Behaviour of the plathelminth Symsagittifera roscoffensis under different light conditions and the consequences for the symbiotic algae Tetraselmis convolutae

Oxygen-dependent photosynthesis is a significant pathway for many organisms. It is suggested that the development of oxygen only occurred once in evolution in the cyanobacteria or late Archaean (Cavalier-Smith, 2006). Later, the pathway was passed over by endosymbiosis to basal eukaryotes, leading to primary, secondary and tertiary plastids of various algal species as well as the plastids of higher plants that evolved from green algae (Johnson, 2011; Taylor, 1973; Trench, 1993). However, there are also many groups of organisms that developed mechanisms to capture photosynthetic products through the formation of symbiotic associations. These include the phyla Porifera (marine and freshwater sponges), Cnidaria (corals, sea anemones and freshwater hydra), Acoelomorpha (marine turbellaria), Mollusca (e.g. giant clams and nudibranchs) and Chordata (marine ascidians) (Serôdio et al., 2014; Trench, 1979; Venn et al., 2008). One special variety of photosynthesis in the animal kingdom occurs in the sea slug taxon Sacoglossa. They feed on algae, sequestering the chloroplasts. Some species of slugs are able to keep these chloroplasts alive within their own cells for several months (Händeler et al., 2009; Kremer, 1976, 1977). A horizontal gene transfer from the host algae to the slugs was suggested, but this suggestion is still controversial and is an area of ongoing research (Bhattacharya et al., 2013; Rumpho et al., 2008; Schwartz et al., 2014; Wägele et al., 2010).

The plathelminth of the order Turbellaria Symsagittifera roscoffensis (Graff 1891), formally known as Convoluta roscoffensis, is a simply organized unsegmented worm, living in symbiosis with the algae Tetraselmis convolutae (Norris et al., 1980; Parke and Manton, 1967). It is 2–4 mm long and contains 20,000–70,000 endosymbiotic cells of the algae within its body cavities (Doonan and Gooday, 1982). These cells are not passed on maternally, but must be acquired by each individual during the first days after hatching (Keeble and Gamble, 1907). This is a critical process, as the symbiosis is obligatory for the animals to survive. Thus, the worm becomes a photoautotrophic organism, consuming nutrients provided by the symbiotic algae (Keeble, 1910; Muscatine et al., 1974). The algae also profit from the symbiosis because they reach a higher rate of carbon fixation, compared with a free-living phytoplankton organism in habitats with tidal changes (Doonan and Gooday, 1982; Gooday, 1970). Doonan and Gooday discussed that the worms may regulate the photosynthesis of their symbionts, but only little is known about the worm's influence. Behavioural and physiological studies are rare but it is known that S. roscoffensis shows a positive phototaxis and escapes into the sandy sediment upon vibration (Keeble, 1910). Artificial light experiments with a light gradient suggest S. roscoffensis optimize photosynthesis by choosing the optimal light conditions for the algae (Serôdio et al., 2011). In general, the eyes of turbellarians are without lenses, consisting of a shading pigment cup and nerve cells (Yamasu, 1991). Nevertheless, this type of eye is capable of assessing the intensity and locating the direction of a light source (Jékely et al., 2008). In order to show whether S. roscoffensis’s phototactic behaviour is directed to optimize light perception of the symbionts, we used light of different wavelengths and intensities to trigger phototaxis.

Oxygen formation was measured using suspensions of algae isolated from the symbionts to gain information about the suitability of the light sources for photosynthesis (Fig. 2). The oxygen concentration of the medium increased with a rate of 0.19±0.12 µmol O₂ l⁻¹ s⁻¹ g⁻¹ biomass µmol⁻¹ photons for blue light and 0.50±0 µmol O₂ l⁻¹ s⁻¹ g⁻¹ biomass µmol⁻¹ photons for red light, respectively. A t-test revealed a significantly higher oxygen evolution rate for red light than for blue light (P=0.043, N=5).

To test whether symbiotic S. roscoffensis preferred illuminated areas over shaded ones, the animals (N=57) were exposed to three different light colours each at a photon flux density of 5 µmol photons m⁻² s⁻¹. The experimental setup is depicted in Fig. 3. A significantly positive response to illumination, i.e. a higher allocation time in illuminated versus shaded areas, was observed for green and blue light but not for red light (Fig. 4). In red light, average stay time was 510±257 s versus 385±255 s in the shaded area (P=0.08), while the allocation time for green light was 566±200 s versus 333±201 s in the shaded area (P=0.00005) and for blue light it was 550±182 s versus 348±183 s in the shaded area (P=0.0002). When the allocation times in areas illuminated by different light sources were compared in an ANOVA on ranks, no effect of light colour could be revealed.

When the animals (N=40) had the choice between all three colours and a shaded sector at the same time, the blue area was the most preferred, with a mean stay time of 455±193 s, and values of 162±114 s for green, 130±99 s for red and 131 s±138 s for shaded sectors (Fig. 5). Even if the blue light was less intense, it was still preferred over a brighter red sector (N=30) (Fig. 6). The allocation time in the blue sector of the arena (600±274 s) was significantly longer than that in the red sector (239±274 s; P=0.006).

The behaviour of S. roscoffensis (N=18) within a light gradient was tested in Petri dishes with 3.2 cm diameter. The arena was divided into two areas of the same size: a highly illuminated part and a part with a gradient of light (the umbra). Allocation times of 18 animals were recorded over 1800 s. The animals tended to prefer the fully illuminated half of the arena, with an average allocation time of 1235±732 s compared with 563±733 s in the umbra (P=0.06) (Fig. 7).

