning stage. A short rest period is Considering all this and due to the high price rates observed in this species that takes place in of this species, it is of interest the study of the cultu- September. re in farms with restocking aims; reason why the reproduction cycle study starts of the same popula- Along the gametogenic cycle, it presents a varia- tion that will be used as parent stock in the farm, tion in the biochemical composition mainly due to that in this work is on a natural bed located at the changes produced in the gonad and no differen- Valdoviño (NOT of Galicia), beach of Vilarrube, ces are observed in the evolutions among the males, exploited by the guild of Cedeira. females and individuals of undifferentiated gender.

Monthly samples have been carried out in this The evolution in the content in carbohydrates bed. Part of these individuals has been processed for keeps an inverse relation with the gametogenic histological, biochemical and pathological analyses, cycle that reduces as it advances. As the carbohydra- the rest are kept in the farm for the obtaining of tes decrease, the lipid component increases. Its stra- spawning that might allow the follow-up of these tegy could follow a conservative model, stores larvae and postlarvae developments of the species. energy reserves (carbohydrates), used during the gametogenesis and that will transform in maturity A scale has been described to define the different and spawning periods in lipid reserves. The proteins stages of the gonadal maturity of the wedge shell: keep an evolution pattern not related with the E0, sexual rest; E1 of the gametogenesis start; E2 of gametogenic development. advanced gametogeneis; E3 of maturity and spaw- ning and E4 of post-spawning. Starting the month of March the spawning phase starts until July, being more abundant in June and The bivalve molluscs present storage cycles and July. The fertilized eggs suffer segmentation proces- use of energy related directly to the gametogenic ses that will give a trochophore larva after some cycle, therefore it has also been studied the varia- hours. After two days they turn into larva D; the tion in the principal biochemical composition (car- umbonated larva is formed after eight days approxi- bohydrates, lipids and proteins) in the gonad and mately and after 20-25 days they turn into pediveli- the diet less gonad of the wedge shell. Its objective ger larvae. Approximately from 7-8 days later, the is to determine the relation that there is between veil will disappear completely giving place to the this variation and the different stages of its gameto- postlarvae. genic cycle in the natural environment. From the two experimental systems of culture in With the results of this experience it can be con- the natural environmental, the torch without subs- cluded that the wedge shell presents a maturity trate are accepted as optimum as the drums with period and spawning that goes from March sand present water recirculation problems though

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the growth starting of the seed is faster. The detec- organisms of the bacterial, protists and metazoans ted parasites do not suppose a threat for the type. Within the found bacteria, the rickettsia exploitations of this species either for its low constitute a potential danger as mortalities asso- intensity and prevalence or for its scarce pathoge- ciated with this pathology are described in this nic power. The wedge shells appear parasited by species.

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SCIENTIFIC ACTIVITY

3.3. ADVISORY SERVICES FOR THE SECTOR AND THE ADMINISTRATION

A.- Reports issued

Title: Analysis of the current situation of the king Title: Pathological conditions of a seed sample of scallop evisceration. grooved carpet shell clam from Olhao (Portugal), Author: J. Blanco; F. Arévalo; J. Correa. which is going to be sowed in Combarro inside the Applicant: INTECMAR project “Regeneration of Combarro bank after the floods in 2006”. Title: Control program developed in 2007 for the Author: López Gómez, C. detection of the protozoan parasite Bonamia exitio- Applicants: Fishing Association of Pontevedra sa in the flat oyster, Ostrea edulis, of Galicia. Authors: Abollo, E.; Villalba, A.; D. Iglesias. Title: Grow-out system of bivalve molluscs seeds in Applicants: Service of Shellfish Gathering and catamaran placed in the harbour area. Fishing, DXRM., CPAM. Author: Guerra, A. Applicant: DXRM. CPAM. Title: Reproductive state of the razor shell (Ensis arcuatus) in Rodas and San Martiño beaches at Cíes Title: Report on restoration of an experimental tray Islands (Pontevedra). in the Aldan Ria (Pontevedra) for the culture of Author: Guerra Díaz, A. molluscs and crustaceans. Applicants: Local Office of CPAM in Vigo, Fishing Author: Guerra A. Association San José of Cangas (Pontevedra). Applicants: DXRM. CPAM.

Title: State of the barnacle in the Fishing Association Title: Pathological analysis of Crassostrea gigas. of Muxía area (A Coruña). Author: Montes Pérez, J. Author: Molares Vila, J. and Giráldez Rivero, R. Applicant: Remastur SA (Asturias) Applicant: Local Office of CPAM in A Coruña. Title: Pathological control in Haliotis tuberculata Title: State of the anemone in the Fishing coccinea. Association of Lira area (A Coruña). Author: Montes Pérez, J. Author: Molares Vila, J. and Giráldez Rivero, R. Applicant: ICCM. Canarias. Applicants: DXRM. CPAM. Title: Pathological analysis of Ostrea edulis. Title: Report on the meeting of the monitoring com- Author: Montes Pérez, J. mittee of lab activities for the characterization of Applicant: Fishing Association of Noia (A Coruña) pathogen strains. Author: López Gómez, C. Applicants: DXIDP. CPAM.

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B.- Participation on scientific or technical commissions

Participation on the monitoring commission of lab Meeting for the preparation of a report in order to activities for the characterization of pathogen apply the change of the legislation about ASP bioto- strains. López Gómez, C. xin control in king scallops to the EC. Blanco Pérez, J.C. Management of the Marine Resource of sectorial projects on R&D in Galicia. Martínez Patiño, D. Meeting for the planning of an research action/con- tract in order to assess the problem of high dose of Member of the assessment commission of sectorial lead in mature dosinia (Dosinia exoleta) and its pos- programs of applied investigation R&D&I in Galicia. sible solutions. Blanco Pérez, J.C. Guerra, A. Member of the Permanent Commission in the Member of the assessment commission of sectorial Galician Net of Biotechnology in Aquaculture programms of applied investigation R&D&I in (ReGABA). Villalba García, A. Galicia. Martínez Patiño, D. Representative of Galicia in the Monitoring Scientific coordinator of the Marine Reserve with Commission of National Plans JACUMAR of the Fishing Interest called “Os Miñarzos”. Molares Vila, General Secretary of Fishing of Madrid. Guerra Díaz, J. A.

