European Commission Research Directorate-General, Programme "Quality of Life and Management of Living Resources"
Marie Curie MCFH-1999-00530 Fellowships:
Contract No QLK5-1999-50530
Marie Curie Training Site
Sustainable exploitation and management of living marine resources:
A holistic approach (SEMLMAR)
This Marie Curie Training Site was run jointly by the University of Aberdeen and Marine Laboratory Aberdeen and ran from September 2000 to February 2005. A total of 108 training months was offered and 15 trainees from EU Member or Associated State spent periods from 3 months to 12 months working in Aberdeen.
Training was offered in topics related to a holistic approach to exploitation and management of living marine resources, i.e. maintaining economically efficient fisheries and aquaculture industries, while preserving the sustainability and diversity of ecosystems, drawing on the exceptionally wide pool of expertise available at Aberdeen including biologists, ecologists, chemists, statisticians, economists and social scientists.
Marie Curie Fellows trained under SEMLMAR: research summaries
Jeannette Dan Møller
Expression of pro-inflammatory cytokines in fish skin during
infection with ectoparasites: The aim of this project was to study the
expression of pro-inflammatory mediators IL-1b, IL-8, TNF-a and COX-2 in the
skin of rainbow trout Oncorhynchus mylkiss after infection with the
monogean Gyrodactykus derjavini. Infections were carried out at the
fellow's home institution and samples brought to Aberdeen for the investigation
of cytokine gene expression. Small pathogen-free naïve fish and fish
just recovered from a primary G. derjavini infection were exposed to detached
G. derjavini parasites for 24h. Infection levels were assessed at regular
intervals. Naïve as well as immune fish were readily infected and a gradual
increase in parasite abundance could be seen within the first week of infection.
Lower infection levels were seen in fish carrying a secondary infection. Samples
for examination of gene expression were taken after 8 and 16 days. Fish were
killed and skin quickly dissected. Skin preparations from 3 fish were pooled
and snap-frozen in liquid nitrogen. Tissues were homogenized in Trizol and
RNA isolated. CDNA was generated from RNA using M-MLV, Rnase H minus, reverse-Transcriptase
and oligo-dT primers. PCR reactions were performed. cDNA encoding b-actin
was amplified as a reference gene. Amplification of genes encoding pro-inflammatory
mediators was subsequently performed using specific primers against IL-1b1,
IL-1b2, IL-8, TNF-a1, TNF-a2 and COX-2. Expression levels of these pro-inflammatory
mediators were measured as values relative to b-actin expression.Expressions
of genes encoding all the examined mediators were found in the skin tissue
preparations. This seems to be the first detection of cytokine expression
in skin or skin-related tissues. As cytokine gene expressions have previously
been detected in gills and intestinal tissues, these findings indicate the
importance of a mucosal component of the immune system in teleosts. Changes
in the macroenvironment (pH, ammonium levels etc) or small injuries could
be sufficient to induce expression of these genes.Regarding expression of
the IL-1b2 isoform, very low levels of specific gene expression were detected
in uninfected fish after 8 days. However, a clear induction of IL-1b2 was
obvious in fish infected with G. derjavini, with a twenty-fold increase
in expression observed in samples from primary infections. A similar, but
less pronounced effect was seen after 8 days in fish carrying a secondary
infection,. No clear differences were seen after 15 days due to induction
of high levels of IL-1b2 expression in uninfected samples. The expression
of IL-1b1 mimicked that for IL-1b2 and this was also the case for the two
isoforms of TNF-a. However, no clear infection-related induction of COX-2
or IL-8 could be observed, primarily because uninfected fish exhibited rather
high levels of expression of these genes, possibly due to effects of handling
stress on the fish. Preliminary studies were also carried out on cytokine
gene expression in rainbow trout infected with the parasitic ciliate Ichthyophthirius
Analysis of stress-induced gene expression in trout red blood cells following TBT exposure: Environmental stresses such as heat shock, toxic metal contamination and hypoxia/anoxia, may be commonly experienced by many aquatic poikilothermic organisms. In order to minimise the potentially detrimental effects of these stresses, organisms are capable of synthesising a group of proteins known as stress proteins or heat-shock proteins. Stress proteins, especially hsp70 and hsp60, have been used as biomarkers in a range of algae, invertebrates, fish, and higher vertebrates. The purpose of this study is to examine the expression of stress proteins, such as Hsp 70, in the RBCs of rainbow trout in response to exposure to organotin compounds. Organotin compounds are pollutants of anthropogenic origin. Their presence in the environment is due to use in many industrial applications and also as agricultural biocides. The main input to water environment depends on their use in marine antifouling paint formulations and especially as stabiliser of PVC. As a consequence, organotins are ubiquitous contaminants in aquatic ecosystems. The toxicity of organotins has been the object of several studies in the last years, resulting in the demonstration that it occurs at several biological levels (i.e. cellular energy production, cell membrane functionality and protein conformation). In general, the toxicity of organotin is determined by the number and nature of the organic substituents on tin (IV). This innovative study is important in elucidating new aspects of stress protein synthesis in nucleated erythrocytes and it could results in the development of application of biosensors to aquatic environment.
