Epilithic biofilms are benthic microbial aggregates formed by an association of autotrophs and heterotrophs, prokaryotes and eukaryotes micro-organisms. With their relatively cohesive organization of micro-organisms, those species agglomerates are quite auspicious for allelochemical interactions development. The aim of this study was to show the existence of this kind of interactions between benthic microalgae in photoautrophic biofilms and to study their role in algal communities structuration through space and nutrients competition.
In a more general context, epilithic biofilm is an example of complex integrated system whose dynamic functioning involves many signalisation and regulation networks. This study will help us to understand how cells can communicate with one another and organize themselves in larger and more complex structures according to their identity or their environment.
Both natural and artificial epilithic biofilms with cyanobacteria and diatoms as main components will be studied to understand the role of allelopathy in their development.
Allelopathy, epilithic biofilm, cyanobacteria, diatoms, confocal microscopy (laser scanning)
Allelopathy is a widespread phenomenon in aquatic environments. It may involve many different organisms: angiosperms, macrophytes, macroalgae, microalgae or cyanobacteria (Gross 2003). Two kinds of aquatic habitat may be considered for the study of allelopathic interactions. First, there are pelagic areas where physical constraints are quite different from terrestrial systems. In such an aqueous environment, allelochemicals need to be sufficiently hydrophilic, the diffusion is less quick because of viscous forces, the dilution is important, and it is a 3D system (Wolfe 2000). The second habitat is the benthos. In this 2D zone, cells are much closer, even touching which allows allelochemicals to be more lipophilic and the dilution is reduced (Jüttner 1999). These properties make the benthos a favourable system for the development of allelopathic interactions. This is heightened by the existence of competition for the available surface (light and nutrient access, anchor zone) in addition of nutrient competition. In freshwater systems, it appeared that many allelochemical producing cyanobacteria where benthic (Smith and Doan 1999; Gross 2003).
Epilithic biofilms are a well-studied example of benthic system. They are microbial aggregates formed by an association of autotroph and heterotroph, prokaryote and eukaryote micro-organisms. They present a heterogeneous spatial organization where micro-organisms are necessarily in interaction (Wimpenny et al. 2000). Jüttner and Wu (2000) demonstrated that 20% of tropical cyanobacterial biofilms presented an allelochemical activity. Nonetheless, allelochemical activity of the others benthic microalgae has not been much studied. Moreover, the effects of the allelochemicals on well-represented benthic algae such as diatoms have not been yet studied. Yet, the role of allelopathy in diatoms-cyanobacteria successions has been demonstrated in planktonic environment (Keating 1977; Keating 1978; Takamo et al. 2003). In biofilms, the sequence of colonization of the substratum contains a succession diatoms-cyanobacteria (Sekar et al. 2004). In this context, the aim of our study was to investigate the interactions between diatoms and cyanobacteria during biofilm development and to understand how these interactions may involve in the spatial structuring of the biofilm. Both natural and artificial biofilms were studied. For this purpose, several algal strains were isolated from the same river and the allelochemical activity of natural biofilms was monitored during one year period.
Epilithic biofilms were sampled in a fixed station of the river Garonne, France. During low-water periods, low current velocities and low depth enable the development of an important epilithic biomass. In these conditions, the specificity of this river which corresponds to a 6th-order river is to be a river with a fixed biomass. Biofilms were sampled during a whole year in order to observe possible variations of the allelochemical activity in relation with species successions.
Every two weeks, biofilms were scrapped off on three pebbles (depth was between 10 and 70 cm). Samples were divided in four subsamples. One was stored at -80°C before molecules extraction. A subsample was fixed with glutaraldehyde (1%) and stored at 4°C for algal identification and counts. Another subsample was used to determine the biomass per surface unit. The last one was centrifugated and stored at -80°C for pigments analysis.
The experimental set-up is based on artificial biofilms, grown in microcosms and composed of a limited number of algae species (cyanobacteria and diatoms isolated for this study). Non destructive techniques of biofilms investigation are used in order to elucidate both spatial structure and functioning (laser-scanning confocal microscopy, micro-electrodes).
Twenty-three strains of diatoms have been isolated from the river Tarn and from the river Garonne: 2 strains of Melosira varians, 9 strains of Nitzschia palea, 3 strains of Fistulifera saprophila, 2 strains of Fragilaria ulna, 1 strain of Diatoma mesodon, 1 Diatoma sp, 1 Cymbella sp, 2 Nitzschia sp., 2 Fragilaria sp. Five strains of cyanobacteria have been isolated from the river Garonne: 4 Oscillatoria sp, a Pseudanabaena sp. Three Phormidium sp, one toxic and two non toxic were also used to test the effects of strain toxicity.
