Garcés, E. (Esther)

Reguera, B. (Beatriz)

Zingone, A.

English

Zingone, A. (Adriana)

Repositorio Institucional Digital del IEO

546BackgroundThe workshop “LIFEHAB: Life history of microalgalspecies causing harmful blooms,” funded by the FifthFramework Programme (Energy, Environment and Sus-tainable Development) of the Commission of the EuropeanCommunities, was held in Calvià (Mallorca, Spain), 24–27October 2001. Complex and heteromorphic life cycles arepart of the adaptive strategies of organisms causing harm-ful algal blooms (HAB). They can influence the intrinsicpotential for growth, persistence and dispersal, allowing thespecies to occupy different ecological niches. Informationabout life cycle strategies are very important for under-standing bloom dynamics and population structure ofHAB species.The workshop report (Garcés et al., 2002) contains ex-tended abstracts, reports of the discussion groups, tables withsummarized information on diatom, dinoflagellate, hap-tophyte and raphidophyte life cycles and a verycomprehensive bibliography. The report is currently avail-able at: http://www.icm.csic.es/bio/projects/lifehab/.The objectives of LIFEHAB were to• review current knowledge on the life cycles of phyto-plankton organisms, focusing on harmful species;• identify the role of heteromorphic life cycles in popula-tion dynamics;• define future HAB research directions to fill existing gapsin knowledge;• debate the most appropriate approaches and methods;• promote the development of cooperative scientific ini-tiatives.The aim of the roundtable held during the Xth Inter-national Conference on Harmful Algae was to expand thediscussion so as to include non-EU scientists who werenot involved on the LIFEHAB Workshop.S. Bates noted that although considerable knowledge hasbeen gained on Pseudo-nitzschia species, little progress hasbeen made on other diatom genera. Advances in species-specific molecular probe design that allow the detection ofdifferent sexual stages in the field and the differentiation ofmale from female gametangia would be most useful, inorder to determine the proportion of these cells in a pop-ulation. Mating compatibility studies could clarify questionsconcerning species definitions. Other approaches, such asthe use of image analysis or the identification of condensedchloroplasts, could prove effective in tracking changes in sizespectra and resting stages. The identification, localizationand physiology of possible overwintering stages are otherkey issues for understanding the population dynamics ofPseudo-nitzschia species.P. Hargraves focused on triggering mechanisms for di-atom auxospore (size, environment, mating types,pheromones) and resting cell (light, nutrients, tempera-ture) formation. Ecological and physiological studies, as wellas monitoring programs, are as good as taxonomic qualityallows them to be. The definition of the species/taxonomicunits is thus another crucial issue, and the correct approachcan be seen as a “three-legged stool” coupling morphology,life cycles, and molecular systematics. Four fields of prior-ity research can be identified in this context: i) relationbetween life cycle events and interspecific competition at thebiochemical level; ii) the role of parasites and pathogens inthe control of blooms; iii) the enhancement of toxicitythrough symbiosis with bacteria; and iv) phylogenetic dis-tribution of toxins, including those affecting organismsother than humans.J. Lewis identified as priority activities: i) investigationof mutation rates in cultures; ii) organization of work-shops on dinoflagellate-culturing techniques; iii)development of markers for gametes and viable cysts; iv)investigation of the role of temporary cysts in life cycles;v) search for overwintering stages of species where cysts arenot known; and vii) improved methodologies for detailedwater-column monitoring and for estimates of in situ ger-mination rates.S. Blackburn illustrated examples of crossing matrixesfor sexual mating, showing that dinoflagellates have rathercomplex mating systems, including multiple mating types.She outlined the importance of sexual compatibility amongstrains, which will ultimately affect cyst production rates andend up in the genetic structure of the population.M. Elbrächter pointed out that little is known about dif-ferent cell division modalities among dinoflagellates andrecalled that in some cases, non-motile stages are involvedin asexual reproduction. He also mentioned aspects of di-noflagellate morphology and life cycle traits that have beenmisinterpreted, as was the case for Pyrocystis and Disso-dinium, the latter with lunate-shaped secondary cysts inwhich up to 8 asexual planospores are formed, or the asex-EU-LIFEHAB: Expanding the Discussion on the Life Cycles of Harmful AlgaeAdriana Zingone1, Esther Garcés2, and Beatriz Reguera31Stazione Zoologica ‘A. Dohrn’, Villa Comunale, Napoli, Italy, 08021;2Institut Ciencies Del Mar CSIC, Passeig Maritim de la Barceloneta No. 37-49, Barcelona, 08039;3Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Aptdo. 