Erythrocytes were obtained from Onchoryncus mykiss. The fish were kept in tanks and fed with commercial fish food. Experiments were done using fish of the same age weighing between 200 and 300 g. Blood was withdrawn with a syringe from the lateral tail vein into an isotonic medium and further treated within 2 h at 4°C. After removal of the plasma and buffy coat, the erythrocytes were washed three times with isotonic phosphate buffer. After washing, the cells were suspended in L-15 medium and kept for 24 hours at 12°C in order to minimise the heat shock response due to handling stress. Cells were further resuspendend in L-15 medium in presence of tributyltinchloride in the range of concentration 1nM / 5mM. for 6hours at 12°C. An aliquot of approximately 1x107 cells was collected hourly, pelleted and immediately frozen in liquid nitrogen. Beside a temperature rise of 10°C, in untreated cells, was used as a positive control and samples were collected between time 0 and 6 hours. Total cellular RNA was obtained using RNAzolB, a guanidine isothiocyanate based extraction medium, isopropanol precipitated and resuspended in DEPC treated water. Purified RNA was quantified by spectrofluorometry using Ribogreen RNA quantification kit from Molecular Probes. Aliquots of 20mg of RNA were electrophoresed on a formaldehide denaturating gel and blotted on a positively charged nylon membrane. Screening of northern blots was performed using a cDNA probe of 280bp, designed on the overlapping region of two isoforms of rainbow trout hsp70 reported in literature (R.K. Kothary, D.Jones, E.P.M Candido Mol.Cell.Biol. 1984, 4:1785-1791). Erythrocytes exposed to TBT were also fixed in glutaraldhehyde/cacodylate buffer, postfixed in osmium tetroxide and finally embedded in resin for transmission electron microscopy analysis.
Erythrocytes were used to measure the relative abundance of hsp70 mRNA following thermal shock and TBT mediated expression. Both stimuli were able to induce an increase in the expression of hsp70. In particular the kinetic of expression in cells submitted to thermal shock presented a peak after 2 hours and a gradual decrease afterwards. TBT presented a complex time and dose dependent response: after one hour of incubation in presence of xenobiotic, mRNA expression profiles presented a clear dose dependent response in the range 1nM/5 mM. Nevertheless the kinetics of expression were different according to the concentration of TBT: at lower concentrations (1-100 nM) a gradual increase between time 0 and 6 hours was evident while at higher range (1 - 5 mM) expression presented a peak after one hour of exposure and decreased afterwards. Transmission electron microscopy investigation revealed a significant mitochondrial perturbation, involving mitochondrial swelling and alteration of mitochondrial cristae ultrastracture. In fact, TBT is known to interfere with cellular respiration, and this could lead to oxidative stress by impairment of the mitochondrial respiratory chain and consequent formation of reactive oxygen species. The last part of the project involves the synthesis of cDNA probes designed on oxidative stress related genes, such as superoxide dismutase and gluthatione peroxidase, for northern blot screening. Furthermore studies are presently in progress in order to compare the response in other in vitro models and tissue specific expression in in vivo experiments.
Extraction, isolation and structural characterisation of secondary metabolites from marine facultative fungi and soft corals: The isolation and identification of new compounds from marine biota is of great interest. Marine metabolites have potential applications in pharmacology and medicine as many have interesting biological activities such as anti-viral activity, anti-cancer properties, antibiotics, etc. They also play, an important role in connection with new developments in nutrition and aquaculture. Marine fungi are rich in hight unsaturated fatty acids (HUFA) and "naturally encapsulate" the long chain fatty acids starting from nonlipid dietary precursors. During the present project the structural elucidation of metabolites extracted from three marine facultative fungi, and two soft corals provided was carried out.
Samples of fungi Paecilomyces variotti and Penicillium roqueforti were isolated in Las Palmas de Gran Canaria, Spain, and identified by CABI, Surrey, and were bulk-cultured in a static bath system with KMV broth in marine water. The fungus Schizochytrium agregatum (American Type Culture Collection 20888) was acquired from Aquafauna Bio-Marine, Hawthorne, California, USA and it has been produced in a 400-L fermenter, following the general procedures in Barclay (1994) and Barclay et al. (1994). The samples of the soft corals Tubipora musica and Melithaea ocracea were collected in Indonesia. The partition method used was a modified Kupchan method (1973). The crude fraction of each sample was extracted with MeOH for 24 h (3x) followed by CH2Cl2 for 24 h (3x), and the concentrated extracts were combined after which the solvent was removed at reduced pressure. The crude extract was partitioned between water and CH2Cl2. The water soluble fraction was partitioned with sec-butanol. The CH2Cl2 fraction was evaporated to give an organic crude extract that was partitioned between hexane and 10 % aqueous MeOH. The MeOH fraction was phase adjusted to 50 % MeOH and partitioned with CH2Cl2. Each fraction obtained, was then subjected to Sephadex LH-20 size-exclusion chromatography (1 : 1, CH2Cl2/ MeOH), and the fractions were subjected to flash chromatography (Hexane/ EtOAc/ MeOH gradient) followed by normal-phase or reversed-phase C-18 HPLC.