Test of allelochemical activity
Molecules were extracted with 80% methanol. The extracts’ activity was tested by addition to a culture of a cyanobacterium, Anabaena PCC7120 which is often used as a model organism for toxicity tests (von Elert and Jüttner 1996; Srivastava et al. 1998). The anti-predator activity of the extracts was analyzed in a bioassay with Thamnocephalus platyurus (Jüttner 2001).
The mutual effects of all these algae were tested by cross-culturing: a species was grown in a medium enriched with cell-free filtrate from another species. Algal growth was followed by optical density.
Variations of the allelochemical activity of the natural biofilms are expected, depending on biofilm development stage, species composition and season. Contrary to our study, Jüttner and Wu (2000) take into account spatial and species composition effect, and no time effect. Authors demonstrated that one biofilm tested over five presented an allelochemical activity. Our study will provide information on the temporal pattern of this activity. Moreover, the biofilms tested here will have a more various composition: diatoms dominated in winter and cyanobacteria in summer. In Jüttner and Wu (2000), only cyanobacteria-dominated biofilms were studied. The species that produce the allelochemicals may be identified thanks to the determination of the biofilm species composition. Transition periods will certainly be the more interesting one. It is the first study concerning the allelochemical activity of several biofilms in a temperate environment, the biofilms studied by Jüttner and Wu (2000) were tropical. The isolated strain with an allelopathic activity will be studied more precisely.
Three strains of Fistulifera saprophila (formerly Navicula saprophila) have been isolated. This species is known to produce the eicosapentaenoic acid (EPA) (Kitano et al. 1997). This polyunsaturated fatty acid has been shown to have an anti-predator activity (Jüttner 2001). This will be tested for the three strains as well as the effect of the presence of predator and others algae on the EPA productivity rate. Moreover, the effect of EPA on the development of a complex biofilm will be tested.
Gross EM (2003). Allelopathy of aquatic autotrophs. Critical Reviews in Plant Sciences 22, 313-339.
Jüttner F (1999). Allelochemical control of natural photoautotrophic biofilms. In 'Biofilms in aquatic environment'. (Eds. C W Keevil, A Godfree, D Holt, C Dow), pp. 43-50.(Royal Society of Chemistry, Cambridge)
Jüttner F (2001). Liberation of 5,8,11,14,17-eicosapentaenoic acid and other polyunsaturated fatty acid from lipids as a grazer defense reaction in epilithic diatom biofilm. Journal of Phycology 37, 744-755.
Jüttner F and Wu JT(2000). Evidence of allelochemical activity in subtropical cyanobacterial biofilms of Taiwan. Archiv für Hydrobiologie 147, 505-517.
Keating KI (1977). Allelopathic influence on blue-green bloom sequence in a eutrophic lake. Science 196, 885-887.
Keating KI (1978). Blue-green algal inhibition of diatom growth: transition from mesotrophic to eutrophic community structure. Science 199, 971-973.
Kitano MR, Matsukawa, et al. (1997). Changes in eicosapentaenoic acid content of Navicula saprophila, Rhodomonas salina and Nitzschia sp under mixotrophic conditions. Journal of Applied Phycology 9, 559-563.
Sekar R, VP Venugopalan, et al. (2004). Early stages of biofilms succession in a lentic freshwater environment. Hydrobiologia 512, 97-108.
Smith GD and NT Doan (1999). Cyanobacterial metabolite with boactivity against photosynthesis in cyanobacteria, algae and higher plants. Journal of Applied Phycology 11, 337-344.
Srivastava AF, Jüttner, et al. (1998). Action of the allelochemical, fischerellin A, on photosystem II. Biochica et Biophysica Acta 1364, 326-336.
Takamo KS, Igarashi, et al. (2003). Causation of reversal simultaneity for diatom biomass and density of Phormidium tenue during the warm season during the warm season in eutrophic Lake Barato, Japan. Limnology 4, 73-78.
von Elert E and F Jüttner (1996). Factors influencing the allelopathic activity of the planktonic cyanobacterium Trichormus doliolum. Phycologia 35, 68-73.
Wimpenny J, W Manz, et al. (2000). Heterogeniety in biofilms. FEMS Microbiology Reviews 24, 661-671.
Wolfe GV (2000). The chemical defense ecology of marine unicellular plankton: constraints, mechanisms, and impacts. Biological Bulletin 198, 225-244.