134 CONSELLERÍA DO MAR Annual Report CIMA 2008 SCIENTIFIC ACTIVITY

C.- Other

Participation as adviser expert of the CPAM in a Technical assessment to the project “Integral study coordination meeting of the seed extraction cam- of Ribadeo, Foz, Viveiro, O Barqueiro e Ortigueira paign of the CIMA mussel (Vilanova de Arousa, Rias: hydrography, dynamics, biogeochemistry, sedi- Pontevedra). Fuentes González, J.M. mentology, ecotoxicology and biology of the areas of shellfish interest”. Molares Vila, J. Assessment as an expert in the preparation of the survey and conclusions of a study about Sea Technical assessment for the project “Integral mana- Cooperativism in charge of the companies Ardora gement of barnacle fishing”. Molares Vila, J. Formación e Servicios. DXIDP de la CPAM. De Coo Martín, A. External scientific consultancy of the research group of Marine Ecology and Biodiversity from the IIM Coordination meetings of research groups for the (CSIC) on the project "Pilot study for the establis- annual report of the National Plan JACUMAR hment of an origin alert technological service of “Development of production technology and clams parasitosis in commercial fisheries". Sectorial culture”. Martínez Patiño, D. Program of Marine Environment INCITE 2007. Code 07MMA015CT. Xunta de Galicia. Coordination meetings of research groups for the development of the National Plan JACUMAR Review of a scientific project presented at the “Culture of new species of interest in hatcheries”. Dirección Xeral de recursos Mariños. López Gómez, C. Martínez Patiño, D. Assessmente of the final repor on a project presen- Coordination meetings of research groups from the ted at the Dirección Xeral de Recursos Mariños. Autonomous Communities that participate in the López Gómez, C. National Plan JACUMAR “reuse of waste waters of marine and auxiliary culture establishments located Review of the scientific project presented at the in terrestrial area”. Guerra Díaz, A. DXRM about the sea urchin. Ojea Martínez, J.

Final Report of the project from the European Montes Pérez, J. (2008). Assessment of the Scientific Initiative INTERREG IIIB: Towards Integrated and Technological Research Project. Ministry of Management of Ensis Stocks Project 206 – TIMES. A. Science, Tecnology and Productive Innovation. Cromie; J. Fernández Tajes; M. Gaspar; F. Da Costa National Agency of Scientific and Technological González; D. Martínez-Patiño; J. Méndez Felpeto; S. Promotion. Buenos Aires (Argentina). Nóvoa; J. Ojea and D. Roberts. Board of staff selection for the CIMA and techni- Planning meetings of the exploitation plan of shell- cians under government sponsored interprise. A. fish banks “Lombos do Ulla” and “O Bohído”. O Guerra. Carril. Molares Vila, J. Board of staff selection for the scholarship awards Planning meetings of the anemone exploitation from the DXIDP in order to write the PhD thesis in plan. Santiago de Compostela. Molares Vila, J. the CIMA. C. López.

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SCIENTIFIC ACTIVITY

3.4. SCIENTIFIC AND TRAINING ACTIVITIES CARRIED OUT BY THE STAFF

3.4.1. ORGANIZATION, CHAIRMANSHIP AND PARTICIPATION IN SCIENTIFIC MEETINGS

11th Forum on Marine and Aquaculture Resources at De Santiago, J. A.; Pouso O.; Graña, L.; Lampon V.; Galician Rias. A Toxa (O Grove). Pontevedra Pombal, M. and Guerra, A. System of trays in rever- sed flow in order to grow-out the pullet carpet shell Guerra, A. Organizing Committee. clam (Venerupis pullastra) outside.

López, M.; Rodríguez,C.; Carrasco, J.F.; Brea, Mª.D.; Fernández, A.; De Santiago, J.A.; Martín, F.; Otero, Pais, R.; Rodríguez, C.J.; Guerrero, S. Experiences of M. and Guerra, A. Grow-out of clam and oyster octopus juvenile grow-out (Octopus vulgaris) with seeds in a system of reversed flow outside. different baits. Marcet, P.; Otero, M.; Asela, F. and Guerra, A. Guerrero, S.; Cremades, J.; Salinas, J.M.; Ocaña, S.; Assessment of effluent waste in fish farms. Fuertes, C.; Sanmartín, J.J.; Devesa, S.; Muiños, A.; Pallarés, A. Development of experiences of integra- Andrés M.C.; Lastres M.A.; Varela, M.; Santamaría, I.; ted multitrophic culture applicable to the Galician Guerra, A. Seeds growth of Haliotis tuberculata L. marine aquaculture. using two kinds of diet.

Cerviño Otero, A.; Da Costa, F.; Ojea, J.; Nóvoa, S.; Solís, L.; Alcalde, A.; Conde, Mª.L.; De Coo, A.; Fariña, Martínez Patiño, D. Embryonic and larval develop- J.; García, A.; García, J.; Poza, G.; Santos, I. The effect ment of three species of bivalves: razor shell (Ensis of predator species on commercial bivalve molluscs arcuatus), sword razor shell (Ensis siliqua) and pullet in Galicia. First samples of populations and prelimi- carpet shell clam (Venerupis pullastra). nary conclusions.

Da Costa, F.; Martínez Patiño, D.; Álvarez, F.; Nóvoa, García, J.; Solis, L.; Alcalde, A.; Conde, Mª. L.; De Coo, S.; Ojea, J. Size of first ripening of the old sword A.; Fariña, J.; García, A.; Poza, G.; Santos, I. A current razor shell Solen marginatus (Pennánt, 1777). vision of predators of commercial bivalve molluscs in Galicia. Louzán, A.; Nóvoa, S.; Ojea, J.; Martínez, D. Evolution of the amount of carbohydrates Turing De Coo A. Shellfish gathering of bivalves and econo- the gametogenic cycle of Donax trunculus (Linné, mic crisis in Galicia. 1758). Alcalde, A.; Conde, Mª.L.; De Coo, A.; Crespo, J.A.; Ojea, J.; Martínez, D.; Nóvoa, S.; Catoira, J.L. Fariña, J.; Fernández, D.; García, A.; García, J.; Poza, Reproduction of the sea urchin Paracentrotus lividus G.; Santos, I. The predator species associated to (Lamarck, 1816) in two natural populations: natural banks and culture parks of bivalve molluscs. Cantabrian Sea (Cedeira) and Atlantic Ocean The point of view of the Galician gathering and cul- (Aguiño). ture sector.

Santamaría, I.; Otero, M., Fernández, M. and A. De Coo, A. XX. Culturing the Sea. O Grove. Guerra. Grow-out of the pullet carpet shell clam, Pontevedra. “Floods of clichés.” Venerupis pullastra (Montagu, 1803) in O Carril (Vilagarcía de Arousa, Pontevedra). 2nd Latin American Forum on marine resources and García, A.; Solís, L.; Alcalde, A.; Conde, Mª. L.; De aquaculture. FIRMA 2008. Coo, A.; Fariña, J.; García, J.; Poza, G.; Santos, I.; Cumaná (Venezuela) Crespo, J.A.; Fernández, D. First results of the preda- tor activity on comercial bivalve molluscs, by fish, Linares Cuerpo, F. International Cooperation on crustaceans, echinoderms and marine gastropods in Fishing and Aquaculture in Galicia. Galicia; action mechanism.