Some "sterols" were identified by NMR and MS techniques.
The remaining fatty acids fractions were hydrolysed, converted in methyl esters
and identifying by GC-MS. During my stay at the University of Aberdeen, working
as a Marie Curie Fellow, I improved my skills in Organic Chemistry working
in a natural products chemistry group. The fellowship was crucial to carry
out part of my Ph-D research and enabled me to process many samples using
a different methodology, and share experiences with experts in this field.
Furthermore, I participated in many taught courses: NMR Spectroscopy (Dr.
Jaspars), Organic Structure Analysis (Dr. Jaspars), Natural Products (Dr.
Jaspars) and Analytical Chemistry - Chromatography Techniques (Dr. Marr),
which have improved my theoretical knowledge in this field.
Evaluation of transport and storage factors which influence the evaluation of toxin levels in shellfish: Since 1998, king scallops (Pecten maximus) obtained from Scottish off-shore sites have been monitored for domoic (DA) and epi-domoic acid (epi-DA); the principal toxic compounds of amnesic shellfish poisoning toxins (AST). Inter-animal variability in toxin concentrations detected in scallops collected from the same geographical location is now well documented. Little information, however, currently exists on the effects of different storage conditions during sample transportation on toxin levels. This type of data is important in terms of both defining quality control criteria for accepting samples during monitoring programmes and for scallop processing. Additionally, not much published information is available on the stability of DA and epi-DA in the solvents (methanol:water (1:1 v/v) and citrate buffer) routinely used for their extraction from shellfish. The stability of these extracts will impact on how sample processing is managed during intense periods of monitoring.
During this fellowship, scallops were transported from the harvesting site and stored under different regimes (in ice, without ice and at 12 °C for upto three days) to mimic conditions which may be used during monitoring transport and storage procedures. The DA and epi-DA content in scallop gonad and combined samples of adductor muscle, gills and hepatopancreas were subsequently determined using HPLC methods. Effects of storage on toxin content in extraction solvents were also investigated. Studies were also commenced to discover the involvement of bacteria in DA production. Bacteria were isolated from diatom cultures (known to produce AST) and tested for autonomous DA production using an Indirect competitive ELISA technique, HPLC and LC-MS. Bacteria demonstrating this trait were subsequently characterised by sequence determination of their 16S rRNA genes. Results from these studies were presented at the 4th Annual Conference on Molluscan Shellfish Safety (Galicia, Spain, 2002).
Population ecology of marine top predators -effects of environment on reproductive success: The program of my study period with the University of Aberdeen SEMLAR programme consisted of following components: 1. Training in scientific research laboratories in Scotland. My main location of operation during the study period in Scotland was department of Zoology of Aberdeen University. I stayed in Aberdeen for the first five weeks of my study period to read scientific literature in departmental and central university libraries, access internet library sources, computerise and process existing original field data gathered during my earlier studies in the Baltic sea. During this phase I also communicated actively with colleagues in Scotland, Europe and North America. In the early phase of my training period I also visited the Sea Mammal Research Unit, University of St.Andrews to communicate with grey seal breeding biologists, discuss research potentials, methodology and prepare participation in a field expedition to Isle of May grey seal breeding colonies. The latter part of my training period was based at the Lighthouse Field Station of Aberdeen University Zoology Department in Cromarty, where I continued work on my main research subject within the group of marine mammal ecologists based there, and made use of the group's comprehensive library. Two world conference poster presentations were composed, designed and printed with the help of colleagues and technical capacities of Aberdeen University.
2. Field work at the Isle of May grey seal breeding colony. In the Baltic Sea, observations of breeding groups of grey seals by human observers are difficult due to the remote location of breeding colonies and cautious behaviour of adult seals. Comparative observations at stable breeding colonies in Scotland therefore help to evaluate objective and subjective factors that may influence measurements and results from the Baltic. I worked for two weeks at the grey seal breeding colony on the Isle of May (Firth of Forth, Scotland) to obtain training and develop methodology for establishing breeding season dynamics from stage-structured pup counts, gather original data on neonatal development of grey seal offspring, make observations on the effect of terrain on adult seal behaviour (e.g. 'open' habitat with access to water in rocky tidal zone versus 'closed' habitat of a flat gully with restricted access to water, density-dependent effects of maternal behaviour, use of available space), and test video equipment to record samples of seal behaviour and habitat utilisation. Using video recorders will facilitate studying several aspects of breeding biology in the Baltic Sea areas, where the presence of humans is logistically complicated or influences directly animal behaviour and outcome of the breeding event.
The fieldwork resulted in series of observations in different habitats. Camera recordings backed up visual observations to enable post hoc observations. The use of video cameras instead of human observers was compared at different sampling rates, and were experimentally tested with synchronous visual observation to enable selection of optimal sampling rates. Although short and experimental in nature, both visual (~ 12 hrs) and recorded (~ 50 samples) can now be used as comparative datasets for specific studies or further method development. As a member of research team on the island I took an active part in all projects that were carried out during the period to gain experience and get acquainted with contemporary study methodology that can also be used in my research in future. Among other new techniques, I gained practical experience in using of ultrasound scanner for exact measurements of body composition (i.e. condition) of grey seals and collecting of tissue samples for DNA analysis. Several recent and ongoing research themes were discussed with experts of the field; some of these can be developed further for comparative research in the Baltic Sea area.