1552, 36280, Vigo, SpainParticipantsDonald M. Anderson, Stephen S. Bates, Susan Blackburn, Chris J. S. Bolch, Barrie Dale, Malte Elbrächter,Paul E. Hargraves, Ichiro Imai, Anke Kremp, Jane M. Lewis, Marina Montresor, Louis Peperzak,Christopher A. Scholin, and Carmelo R. Tomas547ual pellicle cysts in Lingulodinium polyedrum. In his view,more attention should be paid to understanding internalclocks, circannual clocks, social behaviour, communica-tion, and chemical signalling mechanisms.L. Peperzak explained that prymnesiophytes are quitecomplex because they include motile/non-motile and hap-lontic/diplontic stages. Molecular probes and flow cytometryare needed to identify species and ploidy levels. The needto develop stage-specific probes and to identify factors in-fluencing life cycle transitions was also mentioned. Hespeculated on the quorum sensing (QS) abilities of mi-croalgae as a way to detect the abundance of the samespecies by secreting species- and strain-specific competenceactivators. In bacteria, QS is involved in genetic transfor-mation and sporulation (see Dummy and Williams, 1999).Hypothetically, QS could be involved in syngamy/meiosisand cyst/colony formation in prymnesiophytes. Prelimi-nary tests with Phaeocystis indicated that high cell densitiesinduced colony formation. Proving the existence of QS, in-cluding among others competence activators, would providea new perspective in the study of HAB dynamics.C. Tomas and I. Imai noted that raphidophytes arenaked pleomorphic species not easy to study because theyare difficult to preserve without deforming or bursting thecells. Here, the combination of morphology, pigment com-position and molecular probes on live and preservedspecimens becomes truly a strong argument. While somelife cycle information occurs in reports of blooms or cul-tures, there has been no concerted effort to define the lifecycle stages of the different raphidophytes. Given their in-creasing importance as HAB species, there is a need tore-examine the life cycle phases using a number of tech-niques now available (gene sequencing, nuclear staining, etc.)as well as traditional ones using clonal cultures. Little isknown about processes undertaken in dark and cold bot-tom waters. Signalling between cells could be through highdensity or through infochemicals. This kind of communi-cation has not been proven for any HAB species, yet itcould be a means for timing life cycle changes in popula-tions capable of forming dense blooms.M. Montresor summarized research priorities for un-derstanding the importance of life cycle events in HABecology: a) role of life history stages in bloom dynamics,e.g., when are resting stages produced? how many? how manyare viable in the sediments? is there an endogenous controlof life-cycle transitions?; b) role of specific life historystages in avoiding predation, preserving genetic diversity andpromoting dispersal; c) single species and life-stage distri-bution through sampling and observational techniques atthe appropriate scale (e.g., microlayers, sediment-water in-terface); d) identification of key areas to be used as “casestudies” and the importance of long-term data sets; and e)species-specific models integrating life cycles.D. Anderson illustrated problematic issues related to thestudy of cyst germination dynamics in situ. Different tech-niques (emergence traps, changes in cyst fluorescence,laboratory incubation of sediments, repeated quantitativecyst enumeration in core samples through time) were usedto estimate in situ germination rates of Alexandriumfundyense in the ECOHAB-Gulf of Maine program, butnone proved successful. Ongoing population dynamic stud-ies have therefore relied on large-scale cyst mapping,parameterization of cyst germination rates using laboratoryincubations, and incorporation of these data into a coupledphysical/biological model. Model runs indicate that lightreaching the sediments is surprisingly not a crucial factorin germination success. Cysts from shallow waters germi-nated at nearly the same rate as those in deeper waters. Thisis because light is rapidly attenuated in bottom sediments,making attenuation due to water depth a minor factor.Layers of sediment above the cysts as thin as several mmmay be enough to inhibit germination. Similarly, germi-nation will likely be inhibited by anoxia even a few mm belowthe sediment surface. In such cases, resuspension by currentsor bioturbation may be important in fostering cyst germi-nation. Overall, this presentation highlighted the difficultiesthat still exist in estimating in situ germination rates.B. Dale introduced a geological time perspective into thedebate. Changes in time-scales up to 50 years are being re-lated with El Niño-like events, whereas changes in the orderof 100-year periods are related to climatic trends. Climatechange could be advantageous to cyst-forming species fromcold or warm coastal