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Martínez, D.; Cerviño Otero, A.; Louzán, A.; Da Da Costa, F.; Ojea, J. Nóvoa, S.; Martínez Patiño, D. Costa, F.; Ojea, J.; Nóvoa, S. Production of species Biochemical study of larval development in the with shellfish gathering interest in the Plant of razor clam Ensis arcuatus. Marine Culture of Ribadeo (CIMA). Da Costa, F.; Ojea, J.; Nóvoa, S.; Martínez Patiño, D. Nóvoa, S.; Ojea, J.; Martínez, D.; Louzán, A. Lipid Fatty acid composition of the razor clam Solen mar- content change in different stages of the gametoge- ginatus during larval development. niccycle of Donax trunculus (Linné, 1758).

Lastres, M.; Andrés, C.; Santamaría, B.; Guerra, A. XX International Congress of Genetics. Berlín, Training Project for aquaculture technicians about Alemania the development of molluscs in hatchery. Martín-Gómez, L.; Villalba, A.; Abollo, E.. Molecular identification of differentially regulated genes of 7th meeting on Microbiology of the Aquatic the European flat oyster (Ostrea edulis) in response Environment in 2008 (Bilbao) to experimental infection by Bonamia ostreae (Haplosporidia). Doce, A.; López, C.; Diéguez, A.L.; González, A.; Balboa, S. and Romalde, J.L. histopatological and X European Multicolloquium of Parasitology (EMOP bacterial study of cultured clams (Ruditapes decussa- X). Paris, Francia tus and Ruditapes philippinarum) in Galicia. Abollo, E., Ramilo, A.; Villalba, A. DNA probes as tools for the specific diagnosis of Bonamia exitiosa IV Sole Workshop. CCMAR. University of the (Haplosporidia) Algarve. Faro. Portugal Pascual, S.; Abollo, E. Myxosporean infection in fro- García de la Banda, I.; Lobo C.; Martín I.; Moriñigo, zen blocks of Patagonian hakes. M.A.; Arijo, S.; León-Rubio, J.M.; Tapia-Paniagua, S.; Chabrillón, M.; Pazos, Spain-Canada AIS Workshop. Vigo. Pontevedra G.; Linares, F.; Moreno-Ventas, X.; Canteras J.C. Pbp11 probiotic use on Solea senegalensis (Kaup) Fuentes J.M. Participation. larval and postlarval culture”. 17th International Pectinid Workshop

Aquaculture Europe 2008. Cracow, Poland. J. Blanco. Preparatory meetings. Member of the organizing committee. Carballal M.J.; Díaz S.; Louzán A.; Martínez D. Parasites and cells of the internal defence system of the bivalve Donax trunculus. Pollicipes International Conference. Sines (Portugal)

López, C., Rodríguez Díaz, R.; Darriba Couñago, S. Molares Vila, J. Member of the Scientific Parasites and pathological condition of two natural Commission. beds of razor clam, Solen marginatus (Pennánt, 1777), in Galicia (NW Spain). Molares Vila, J. Effect of oil spill from tanker “Prestige” upon goose barnacle populations in Cerviño Otero, A.; Da Costa, F.; Ojea, J.; Nóvoa, S.; Galicia (NW Spain). Martínez Patiño, D. Embryonic, larval and post-larval development in the pullet carpet shell, Venerupis Molares Vila, J. Management of the barnacle in pullastra (Montagu, 1803). Galicia.

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100th National Shellfisheries Association Annual Identifying familiar relationships in cohorts of Meeting, Providence (RI, EE. UU.) April 2008 Ostrea edulis using microsatellite loci.

Martin-Gómez L.; Abollo E.; Villalba A. Identification Cao A., Abollo E.; Pardo, BG; Martínez, P.; Villalba, A. of differentially expressed genes in the European Identification of the Perkinsus spp., occurring in the flat oyster (Ostrea edulis) in response to infection by Spanish coast and evaluation of their intraspecific the haplosporidian Bonamia ostreae. variability . Pardo BG, Castro J; López, A.; Pino-Querido, A.; Bouza, C.; Fuentes, J.; Villalba, A.; Martínez, P.

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3.4.2. SUPERVISION OF DOCTORAL THESIS AND INTERN TRAINING

• Higher qualified degree

A.- PhD Thesis

In CIMA

Issue: Production of high quality mussel seeds in hat- Issue: Study of a disseminated neoplasia and other chery. pathological changes that affect the populations of PhD student: Domínguez Pérez,L cockle, Cerastoderma edule, in Galicia Degree: Graduate in Biology PhD student: Díaz Costas, S. Tutor and supervisor: Fuentes González, J.M. (tutor); Degree: Graduate in Sea Sciences Fuentes González and Villalba García, A. (supervi- Tutor and supervisor: Carballal Durán, M.J. (tutor sors). and co-supervisor), Villalba García, A. (co-supervi- sor.) Issue: Accumulation of ASP toxins in the King Scallop Pecten maximus Issue: Molecular characterization of genes involved PhD student: Mauríz Pereira, A. in the immune response of the flat oyster (Ostrea Degree: Graduate in Sea Sciences edulis) and the Pacific (Crassostrea gigas) against Tutor and supervisor: Blanco Pérez, J. pathogens. PhD student: Martín Gómez, L. Issue: Accumulation of DSP toxins in the mussel Degree: Graduate in Biology Mytilus edulis Tutor and supervisor: Abollo Rodríguez, E (tutor and PhD student: Escudeiro Rossignoli, A. co-supervisora), Villalba García, A. (co-supervisor) Degree: Graduate in Biology Tutor and supervisor: Blanco Pérez, J. Issue: Perkinsosis in the Spanish coast: characteriza- tion of the taxonomic variants of the parasite, of its Issue: Toxic and harmful episodes because of phyto- life cycle and its immunological answer to the host. plankton in Chile PhD student: Fernández Boo, S. PhD student: Álvarez Vergara, G. Degree: Graduate in Biology Degree: Aquaculture expert Tutor and supervisor: Villalba García, A.(tutor and Tutor and supervisor: Blanco Pérez, J. co-supervisor), Cao Hermida , A. (co-supervisor), Martínez Portela, P. (co-supervisor). Issue: Pathology of the sword razor shell (Ensis sili- qua) in Galician coast. Study of the neoplasia in soli- Issue: Culture of new species of molluscs with com- nedae in Galicia. mercial interest. Culture of Donax trunculus. PhD student: Ruíz Pérez, M. PhD student: Louzán Pérez, A Degree: Graduate in Sea Sciences Degree: Graduate in Sea Biology Tutor and supervisor: López Gómez, C. (tutor and co- Tutor and supervisor: Sánchez López, J.L.; Martínez supervisor) and Tajes, J. (co-supervisor, UC) Patiño, D; Nóvoa Vázquez, S.