3. Biennal World Marine Mammal Science Conference, Vancouver, Canada, 28.11. - 03.12.2001. I presented two poster presentations at the conference, namely: (1) Effect of habitat on Baltic grey seal breeding success by Mart Jüssi, Tero Härkönen, Eero Helle and Ivar Jüssi, and (2) Activity patterns of satellite tagged Baltic ringed seals (Phoca hispida botnica) by Tero Härkönen, Mart Jüssi, Karin Harding, Ivar Jüssi and Eero HelleBoth posters were produced during the training period in Aberdeen. Participation in the event was supported by the Marie Curie Fellowship (travel in UK and accommodation in Vancouver, printing of posters), University of Tartu, Estonia (Intercontinental flight), conference fee and daily costs were covered from personal resources. The conference programme offered good possibilities to learn about recent achievements in marine mammal science and discuss the specifics of my research projects with several colleagues. I benefited from spontaneous group discussions and a special meeting of ringed seal scientists that was aimed to establish an international research network of the species.
4. Sea Mammal Research Unit & University of Aberdeen Postgraduate workshop. At the end of January, I returned to the UK to attend a joint workshop involving Postgraduate students studying marine mammal biology from the Universities of Aberdeen and St Andrews. During the meeting I gave a lecture on recent studies of grey seals in the Baltic Sea, attended presentation by other students, and joined discussion groups on current scientific issues in the field.
The Marie Curie research and training fellowship contributed
significantly to my studies and research. Working in different research groups
provided invaluable experience, and meeting colleagues in Scotland and Canada
opened several new perspectives through new methodology and potential joint
Growth rates of squid Loligo forbesi in Scottish and French waters: Populations of cephalopods have large abundance fluctuations, often considered like the consequence of variable dynamic parameters. Characteristics of their life cycle cause inter-annual fluctuations. For many cephalopods stocks, the fishery productivity depend on the annual cohort recruitment. For those species, recruitment is the key factor of stocks variation. The hatching period of Loligo forbesi spread out in time, often on several months. This period suggest heterogeneous growth conditions during the pre-recruitment phase. Temporal and spatial growth variations are studied in those juvenile squids (mantle length < 20 cm). The study of this growth variability is possible with the individual age determination technique through the quantification of statolith increments.We decided to compare the early growth phase between two areas: the English Channel and the North Sea for 2 years : 1993 and 1998. In these 2 years, smaller periods of study were chosen in relation with the peaks of recruitment: from April to September in the English Channel and from February to August in the North Sea. During the first 4 months, 380 samples were prepared and age readings were completed on half of these. First results confirmed that the hatching period spread out over the year in the two areas.
Loligo forbesi -age at recruitment and growth variations in the English Channel and Scottish waters: Practical work followed on from that completed under training period QLK5-GH-99-50530-07. The main conclusions from the work are as follows. The individual age determination method gave information about the age at recruitment of small squids Loligo forbesi (about 8 or 9 months) in the English Channel and Scottish waters. These results are consistent with a 16-month to 18-month life-cycle or a one-year life-cycle with a long juvenile phase and a fast growing adult phase. A wide range of ages was found for each range of size. Except for range I in the English Channel 1998, all ranges of size covered several months, indicating a high individual growth variation. However, ages also showed month-to-month progression as might be expected.
Growth models were fitted to length-at-age data collected for juveniles in the English Channel and Scottish waters of cohorts 1993 and 1998. Inter-annual differences were identified in growth of pre-recruits in the English Channel and in Scottish waters. Spatial comparisons revealed no significant difference between areas, in either 1993 or 1998. The pattern of growth was similar in both areas: growth rates were higher in 1998 (24.9 mm.m-1 in the English Channel and 19.2 mm.m-1 in Scotland).
Growth comparisons were also made between micro-cohorts within each cohort. A high growth rate is observed in the English Channel in June (41 mm/month) in 1993 and in July, August (64 and 58 mm/month) in 1998. In Scottish waters, animals born in May also have a high growth rate (35 mm/month). In spite of these apparent differences, the model could not detect any influence of the hatching month on the growth of pre-recruits from English Channel and Scottish waters. There is no significant difference of growth between micro-cohorts. High individual variation and small sample sizes may however have resulted in differences being undetected.
Results from this work were presented at a conference in February 2003 [Challier L., Royer J., Pierce G.J. & Robin J.P., 2003. Relationships between early growth variation and recruitment in English Channel and Scottish waters Loligo forbesi. Cephalopod International Advisory Council Triennial Conference, Phuket, February 2003].