waters, allowing them to better exploitthe time shift in seasonal patterns. He criticized the use ofthe term “harsh environment,” which reflects an anthro-pocentric point of view.C. Bolch celebrated that probes are solving lots of oldproblems. Twenty out of 90 attendees to this round tableare currently using molecular probes in their research. Heemphasized the need to present more risky proposals, andto apply innovations in sampling strategies and moleculardesigns.C. Scholin noted that probes can be applied to intact cellsas well as cell homogenates. It is essential that developmentof these methods remain tightly integrated with traditionalmicroscopy-based species identification techniques. Sys-tems that enable use of molecular probes for near real-timedetection of HAB species, in situ, are fast becoming a re-ality. Many approaches that rely on cell-free detectionformats offer incredible sensitivity and speed, and can bepackaged in very small platforms such as hand-held devices.However, whole cell and cell-free detection methods can yielddifferent answers as to what species are apparently presentin a given sample. Working with natural samples, speciesidentifications based on cell-free methods will likely revealthat target organisms are present with greater frequency thanthose estimates based on intact cells. Key issues to resolveare creating standards for reporting cell presence/abun-dance using cell-free formats, and documenting therelationship between molecular signatures as seen in intactversus homogenized material.A. Kremp recalled that, in addition to nucleic acids,cell surface molecules are a group of potential target mol-ecules to improve our understanding of life cycle processes.548Studies of Chlamydomonas and ciliate sexual reproduc-tion have shown that cell wall molecules, and changes in theirstructure and composition, largely mediate gamete recog-nition and fusion. Thus, looking at cell wall proteins orglycoconjugates may help to characterize sexual cells andthe physiological processes involved in the sexual repro-duction of HAB species. Immunological techniques andproteomic approaches should be explored to identify spe-cific surface molecules and to characterize proteins.Carbohydrates could be targeted with complementarylectins. Fluorescent probes targeting specific cell surfacestructures can be developed and optimised for detection oflife cycle stages in the field, in conjunction with flow cy-tometry. Recognition and adhesion molecules on the cellsurface can also be useful for inhibition experiments tostudy the function of signalling, recognition and adhesionof gametes.Conclusions From the general discussion, several commonalities emergedfor HAB species research priorities. Complementing thoseoutlined by LIFEHAB, research should be focused on thefollowing knowledge gaps:• Inadequate knowledge of life cycle events for many of theimportant HAB species. The present state of knowledgedoes not allow for the formulation of general paradigms.• Social behaviour, active aggregation mechanisms.• Signalling (Quorum Sensing), infochemicals, species-spe-cific features.• Circadian and circannual clocks. Endogenous regula-tion of life cycle events. Role of photoperiod orphotoperiod fluctuations, temperature or temperaturefluctuations, in driving life-cycle transitions.Major gaps related to the ecological role of life cycles wererecognized to be caused by methodological and observa-tional constraints in field studies. The limited time-scale ofour data sets is also a problem which should be circumventedby a more extended use of sedimentological record, wherepossible, and by the support to long-term observationalprograms. Additional suggestions on new approaches,methodologies and research strategies were as follows:• Couple traditional techniques with advanced/moleculartools, in a bold and creative fashion.• Complement/substitute observation with adequate mod-els, to be developed.• Exploit knowledge and experience from different fields(e.g., microbiology, limnology, genetics of non-microal-gal organisms).• Coordinate efforts among scientists and promote cross-validation of methodologies and results.• Compare the behaviour of a species over its geographicrange through international cooperative research.• Improve the quality of species identification in field stud-ies through the cooperation of classical taxonomists,molecular biologists and ecologists.ReferencesG.M. Dunny and S.C. Winans, eds., American Society for Mi-crobiology, Washington, D.C., 1–367 (1999).E. Garcés, A. Zingone, M. Montresor, B. Reguera and B. Dale,eds., Proceedings of the LIFEHAB Workshop: Life history ofmicroalgal species causing harmful algal blooms. Commis-sion of the European Community, 1–189 (2002).

EU-LIFEHAB: Expanding the discussion on the life cycles of harmful algae

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EU-LIFEHAB: Expanding the discussion on the life cycles of harmful algae

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