Issue: The exploitation of the cockle Cerastoderma Issue: Culture of three species of solinedae with edule in Galicia. Biology, ecology and Management commercial interest in Galicia. of natural banks. PhD student: da Costa González, F. PhD student: Santos Piñeiro, I. Degree: Graduate in Biology Degree: Graduate in Biology Tutor and supervisor: Sánchez López, J.L. (tutor); Tutor and supervisor: Molares Vila, J Martínez Patiño, D;

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Issue: Optimization of pullet carpet shell clam cultu- Specialization: Specialist Technician in Pathological re Venerupis pullastra. Anatomy PhD student: Cerviño Otero, A Degree: Graduate in Biology Tutor: Ojea Martínez, J. Tutor and supervisor: Martínez Patiño, D Vocational intern: Costa Costa, D. Specialization: Advanced Technician of Fish Farming Title: Study of the reproduction in a natural envi- Production ronment of the grooved carpet shell clam Ruditapes decussatus and its fitting-out in a hat- Tutor: Ojea Martínez, J. chery.. Vocational intern: Rodríguez Castro, B. PhD student: Ojea Martínez, J. Specialization: Advanced Technician of Fish Farming Degree: Graduate in Biology Production Supervisor: Lina Abad (UC) and Martínez Patiño, D. Tutor: Nóvoa Vázquez, S. Vocational intern: Cardoso Loureiro, I. In other centers Specialization: Advanced Technician of Analysis and Monitoring Issue: Accumulation of metal in bivalve molluscs. PhD student: Saavedra Yánez, Y. Tutor: Nóvoa Vázquez, S. Degree: Graduate in Chemistry Vocational intern: Brión Bello, R. Workplace: INTECMAR Specialization: Advanced Technician of Analysis and Supervisor: Blanco Pérez, J. and González, A. (USC) Monitoring

Issue: Effect of cooking processes and preservation Tutor: Villalba García, A. of toxin concentration in molluscs. Vocational intern: Vicente Fernández, R. PhD student: Vidal, A. Specialization: Advanced Technician of Analysis and Degree: Graduate in Chemistry Monitoring Workplace: INTECMAR Supervisor: Blanco Pérez, J.C. Tutor: Villalba García, A. Vocational intern: Ramos Caamaño, J.M. Issue: Immunodetection of mussel larvae in Specialization: Advanced Technician of Analysis and Galician coast through monoclonal antibodies Monitoring M22.8 and M36.5. PhD student: Pérez Estévez, D. Tutor: López Gómez, C. Degree: Graduate in Biology Vocational intern: de Castro Puente, M. Workplace: Immunology area, Faculty of Sciences, Specialization: Specialist Technician in Pathological University of Vigo Anatomy Supervisor: González-Fernández, A. and Fuentes Tutor: Montes Pérez, J. González, J.M. Vocational intern: Romanos Mondragón, L. Specialization: Specialist Technician in Pathological Anatomy • Vocational interns Tutor: Montes Pérez, J. Tutor: Carballal Durán M. J. Vocational intern: Balsa Freire, S. Vocational intern: Riveiro Arjomil, F. Specialization: Specialist Technician in Pathological Specialization: Specialist Technician in Pathological Anatomy Anatomy

Tutor: Carballal Durán M. J. Vocational intern: Rodríguez Galván B.

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• On-the-job training Student: Castelo Triñanes, A.M. Place: IES Fogar Prov. Principe Felipe A.- University degrees Length: 710 hours Tutor: Guerra Díaz, A. Student: Vega Aramburu, J. Place: USC Student: Chauvet, A. Length: 2 months Place: Lycée de la Mer et du Litoral. University of la Tutor: Martínez Patiño, D. Rochelle. France Length: 96 hours Student: Vilas Arrondo, N. Tutor: Guerra Díaz, A. Place: USC Length: 2 months Student: Castro Otero, M.T. Tutor: Nóvoa Vázquez, S . Place: IGAFA Length: 440 hours Student: Veiga Suárez, J.A. Tutor: Martínez Patiño, D. Place: USC Length: 2 months Student: Gutiérrez Alonso, S. Tutor: Ojea Martínez, J. Place: IES Lucus Augusti- Analysis and Monitoring Length: 380 hours Tutor: Martínez Patiño, D. B.- Technicians Student: Miranda Teijeiro, P Student: Romero Suárez, A. Place: IES Lucus Augusti- Analysis and Monitoring Place: IES Fogar Prov. Principe Felipe Length: 190 hours Length: 710 hours Tutor: Martínez Patiño, D. Tutor: Guerra Díaz, A.

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3.4.3. COURSES GIVEN

3rd University Cycle Program: Marine Biology and Parada Encisa, J.M.; Sánchez Mata, A.; Molares Vila, Aquaculture. USC (Interuniversity Program). J.

Course: Ficotoxicity episodes in molluscs. Blanco, J. International University Menéndez Pelayo. Vilagarcía de Arousa. Pontevedra. Research on oce- Course: Pathological changes and immune system in anography of Galicia waters: present and futureo. A bivalve molluscs. view of the phytoplankton through pigments. Abollo Rodríguez, E.; Carballal Durán M. J.; Villalba Zapata Gago, M. García A. Worshop: Le conscenza attuali per una gestione sos- Course: Molluscs culture techniques. Study of the tenibile della mitilicoltura off-shore in Altro bonamiosis impact. Adriático. Instituto Centrale per la Ricerca Scientifica Guerra Díaz, A.; Montes Pérez, J. e Tecnologia Applicata al Mare (ICRAM), Universita Ca`Foscardi di Venezia. Course: European Master of Science in Marine Guerra, A. y Grassia, L. The organization of mussel Environment and Resources”. Villalba. A. culture in Galicia.

Course: Histology and Histopathology of Aquatic Course of Development Cooperation and Animals. University of the Basque Country. Aquaculture. CETMAR- IGAFA Given seminars: Title: Influence of molluscs hatcheries as a boost to • General Histopathology of Molluscs the development of molluscs aquaculture. Guerra, • Microscopic observations of general mollusc his- A. topathology • Neoplastic lesions in molluscs 2nd Latin American Forum of Marine Resources and • Identification of neoplastic lesions Aquaculture (II FIRMA). University of Oriente (UDO). • Round Table: Mollusc and Fish Histopathology in ecosystem health assessment. Minicourse: Bivalve molluscs culture: Aquaculture A Villalba. without feeding stuffs. Guerra, A.

Course: EGAP: Sampling in sea environment. Data Workshop. “Management and culture of the sea processing and computer uses.[FC08134]. urchin” Director-responsible: Guerra, A. Martínez Patiño, D. Professors from the CIMA: Parada Encisa, J.M.; Sánchez Mata, A.; Molares Vila, J.; C. López; Guerra, 2nd REGABA Course of Molecular Phylogeny (10 h). A. Organized by the Galician Net of Biotechnology in Aquaculture. Santiago de Compostela. Abollo, E. Course: Scientific and technical assessment to shell- fish gathering: Sampling and data analysis. Summer Course UO. The aquaculture, a growth acti- Delegación Territorial de la Consellería de Pesca vity. Martínez Patiño, D.; Nóvoa Vázquez, S. y Ojea (Celeiro)-CIMA. Martínez, J.