Information conveyed by the sounds of haddock (Melanogrammus aeglefinus) - results of a playback experiment: The haddock (Melanogrammus aeglefinus) is a common food fish, which in some areas, including the North Sea, is exploited beyond safe biological limits. This specie is also are well known to be soniferous fish, producing a wide repertoire of sounds. Studies in captivity demonstrated that during the spawning period (February to April), mature haddock males produce long sequences of regularly spaced knocks, lasting up to 20 minutes and emitted for several hours, accompanied by a peculiar visual display, defined 'Solitary Display' (Hawkins & Amorim, 2000; Casaretto & Hawkins 2002): an individual male fish swims in tight circles or in a figure of eight pattern on the bottom, occupying a peculiar area with no other fish swimming around. This points towards the SD as a territorial signal, the acoustic display being the main broadband signal. Following Casaretto & Hawkins (2002), the features of the knocks are different for individual males, suggesting morphological differences in the drumming muscles, i.e. the muscle responsible of the production of the sounds. Therefore the authors suggest that the SD sound provides 'honest information' (sensu Zahavi's principles, 1975) about the sender. In its turn, this might enable males to judge the fitness of their neighbours and females to choose their mates.
The specific goal of the present study was to test the role of the SD sound as territorial and mate-attraction signals, examining the phonotactic response of mature haddock males and females to the projected SD call. In order to achieve our goal, we used the technique of playback. This technique consists in projecting appropriate acoustic signals through underwater loudspeakers to one or more receiving animals, measuring afterwards any modification of their behaviour (McGregor 1992).
Twenty-five haddock (12 females, 9 males and 3 immature) were tested by projecting the SD sound recorded from a big mature male, not actually present in the experimental tank. A total of 18 trials took place during haddock breeding season, from late afternoon to night (17:20 - 01:40), when haddock are normally most active. The fish were video- and acoustically recorded from one hour before to one hour after each playback session, consisting in projected the SD sound for a continuous period of 30 min.
Our results showed no clear reaction of the fish to the playback of SD sound. The response of the animals, however, appeared to be conditioned by the presence of dominant males in the tank: when a male was actively displaying close to the projector, this fish inspected the loudspeaker during the trial, emitting aggressive sounds. Conversely, during the presentation of a neutral acoustic stimulus (as the background noise playback) no display toward the sound source was observed. This demonstrated that SD vocalisations are effective in modifying the behaviour of the 'receiver', involving a communication exchange. The played-back sound elicited the aggressive approach of the 'dominant' male for only one time during each trial, even if the playback treatment ran continuously for 30 minutes. Once inspected the loudspeaker, the animal moved away from the area after a couple of seconds. This would suggest that even if the SD sound, per se, provides an activation of the motivational state of 'rival' males, visual or tactile stimulations are necessary to arise a complete behavioural response. The presentation of a sound from a loudspeaker in the tank, in fact, may be considered an imperfect analogue of a situation where a settled male detects sounds emitted by a conspecific with whom he is not in visual contact. It is well known that water is an excellent medium for sound propagation and territorial haddocks may be exposed to many sounds coming from adjacent territories. Therefore not responding to sound signals unless additional visual information is available (as the physical presence of the displaying male) could be a highly adaptive strategy for a territorial male: in this way they would avoid being continually involved in aggressive encounters taking place outside their territory.
In conclusion, this playback experiment demonstrates that the SD sounds do provide informations on the species, sex and location of the vocal fish. Nevertheless visual and other stimuli are necessary for eliciting a full range of behavioural responses from the other individuals. The results obtained from this work were presented at the ICES symposium on fish behaviour in exploited ecosystems 23rd -26th June 2003 Bergen Norway with the title 'Information conveyed by the sounds of haddock: results of a playback experiment' by M. Picciulin & A.D. Hawkins.
Energetics and food consumption of summering atlantic walruses (Odobenus rosmarus rosmarus) in North East Greenland: The ecology of Atlantic walruses (Odobenus rosmarus rosmarus) is poorly understood due to the remoteness of these animals' range to their secluded lifestyle. Energy expenditure, time-activity budgets are essential but still obscure parameters to gain deeper understanding of these pinnipeds' ecology. Data from other pinniped species though cannot be used directly to infer these parameters because of basic morphological and behavioural differences. An experimental setup that couples the use of isotopic techniques (doubly labelled water) for the understanding of the actual energetics of these animals in the field with time and area use data gathered by ARGOS satellite telemetry and the application and retrieval of Time Depth Recorders and direct observation of feeding activity will provide the best possible basis to date to obtain insight in the walrus' life.
The samples needed to measure energy expenditure by the doubly labelled water technique have been gathered in the Summer 2001 and in the period covered by this report have been prepared by distillation and analyzed at the Mass Spectrometry facility at Aberdeen University. Instrumental data from satellite tags and dive recorders has also been analyzed. Food consumption has been estimated through direct observation of feeding activity both above and below the water surface.
The results obtained during the period covered by this report have provided considerable contribution to the following:
Born, E.W., Rysgaard, S., Ehlmé, G., Sejr, M., Acquarone, M. & Levermann, N. 2003: Underwater observations of foraging free-living Atlantic walruses (Odobenus rosmarus rosmarus) and estimates of their food consumption. - Polar Biology 26(5): 348-357.
Acquarone, M., Levermann, N. & Born, E.W.: Diving and haul-out activity of free-living, summering Atlantic walruses (Odobenus rosmarus rosmarus). 10. Arktisk Biologisk Forskermøde / 10th Arctic Biological Forum, Zoological Institute, Copenhagen, Denmark, 24th January 2003.
Acquarone, M., Levermann, N. & Born, E.W.: Walruses and Carbon. NARP Workshop C Flux and Climate Change: The Nordic Contribution to a Panarctic Perspective, Sigulda, Latvia, 1-7 November 2002.
Characterisation of the surface structures of Flavobacterium psychrophila by electron microscopy: The non-virulent typestrain as well as a virulent strain of the fish pathogenic bacteria F.psychrophilum have been examined for capsular material following stabilisation with antibody (Jacques & Foiry, 1987). Both strains possessed a thin capsular layer compared to other known capsulated bacteria. The virulent strain expressed slightly more capsule than the non-virulent strain. Likewise did growth on serum-supplemented media slightly increase the amount of capsule detected.
Several staining methods were tested to show pili (fimbriae) expression on F. psychrophilum. Using uranyl acetate pili expression was studied on the non-virulent typestrain as well as a virulent strain. Pili or pili-like structures were observed on both strains. The percentage of cells expressing pili was recorded and the virulent strain was shown to have a higher expression rate than the non-virulent strain. The non-virulent strain did however show a significant increase in the expression rate when cultured on media with limited iron availability and the two strains thus have similar expression rates when grown on iron-limited media. The observed pili sizes were approximately 0.03 µM in diameter and 0.2-2 µM long. Siderophore production was tested on CAS-agar for different strains of F. psychrophilum. The results divided the tested strains into siderophore producing and non-siderophore producing strains. Ongoing studies will show the importance of these siderophores for growth in iron-limited media as well as in serum. Work to characterise of the type of siderophore released by F. psychrophilum was initiated and is being continued.
Spatial and temporal patterns of decapod larval abundance: Most studies of zooplankton in the North Sea and Scottish coast are focused on copepods, which reach up to 90% of zooplankton biomass. Studies of decapod larvae are relatively rare due to their relatively low abundance in the zooplankton in these latitudes and the difficulty of their taxonomic identification, which leads to their grouping generically as "decapod larvae" in most zooplankton studies. The relatively small number of studies on decapod larvae in this region does not mean that they are not an important constituent of zooplankton, especially in coastal ecosystems, where their meroplanktonic larvae play an important role in the food web and ecosystems dynamics. Besides, many species of decapods support important fisheries around the world, with a great socioeconomic importance and the same is the case in the North Sea.
The samples analysed in this project were collected from January 2002 to December 2003 at a weekly monitoring station off Stonehaven, south of Aberdeen, Scotland (56º 57.8' N, 02º 06.2' W), 3 km offshore. Time series sampling for plankton, fish larvae and environmental variables is carried out there as part of the environmental monitoring work of FRS. The student analysed two or three samples per month (more in warmer months, when larvae were present) to as fine a taxonomic level as possible, and related the patterns of species abundance to environmental conditions. A spatial comparatison was carried out between the Stonehaven samples and those collected at another monitoring station on the west coast of Scotland (Loch Ewe, 57º 50.99' N, 05º 38.97' W), over a period of 1 year.
The student benefited from the period of Marie Curie funding in a wide variety of ways. She perfected her taxonomic skills to a very high level, becoming an expert in the field, and has trained other analysts in FRS and even produced an illustrated taxonomic key of the species found in the samples for future reference and taxonomic training. The student has received training in advanced statistical techniques of direct applicability to her PhD research. The spatial and temporal studies should produce 2 publications in peer-reviewed journals, describing temporal and spatial patterns of decapod larval abundance and relating these to environmental factors. One of these publications is in a very advanced state of writing and all relevant results for the other one have been obtained.
Ecology of harbour seals at contrasting spatial scales: Harbour seals have rarely been studied in Irish waters and my work at University of Gallway represents the first detailed ecological study of harbour seals in this country. While at Aberdeen University I received training in several aspects of marine mammal research. This included the opportunity to participate in fieldwork both on harbour seals and bottlenose dolphins. In particular, I worked on a seal photo-identification project, using pelage patterns to recognise seals and use this information can then be used to investigate dispersal, survival and pup production. During my time at the Lighthouse, I also got the opportunity to assist the Sea Mammal Research Unit with fieldwork, undergoing training in seal capture and handling techniques. Seals were then immobilised and various samples including blood and blubber were taken. The aim of this work was to assess the level of contact with the PDV virus that spread through the European seal population last year.
A key aspect of my time at the Lighthouse was to begin work on a spatial analysis of harbour and grey seal haul-out distribution. I developed new skills in GIS software and analysis, and haul-out sites have been entered into a GIS database. This portion of the work is a collaboration, initiated by me, with Dr. Nicolas Ray of Melbourne University. A 'least-cost' path approach is being applied to look at distances by sea between sites. This information will then be used to cluster the sites into probable meta-populations. The hypotheses this analysis generates will then be tested later on during my Ph.D. It is hoped that this may help guide survey strategies and help in defining sampling locations for genetic work.