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3.4.4. PARTICIPATION ON EXAMINING BOARDS TO JUDGE THESIS

J. Blanco Perez -PhD student: Comesaña Lestayo M.P. -Title: “Estudio, mediante LC-MS, de la variabilidad -Aquaculture Institute. USC del perfil y contenido de toxinas, en poblaciones de Dinophysis spp., agentes de episodios DSP en las Rías Villalba Garcia, A. Baixas Gallegas.” -Title: “Investigations into the life cycle of the protis- -PhD student: Pizarro Nova, G. tan Bonamia ostreae and the possible role of -University of Vigo carriers.” -PhD student: Sharon Lynch Martínez Patiño, D. y Guerra Díaz, A. -University Collage Cork, Irlanda -Title: Metabolismo lipídico, ácidos grasos en el cul- tivo larvario de almeja babosa, Venerupis pullastra Villalba Garcia, A. (Montagu, 1803) “Calidad ovocitaria, larvaria y -Title: “Principales patologías de la almeja fina, nutricional con una aproximación al uso de la micro- Ruditapes decussatus (Linnaeus, 1758), del litoral encapsulación lipidica.” onubense con especial referencia a la parasitosis por -PhD student: Nóvoa Vázquez, S. Perkinsus olseni Lester y Davis, 1981: diagnóstico, -University of Santiago de Compostela transmisisón y efectos sobre la almeja.” -PhD student: Navas Triano, J.I. Carballal Durán, M. J. -Faculty of Biology, University of Sevilla -Title: “Comparación de parámetros inmunológicos entre poblaciones de Ostrea edulis con diferente susceptibilidad a la bonamiosis y la especie resisten- te Crassostrea gigas”.

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3.4.5. EDICIÓN DE PUBLICACIONES CIENTÍFICAS

Blanco Pérez, J.C. Editor of the “Revista Galega dos Villalba García A. (member of the Publishing Recursos Mariños” (4 series) Committee). Journal on Marine Biology and Oceanography.Faculty of Sea Sciences, University of Villalba García, A. (Editor). Workshop for the analy- Valparaíso, Chile. sis of the impact of perkinsosis to the European shellfish industry. CIMA. Consellería de Pesca e Guerra Díaz, A. (member of the Publishing Asuntos Marítimos da Xunta de Galicia, Vilanova de Committee). X Foro de Rec. Mar. e da Ac. das Rías Arousa, Spain. Centro Tecnológico del Mar – Gal. e I Foro Iberoamericano de Rec. Mar. y Acuic. 10. Fundación CETMAR, Vigo, Spain. 164 p. Santiago de Compostela. ISBN 978-84-608-0755-I. 710 pp. Villalba García A. (editor checker). Diseases of Aquatic Organisms. INTER RESEARCH, Oldendroff/Luhe, Germany.

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3.4.6. REVIEW OF ARTICLES IN SCIENTIFIC PUBLICATIONS

Toxicon. Blanco Pérez, J.C. Assessment in a research project of "Livestock and Fishing Technology” area, from the National Agency Boreal Environment Research. Blanco Pérez, J.C. of Scientific and Technological Promotion (ANPCYT) in Argentine. Villalba García, A. Emergencias. Blanco Pérez, J.C. Journal of Invertebrate Pathology: 1 manuscript. Marine Druggs. Blanco Pérez, J.C. Villalba García, A.

Harmful Algae. Blanco Pérez, J.C. Journal of Marine Biology and Oceanography: 1 manuscript. Villalba García, A. Journal Invertebrate Pathology. Carballal Durán M.J. (2 artículos) Review of two chapters of the “Manual of Diagnostic Tests for Aquatic Animals, Sixth Edition", Diseases of Aquatic Organisms. Carballal Durán M.J. which is published by the World Organization of Animal Health. Villalba García, A. Ciencias Marinas. Guerra, A.

Diseases of Aquatic Organisms: 5 manuscripts. Villalba García, A.

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3.5. STAFF TRAINING

3.5.1. VISITS TO OTHER CENTRES

Molares Vila, J. Fernández Boo, S. -Issue: Layout of cirripedia larvae in the - Issue: Analysis of the loci microsatélite variabi- Oceanographycal Campaign about the study of lity of the protozoo genome Perkinsus olseni, bathyal bottoms of carbonated crusts in the bivalve molluscs parasite. Selva (NW Galicia). - Length: 1 week, November 2008 -Length: 28 days - Centre: Department of Genetics, Faculty of -Centre: Sarmiento de Gamboa Veterinary, University of Santiago de Compostela, Campus de Lugo Martín Gómez, L. -Issue: General aspects of Microarray technology Louzán Pérez, A. and its application in the study of infectious -Issue: Culture of Donax clam (Donax trunculus), processes of Ostrea edulis. reproductive cycle, culture stages and extraction -Length: 1 month from the natural environment. -Centre: The Netherlands Cancer Institute. -Length: 15-19 September 2008 Amsterdam. Netherlands -Centre: IFAPA “Agua del Pino”. Cartaya (Huelva) Balsa Freire, S. -Issue: Stay in the Department of Microbiology of the Faculty of de Biology -Length:15 May – 15 June 2008. -Centre: University of Santiago de Compostela.

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3.5.2. COURSE ATTENDANCE

EGAP Course. Toma de mostras no medio Mariño. “Dinámica de Sistemas” (on line). Universidad Tratamento de datos e aplicacións informáticas. Politécnica de Catalunya. Santos, I. [FC08134]. CIMA (Vilanova de Arousa) Campaña Ferro, E.; Gregorio Chenlo, M.V.; Penas “Creación de modelos en Ecología y Gestión de Pampin, E.. Recursos Naturales” (on line). Universidad Politécnica de Catalunya. Santos, I. “Curso de Filogenia”. REGABA. USC. Santiago de Compostela. “Microscopía óptica básica”. IZASA. Santander. Díaz Costa, S. Cerviño Otero, A.; Ojea, J.; Louzán, A.

“Introducción a la bioestadística”. REGABA. USC. “Curso teórico-práctico de Estadística Descriptiva”. Santiago de Compostela. EGAP. Santiago de Compostela. Cerviño Otero, A.; da Díaz Costa, S.; Domínguez, L. Costa González, F.; Domínguez, L.