I also attended several conferences and workshops during my training period. This included the European Cetacean Society Conference in Las Palmas de Gran Canaria, where I also participated in the Phocine Distemper Workshop. It was a great opportunity to meet other biologists working on PDV and seals. I gave talks at both the University of Aberdeen and Sea Mammal Research Unit postgraduate conferences and attended seminars and discussions on scientific issues and various aspects of career development.
Methods for age determination in small cetaceans: Age determination of individuals is an important tool for research into aspects of the biology, ecology and physiology of marine mammals. Age is important to describe life history strategy and support some biological parameters (Hohn, 1990). Different methods are used for determining age in marine mammals. Counting growth layer groups (GLGs; sensu Perrin & Myrick, 1980) present in the teeth (dentine and cement) is considered the most reliable (Klevezal & Kleinenberg, 1967; Perrin & Myrick, 1980, Hohn, et al.,1989, Lockyer, 1995c; Hohn, 2002). The technique used for preparing teeth and how to determine which would provide the most successful count of the laminae is still under discussion. Moreover, according to Hohn, et al., (1999), , the techniques for preparing and sectioning teeth can also introduce marked biases into the interpretation of age. The aim of this study is the comparison of two common histology techniques, the paraffin and cryostat technique, for age determination in different species of odontocetes.
Materials & Methods: Teeth from 67 stranded animals, representing seven species in two geographical locations in the North Atlantic Ocean (Scotland and Canary Islands), were prepared using these two techniques. 2-3 teeth were removed from the middle of the lower jaw and preserved in 10% neutral formalin, then decalcified in a rapid decalcifying agent (RDO®). Particular investigations regarding staining method and thickness of the sections were carried out within techniques. Thus the paraffin technique was a modification of a published protocol for Hector´s dolphin (Slooten, 1991) whereby the teeth were sectioned at 8µm thick, whereas the cryostat technique was adapted from Hohn & Lockyer (1995) and Lockyer (1995c). Three different thicknesses were used (8µm, 16µm, 24µm). The most central sections were stained using four different staining methods: Mayer's haematoxylin, Ehlrich ´s haematoxylin, Toluidine blue and Giemsa. Finally, stained sections were mounted and permanent slides were prepared using DPX-mountant. Stained sections were examined by binocular microscope at 10-50x magnification. Age was estimated by counting the GLGs present in the dentine (Perrin & Myrick, 1980) by two independent readers without any reference to biological data (body length, sex, etc.). The readings were achieved taking into account the quality of preparation (presence and absence of the neonatal line, closure of pulp cavity and occurrence of features such as mineralization anomalies, accessory layers, marker lines, etc.,) and compared to an approximated known biological age based on other biological data recorded.
Results: In the paraffin technique I investigated differences in estimated age by using these three staining methods. Results showed a high correlation in the estimates. A linear regression model using age (GLG) as a response variable and these three stains as explanatory variables showed a significant difference regarding stain used. In the cryostat technique I investigated differences in estimated age from preparations at three thickness within each staining method. Again results showed high correlations for any combination. A linear regression model showed significant effects for Ehlrich´s haematoxylin regarding different thicknesses. Based on sections at 24 µm thicknesses, no difference was found between staining methods used.Using both techniques and based on Mayer´s haematoxylin stained longitudinal sections at 8 µm thickness (80% of sections were categorised as good quality), estimated ages were highly correlated. Paired t-tests showed that there was no significant difference in the estimated age. A linear regression model showed no significant difference between estimated ages from both techniques. To find out whether the species could also be a key variable affecting the accuracy of the technique, I worked with harbour porpoise samples and no difference was found.
In conclusion this experiment shows there is no significant difference fin the estimated age from both technique and therefore confirms the viability of their use for preparation teeth in terms of age determination in small dolphins and porpoises. Nevertheless some differences were found within technique regarding staining method and thicknesses, which have to be considered. In terms of absolute verification of the age and therefore the technique itself, we recommend a further investigation; thus the technique should be tested on teeth of known age individuals. The results obtained from this work were presented at the European Cetacean Society (2-7 April 2005, La Rochelle, France).
Modelling the growth of sandeel larvae as a function of temperature and zooplankton concentration: The larval stages of marine fishes are characterized by high mortality rates and it is therefore generally acknowledged that rapid growth through the larval period enhances survival. Larval growth is often modelled as a function of seawater temperature, which is used as a proxy for overall environmental variability since in general temperature displays a similar pattern to seasonal productivity in the sea. However this approach is often criticized by the fact that "fish do not feed on temperature" and the effect of food availability on growth should be modelled explicitly. The primary objectives during my stay at the FRS Marine Laboratory, Aberdeen were: to model the growth of sandeel larvae, a species of high economic and ecological importance; to estimate suitable prey fields from existing data sources; to incorporate those data into existing particle tracking methodology and to develop my programming and data analyzing skills.