“Bioinfomática: “¿Cómo puede ayudarme el INB a tra- “Cromatografía de gases”. EGAP. Bergondo. A Coruña. bajar con mis datos?” REGABA. USC, Santiago de Cerviño Otero, A.; Nóvoa, S. Compostela. Comesaña Lestayo M. P., Ramilo Álvarez A. “Cromatografía líquida de alta resolución, HPLC, aco- plada a espectrometría de masas LC-MS”. EGAP. “Introducción a la inferencia de filogenias molecula- Bergondo, A Coruña. res”. REGABA. USC. Santiago de Compostela. Nóvoa, S. Abollo Rodríguez E., Comesaña Lestayo M.P., Díaz Costas S., Ramilo Álvarez A. “PhotoShop CS y PowerPoint”. Ribadeo. Lugo. Álvarez, A.; Bouzamayor, V.; Brión, R.; Cerviño Otero, “Epidemiología en Acuicultura”. REGABA. USC, A.; Costa, D.; da Costa, F.; Louzán, A.; Martínez, D.; Santiago de Compostela. Neira, C.; Nóvoa, S.; Ojea, J. Comesaña Lestayo M.P., Villalba García A. “Curso Básico de Patología en Acuicultura y Patología “ Introducción a la inmunología de peces y moluscos”. de Piscicultura”. USC, Santiago de Compostela. REGABA. USC, Santiago de Compostela. Balsa Freire, S. Comesaña Lestayo M.P., Fernández Boo S., Martín Gómez L. “Control y Prevención en Acuicultura”. USC, Santiago de Compostela. “Citometría de flujo para usuarios”. REGABA. UV, Vigo. Balsa Freire, S Ramilo Álvarez, A. Seminario galego “Mecanismos implicados en los pro- “Introducción a la Bioestadística para investigadores”. cesos de oxidación lipídica y la relevancia de los antio- REGABA. USC Santiago de Compostela. xidantes”. IIM (CSIC), Vigo. Díaz Costas S.; Ramilo Álvarez A. Cores González, M.J.; Pazos Sieira, G.

“I Curso REGABA de Immunología”. REGABA. USC, “Offshore Mariculture 2008”. Alicante. Santiago de Compostela. Pazos Sieira, G. Martín Gómez L.; Domínguez, L. VII Curso teórico práctico de citometría de flujo. “Curso básico de Patología en Acuicultura: Patología Madrid. CIEMAT piscicultura”. INTECMAR. Santiago de Compostela. Ruíz, M. Díaz Costa, S.

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Curso Aplicado de SPSS y R. Facultad de Ingeniería “Curso Básico de Prevención y Control. Patología de Industrial de la Universidad de Vigo. Organizado por la Peces”. Instituto de Acuicultura, USC, Santiago de Asociación de Oceanógrafos de Galicia y UV. Compostela. Ruíz, M. Pazos Sieira, G.; Soto Fraga, N.; Álvarez Fernández, M.J.

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3.6. COLLABORATION AGREEMENTS

Collaboration agreement with OPMEGA for the deve- da denominación de orixe Mexillón de Galicia, in lopment of the project RITOX. order to start the research project: “Busca de marca- dores genéticos para la identificación del mejillón de Collaboration agreement with the Consellería de Galicia”. Innovación e Industria, CPAM, Fundación CETMAR, INTECMAR and USC for the development of the action Collaboration agreement between the Xunta de EPITOX. Galicia through the CPAM and the USC in order to star the research project: National Plan JACUMAR Collaboration agreement with INTECMAR and the “Cultivo y Gestión del erizo de mar”. Consello Regulador de la Denominación de Origen Mejillón de Galicia, for the development of a JACU- Collaboration agreement between the Xunta de MAR subproject (Jacumar-mejillón). Galicia through the CPAM and the UDC in order to start the research project: National Plan JACUMAR Collaboration agreement with INTECMAR and CSIC, “Optimización del cultivo de la almeja en criadero." for the development of a subproject of JACUMAR (Jacumar-PSP). Collaboration agreement between the Xunta de Galicia through the CPAM and the USC in order to Collaboration agreement with the Universidad start the research action: “Control microbiológico de Católica del Norte, inside the Collaboration of this los criaderos de moluscos.” Institución with the CPAM. Collaboration agreement between the Xunta de Collaboration agreement between the CPAM and the Galicia through the CPAM and the IEO in order to CETMAR for the development of a research action: start the research action: “Puesta a punto de un siste- “Plan de actuación para la recuperación de los bancos ma de cultivo de paralarvas del pulpu Octopus vulga- marisqueros “Lombos do Ulla” y “O Bohído” (Ría de ris e importancia de la composición bioquímica en la Arousa).” dieta larvaria.”

Collaboration agreement between the Xunta de Collaboration agreement between the Xunta de Galicia, the USC and the IFAPA in order to start the Galicia through the CPAM and the IEO in order to research project “La perkinsosis en el litoral español: start the research action: “Captación, preengorde y caracterización de variantes taxonómicas del parásito, cultivo de volandeira (Aequipecten opercularis) y viei- de su ciclo de vida y de la respuesta inmunitaria del ra (Pecten maximus).” hospedador.” Collaboration agreement between the Xunta de Collaboration agreement between the Xunta de Galicia through the CPAM and the Asociación de Galicia through the CPAM and the USC in order to Acuicultores “Illa do Santo” de Bueu (Pontevedra) for start the research action: “Estudio de factores ambien- the culture of fish in cages. tales y posibles patógenos que afectan al cultivo de almeja fina (Ruditapes decussatus) en la Ría de Collaboration agreement between the Xunta de Pontevedra.” Galicia and the USC in order to start the research action: “Estudio de factores ambientales y posibles Collaboration agreement between the Xunta de patógenos que afectan al cultivo de la almeja fina Galicia through the CPAM an the Consello Regulador (Ruditapes decussatus) en la Ría de Pontevedra”.

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3.7. OUTSIDE VISITORS

CIMA from Corón CIMA from Ribadeo

Filomena Livramento González Henríquez, N. and Moreno Batet, E. Universidade Agostinho Neto. Luanda. Angola. Instituto Canario de Ciencias Marinas. Gran Canaria Preparation o PhD thesis. Analysis of toxins in phyto- Culture of sea urchin Paracentrotus lividus in hat- plankton and bivalves from Angola. chery.

Dra. Guisla Boehs Nuno Miguel Campos Mendes Sousa Pereira Professor of the “Universidade Estadual de Santa Universidade dos Açores. Portugal Cruz” (Ilhéus, Bahía, Brasil) Culture of grooved carpet shell clam Tapes decussa- Parasites and sea molluscs histopathology of econo- tus in hatchery.. mic interest in estuarine areas in the South of Bahía, Brasil. Marisol Izquierdo López and Gercende Courtois de Viçose Consejería de Educación, Cultura y Deportes. Instituto Canario de Ciencias Marinas. Department: Aquaculture. Canary Government. Culture and management of the perlemoen abalone H. tuberculata spp.