To achieve those goals I had to prepare a Generalised Additive Model (GAM) of four zooplankton classes: Calanus 1-4, Calanus finmarchicus 5-6, Calanus helgolandicus 5-6, Pseudocalanus and interpolate their abundance over a grid with spatial and temporal resolution used by particle tracking model. I used CPR (Constant Plankton Recorder) data provided by Sir Alister Hardy Foundation for Ocean Sciences (SAHFOS) based in Plymouth, United Kingdom. In addition to CPR data I used spatial zooplankton data collected during FRV Scotia cruise in April 1992 and zooplankton samples collected weekly since 1997 in Stonehaven (south of Aberdeen) as part of an Ecosystem Monitoring Programme, carried out by FRS Marine Laboratory. The data obtained from GAM model were used in forward tracking individual based model of sandeel early life history, to diagnose space-time variability in growth and survival. The results showed that there was a strong correlation between temperature and zooplankton concentration. A model with explicit temperature and food terms explained only slightly more variability in the data than simpler models with single food or temperature terms. As a part of my work in Marine Laboratory, I developed particle-tracking code to incorporate zooplankton monthly fields. I attended to MS Access course, statistics course and daily Fortran, S-Plus and C-shell programming training. The preliminary results of my work were presented in March 2005 in the Lecture Theatre of the Marine Laboratory, Aberdeen. A further anticipated output of this work is peer-reviewed publication and oral presentation at the ICES Annual Science Conference in September 2005.
Fish immunology using molecular techniques: During my period in Aberdeen as a Marie Curie Fellowship, my project focused on fish immunology using molecular biology techniques. The animal model was sea bass (Dicentrarchus labrax), one of the most important fish species in the Mediterranean aquaculture. The group in Italy, where I am working for my PhD thesis, has recently cloned IL-1b from this species (Scapigliati et al., 2001) and expressed it in E. coli (Buonocore et al, 2004, subject area of my pre-doctoral university diploma), showed its peculiar gene organization (Buonocore et al., 2003), and resolved its three dimensional molecular structure (Scapigliati et al., 2004).The aim of this Marie Curie Fellowship's Project was to clone important genes for immune system and, in particular, was focused on two cytokines: IL-10 and IL-2. Cytokines play a significant role in initiating and regulating the inflammatory process, which is an important defense system in innate immunity. As innate immunity is known to be important in the defense against pathogens, isolation and characterization of cytokines is of fundamental importance. Only a few cytokines and chemokines are known in fish, where they have been cloned either by expressed sequence tag (EST) analysis or by PCR-mediated homology cloning. Cytokines are subdivided into families such as interleukins (ILs), lymphokines, growth factors, interferons (IFNs) and chemokines. The main function of IL-10 seems to be the regulation of immunity and inflammatory response, thereby minimizing damage to the host induced by response to a pathogen or by the self-immune system. The IL-2 is a soluble growth factor especially for T-cell. In these three months, I learnt the tools to continue the project in my laboratory in Italy.
Metal speciation in marine invertebrates: The aim of the research was to determinate the metal concentrations in extracts of marine invertebrates: sea-squirts and sponges, and to separate, purify and identify compound that have the capacity to bind metals. We were particular interested in the following metals: V, Cr, Mn, Fe, Co, Ni, Zn, Cu, As, Se, Mo, Cd. During the work, I analysed 14 samples of sea squirts and ascidians, and the results showed high concentration of V (only in sea-squirts), Fe, Cu, Zn, Se, As. I purified and identified 5 compounds, while the other 9 are still being analysed for elucidation of structure. Most of them have the ability to bind metals effectively.Applications of such compounds can be in the fields of industrial chemistry for example as catalysts. Furthermore, they can be used as bio-molecular tools for the study of disease-cellular mechanisms and more importantly as pharmaceutical agents. Applications include antibiotics, anticancer and the treatment of diseases in which metal homeostasis has become unregulated, such as Wilson's disease and Alzheimer's disease. The results of the research have the potential to be used for publication, and also presented in talks, conferences etc.
During my stay in Aberdeen University, I was trained in trace element analysis, as well as in marine natural product chemistry. The training involved using various instruments and techniques, such as sample extraction and sample digestion, separation of compounds using size exclusion chromatography, flush chromatography, HPLC, structure elucidation using NMR and MS, metal analysis using ICP-MS and identification of metal-containing-biomolecules using the coupling LC-MS/MS. During the training I participated in courses in the field, and I received training and help from the professors, technicians, postdoctoral researchers and colleagues from the University. I was very pleased to have access to use the laboratories and facilities of the University of Aberdeen. I also want to thank the European Union for financing the project, which gave me the possibility to be trained in this modern state of the art science techniques, unavailable in my country, to improve my skills, and also to prepare myself for a future career in science.
Results of this work were presented as a poster at the European Forum for Early Career Researchers in Slovenia in May 2005. Daniela's poster on "Investigations of metal levels and metal-binding compounds in marine invertebrates" won one of the two "best poster" awards. [Further information appears on the conference web page.]
Above: Daniela Blihoghe is presented with the "best poster"
award at the European forum for Early Career Researchers.