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3.8. PUBLICATIONS

3.8.1. ARTICLES IN JOURNALS LISTED IN THE SCI (Science Citation Index)

Abollo E.; Ramilo A.; Casas S.M.; Comesaña P.; Cao Da Silva, P.M.; Comesaña, P.; Fuentes, J.; Villalba, A. A.; Carballal M.J.; Villalba A. (2008). First detection (2008). Variability of haemocyte and haemolymph of the protozoan parasite Bonamia exitiosa parameters in European flat oyster Ostrea edulis (Haplosporidia) infecting flat oyster Ostrea edulis on families obtained from brood stocks of different grown in European waters. Aquaculture. 274:201- geographical origins and relation with infection by 207. the protozoan Bonamia ostreae. Fish and Shellfish Immunology, 24:551-563. Azevedo, C.; Casal, G.; Montes, J. (2008). Ultrastructural life cycle of Haplosporidium mont- Da Silva, P.M.; Fuentes, J.; Villalba, A. (En prensa). forti (Phylum Haplosporidia), a parasite of the far- Differences in gametogenic cycle among strains of the med abalone, Haliotis tuberculata (Gastropoda). European flat oyster Ostrea edulis and relationship Journal of Parasitology, 94: 137-142. between gametogenesis and bonamiosis. Aquacul- ture, 287:253-265. Boehs, G.; Lenz, T.M.; Villalba, A. (En prensa). Xenomas in Crassostrea rhizophorae (Ostreidae) Fernandez-Tajes, J.; Martínez-Lage, A.; Freire, R.; from Camamu Bay, Bahía, Brazil. Brazilian Journal of Guerra, A.; Méndez, J. 2008. Genome sizes and kar- Biology. yotypes in the razor clams Ensis arcuatus (Jeffreys, 1865) and E. siliqua (Linnaeus, 1758). Cah. Biol. Mar. Cao, A.; Abollo, E.; Pardo, B.G.; Martínez, P; Villalba, 49: 79-85. A. Identification of the Perkinsus spp., occurring in the Spanish coast and evaluation of their intraspeci- Fraga, S.; Penna, A.; Biancon, I.; Paz, B.; Zapata, M. fic variability. Journal of Shellfish Research, 27 (4): (2008). Coolia canariensis sp. nov. (Dinophyceae), a 994. new epiphytic benthic dinoflagellate from the Canary Islands. Journal of Phycology, 44: 1060-1070. Casas, S.M.; Reece, K.S.; Villalba, A.; La Peyre, J.F. (2008). Continuous Culture of Perkinsus mediterra- Garrido, J.L.; Rodríguez, F.; Zapata, M. (en prensa). neus, a Parasite of the European Flat Oyster Ostrea Occurrence of loroxanthin, loroxanthin decenoate edulis, and Characterization of its Morphology, and loroxanthin dodecenoate in Tetraselmis species Propagation and Extracellular Proteins In Vitro. (Prasinophyceae, Chlorophyta). Journal of Journal of Eukaryotic Microbiology, 55:34-43. Phycology.

Da Costa, F.; Darriba, S. & Martínez-Patiño, D. 2008. La Peyre, M.K.; Casas, S.M.; Villalba, A.; La Peyre, J.F. Embryonic and larval development of Ensis arcuatus (2008). Determination of the effects of temperature (Jeffreys, 1865) (Bivalvia: Pharidae). J. Moll. Stud. 74: on viability, metabolic activity and proliferation of 103-109. two Perkinsus species, and its significance to unders- tanding seasonal cycles of perkinsosis. Parasitology, Da Costa, F. & Martínez-Patiño, D. (en prensa). 135:505-519. Culture potential of the razor clam Solen margina- tus (Pennánt, 1777). Aquaculture. doi: 10.1016. J. López-Flores. I.; Robles, F.; Valencia, J.M.; Grau, A.; Aquaculture.2008.11.001. Villalba, A.; De La Herrán, R.; Garrido-Ramos, M.A.; Ruiz-Rejón, C.; Ruiz-Rejón, M.; Navas, J.I. (2008). Da Silva, P.M.; T. Renault; J. Fuentes; A. Villalba. Detection of Marteilia refringens using nested-PCR (2008). Herpesvirus infection in families of European and in situ hybridisation in Chamelea gallina from flat oysters Ostrea edulis obtained from brood the Balearic Islands (Spain). Diseases of Aquatic stocks of different geographical origins, through on Organisms, 82:79-87. growing in Galicia (NW Spain). Diseases of Aquatic Organisms 78:181-188.

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Martín-Gómez, L.; Abollo, E; Villalba, A. Pascual, S.; Abollo, E. Myxosporean infection in fro- Identification of differentially expressed genes in zen blocks of Patagonian hakes. Journal of Food the European flat oyster (Ostrea edulis) in response Protection, 71 (11):2316-2327. to infection by the haplosporidian Bonamia ostreae. Journal of Shellfish Research, 27 (4): 1030. Rossignoli, A. & Blanco, J. 2008. Cellular distribution of okadaic acid in the digestive gland of Mytilus Ojea, J.; Pazos, A.J.; Martínez, D.; Nóvoa, S.; García- galloprovincialis (Lamarck, 1819). Toxicon, 52(8): Martínez, P.; Sánchez, J.L. and Abad, M. 2008. Effects 957–959. of temperature regime on broodstock conditioning of Ruditapes decussatus. Journal of Shellfish Saavedra, Y., Gonzalez, A., & Blanco, J. 2008. Research, 27 (5): 1093-1100. Anatomical distribution of heavy metals in the sca- llop Pecten maximus. Food Additives and Parada, J.M.; Molares, J. 2008 Natural mortality of Contaminants, 25, 1339–1344. the cockle Cerastoderma edule (L.) from the Ría of Arousa (NW Spain) intertidal zone. Revista de Biología Marina and Oceanografía 43(3): 501-511.

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3.8.2. ARTICLES IN JOURNALS NOT LISTED IN THE SCI

Da Costa F.; Ojea, J.; Nóvoa, S.; Martínez Patiño, D. Lastres, M.A.; Andrés, M.C.; Santamaría, I.; Rubirosa, Biochemical study of larval development in the M.J.; Ancosmede, C.; Pérez, N.y Guerra, A. Cultivo razor clam Ensis arcuatus. Resource Management. and gestión de la oreja de mar Haliotis tuberculata Aquaculture Europe 2008. 167-168. spp. X Foro Rec. Mar. Acuic. Rías Gal. 10: 461-465.

Da Costa F.; Ojea, J.; Nóvoa, S.; Martínez Patiño, D. Louzán, A.; Cerviño-Otero, A.; Nóvoa, S.; Ojea, J.; Fatty acid composition of the razor clam Solen mar- Martínez, D. Seguimiento del ciclo reproductor and ginatus during larval development. Resource desarrollo larvario de una población de coquina Management. Aquaculture Europe 2008. 165-166. Donax trunculus (Linné, 1758). X Foro Rec. Mar. Acuic. Rías Gal. 10: 475-481. De Santiago, J.A.; Fernández, A.; Ruíz, M. y Guerra, A. Preengorde de almeja babosa (Venerupis pullas- Macías, J.C.; F. Aguado, N.; Henríquez, S.; Guerrero, tra, Montagu, 1803), almeja fina (Ruditapes decussa- A.; Estévez, J. Mª; Valencia, J. Cremades. Acuicultura tus, Linné, 1758) y almeja japonesa (Ruditapes phi- Integrada: desarrollo de experiencias de cultivos lippinarum, Adams & Revé, 1850) en tres sistemas de multitróficos en la costa española. X Foro Ac. Rec. Preengorde. X Foro Rec. Mar. Acuic. Rías Gal. 10: Mar. Rías Gal. 10: 483-490. 2008. 381-389. Molares, J.; Parada, J.M.; Navarro-Pérez, E.; Carballal Durán, M. J.; Díaz, S.; Louzán, A.; Martínez, Fernández, A. (2008). Variabilidad interanual de las D. Parasites and cells of the internal defence system ventas de los principales recursos marisqueros de of the bivalve Donax trunculus. European Galicia and su relación con las condiciones ambien- Aquaculture Society, Special Publication Nº 37, pági- tales. Revista Galega dos Recursos Mariños (Art. Inf. nas 124-125. Tecn.) 2(1): 1-42.

Cerviño Otero, A.; Da Costa, F.; Ojea, J.; Nóvoa, S.; Ojea, J.; Martínez, D.; Nóvoa, S.; Cerviño-Otero, A.; Martínez Patiño, D. Embryonic, larval and post-larval Catoira, J.L. Datos biométricos e índices de condición development in the pullet carpet shell, Venerupis del erizo de mar (Paracentrotus lividus, Lamarck pullastra (Montagu, 1803). Resource Management. 1816) en cuatro localidades de Galicia. X Foro Rec. Aquaculture Europe 2008.133-134. Mar. Acuic. Rías Gal. 10: 525-530.

Gabin, C.; Otero, M.; y Guerra, A. Navajas and lon- gueirones en Galicia (N.O. de España). X Foro Rec. Mar. Acuic. Rías Gal. 10: 415-423.

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3.8.3. CONTRIBUTIONS TO BOOKS

Patiño, M. 2008. Centro de Cultivos Marinos de de Galicia, Vilanova de Arousa, Spain. Centro Ribadeo (Lugo): Tecnología del mar en Galicia. En: Tecnológico del Mar-Fundación CETMAR, Vigo, Lucensia. Miscelánea de cultura e investigación. Spain. pp: 78-109. Biblioteca Seminario Diocesano (Ed.). Lugo. SIN 1130-6831. Nº 37: (Vol. XVIII). pp: 251-265. Villalba, A. (2008). Perkinsosis in molluscs: epizootio- logical aspects. En: A. Villalba (ed.) Workshop for the Soudant, P.; R. Leite; F.-L. E. Chu; A. Villalba; L. analysis of the impact of perkinsosis to the European Cancela. (2008). Bivalves-Perkinsus spp., interactions. shellfish industry. Centro de Investigacións Mariñas, En: A. Villalba (ed.) Workshop for the analysis of the Consellería de Pesca e Asuntos Marítimos da Xunta impact of perkinsosis to the European shellfish de Galicia, Vilanova de Arousa, Spain. Centro industry. Centro de Investigacións Mariñas, Tecnológico del Mar – Fundación CETMAR, Vigo, Consellería de Pesca e Asuntos Marítimos da Xunta Spain. pp: 44-60.

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4. SCIENTIFIC MEETINGS AND TRAINING ORGANIZED BY THE CIMA

SCIENTIFIC MEETINGS AND TRAINING ORGANIZED BY THE CIMA

4.1. SEMINARIOS

Dr. Josianne G. Støttrup Dr. Profesor Jorge Eiras Logical foundations for the improvement of turbot Mesozoas, fish parasites: aspects of biology and its and codfish stocks or restocking in local systems. importante in fish farming. Danish Institute for Fisheries Research. Denmark. Dept of de Zoology and e Anthropology. Faculty of Marzo, 2008. Sciences. Universidade do Porto. Portugal. July, 2008.

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Previous annual report covers: Years 1998-2008

5. ANNEX

ANNEX

2.1 ABBREVIATIONS USED

AC Autonomic Community ANFACO Asociación Nacional de Fabricantes de Conservas y Mariscos de España AZTI Instituto Tecnológico Pesquero y Alimentario CCMAR Centro de Ciências do Mar do Algarve CCMM Centro de Control de Calidade do Medio Mariño CEP Centro de Experimentación Pesquera CETGA Centro Tecnológico Gallego de Acuicultura CETMAR Centro Tecnológico del Mar CIMA Centro de Investigacións Mariñas CO Centro Oceanográfico CPAM Consellería de Pesca e Asuntos Marítimos CSIC Consejo Superior de Investigaciones Científicas CTAG Centro Tecnológico de Automoción de Galicia DXIDP Dirección Xeral de Innovación e Desenvolvemento Pesqueiro DXRM Dirección Xeral de Recursos Mariños EGAP Escola Galega de Administración Pública FAO Food and Agriculture Organization IEO Instituto Español de Oceanografía IFAPA Instituto Andaluz de Investigación y Formación Agraria, Pesquera, Alimentaria y de la Producción Ecológica. IFREMER Institut Français de Recherche por l´Exploitation de la Mer IGAFA Instituto Galego de Formación en Acuicultura IMARES Institute for Marine Resources & Ecosystem Studies (antes RIVO) IMIDA Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario IIM Instituto de Investigaciones Marinas (del CSIC). INTERREG Iniciativa Comunitaria de Cooperación Transfronteriza INTECMAR Instituto Tecnolóxico de Control do Medio Mariño IRTA Institut de Recerca i Tecnologia Agroalimentàries JACUMAR Junta Asesora de Cultivos Marinos MAPA Ministerio de Agricultura Pesca y Alimentación. NOAA National Oceanic and Atmospheric Administration OIE Oficina Internacional de Epizootias OPIDI Oficina de Programas Internaciones I+D+I OPMEGA Organización de Productores de Mejillón de Galicia OTRIS Oficina de Transferencia de Resultados de la Investigación PGDIT Plan Galego de Investigación, Desenvolvemento e Innovación Tecnolóxica SIGREMAR Sistema de Información Geográfica Orientado a la Gestión de Recursos Marinos SITGA Sistema de Información Territorial de Galicia UTPB Unidad Técnica de Pesca de Baixura UCM Universidad Complutense de Madrid USC Universidade de Santiago de Compostela UDC Universidade da Coruña UO Universidad de Oviedo UV Universidade de Vigo

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