All data from the previous experiments were analysed to characterize the movement behaviour of S. roscoffensis. During 71 h observation time in total, the animals showed measureable movement for 34 h. The overall distance travelled by all animals was 135.3 m, resulting in an average speed of 1.1 mm s⁻¹ (3.9 m h⁻¹). With a probability of 99%, the speed of motion was about 4.5 mm s⁻¹ (17.55 m h⁻¹) or below (Fig. 8).

The aim of this study was to investigate the interaction between the host S. roscoffensis and symbiont T. convolutae, in order to find out whether the host provides an optimal environment for photosynthetic activity of the algae. Regarding the light sources used for the experiment, the blue and red LEDs were suitable for algal photosynthesis, and an oxygen development was detectable. However, the blue light was exceptionally less efficient and the red light is the optimal colour for the green algae photosystems (Butler, 1978; Emerson and Lewis, 1943). Concerning light intensity, T. convolutae reaches its optimal electron transfer rate at relatively low light doses. This was also observed in this study for the closely related species T. striata and T. suecia. Other common zooxanthellae such as diatoms or dinoflagellates reach their peak activity at an illumination level of 500–1500 µmol photons m⁻² s⁻¹ (Geel et al., 1997; Guarini and Moritz, 2009) or 600 to 800 µmol photons m⁻² s⁻¹ (Rodríguez-Román and Iglesias-Prieto, 2005), respectively.

The eyes of plathelminthes are very simple organs, built of only one type of receptor cell half shaded by a pigment cup cell. The receptor cells do not have enlargement of the membrane surface like cilia or microvilli (Nilsson, 2009; Yamasu, 1991). These simple eyes are capable of detecting the direction of a light source (Jékely et al., 2008). Nilsson showed by simulations that eyes of this kind are able to find the direction of a light source in water up to a depth of 200 m. Photoreceptors are proposed to originate from a single common ancestor and are all based on the pigment rhodopsin. They detect one specific wavelength according to rhodopsin's absorption spectrum (Arendt, 2003; Shichida and Matsuyama, 2009). It is very likely that S. roscoffensis only expresses one type of receptor and therefore is limited in its viable light spectrum. Depending on the water conditions, blue or green light will reach deeper areas in the sea, which makes light of shorter wavelength more important for phototaxis. Symsagittifera roscoffensis lives in a tidal environment in a water depth of about 0–4 m (Doonan and Gooday, 1982). The average light intensity in summer months in 4 m deep water is about 5.47% compared with the surface, but there is a significant difference in the transmission of different wavelengths depending on the water type: the observed light transmissions were 67, 52 and 55% m⁻¹ for green, blue and red light, respectively, in summer in a tidal environment (Lüning and Dring, 1979).

Our experiment revealed that the host's reaction to light stimuli is not adjusted to the symbionts' needs. Symsagittifera roscoffensis did not show a reaction to red light, but did to blue and green light, while the symbiotic algae show the highest photosynthetic rate for red light.

Our understanding is that S. roscoffensis does not react to red light, because it is not able to detect it and therefore its eyes are not suitable for measuring the exact photosynthetically active radiation to the benefit of the photosymbionts. Nevertheless, perception of wavelengths that are not dominant in a specific depth may be useful for the detection of fluorescent light, e.g. for communication, which has been observed in different fish species (Michiels et al., 2008; Shcherbakov et al., 2013, 2012).

Considering light intensities, the animals moved in the areas with an illumination higher than optimal for the algae. Possibly, these light intensities may even be harmful for the photosynthetic apparatus, though they may use photoprotectants to avoid oxidative stress (Cruz et al., 2013, 2015; Peers et al., 2009). However, S. roscoffensis is very mobile with speeds of up to 17.5 m h⁻¹ and would be able to reach water depths providing T. convolutae with optimal light by phototaxis. A previously published study by Serôdio et al. suggests an active regulation of the photosynthetic activity (Serôdio et al., 2011). However, in their setup, the light came from below with the gradient generated by a linear attenuation filter. Therefore, the animals trying to approach the light source would estimate it as being near the centre of the arena, maybe with a shift from the dark to bright side of the arena. This could in itself explain the result presented by Serôdio et al. (2011) without any active photosynthesis regulation. We avoided this problem with our system, which is closer to the situation in nature, with our light gradient generated with a light source illuminating the arena at an angle of 70 deg. In our setup, the animals did not avoid high illumination, but further studies are necessary to determine whether the host's behaviour may even harm the photosynthetic symbionts in case of a very high illumination level on bright summer days. The saturation of the ETR was observed as for Serôdio et al. (2011) for individual animals, with their results in the same range as our measurements with the separated algae.

Avoidance of high levels of illumination is known for other symbiotic animals: for example, negative and positive phototaxis is known in symbiotic sea anemones (Actiniaria spp.) depending on light intensities, e.g. negative phototaxis at a high illumination of 700 foot candle and positive phototaxis at 250 foot candle, which is about 140 PAR and 50 PAR, respectively (Pearse, 1974). Pearse noted that non-symbiotic anemones showed negative or indefinite reactions to light stimuli, which implies a relationship between host behaviour and photosynthesis of the symbiotic algae, but did not give any data about photosynthesis of the symbiotic algae. According to measurements made by other authors, the high illumination is below the photosynthetic optimum of common zooxanthellae (Geel et al., 1997; Guarini and Moritz, 2009; Rodríguez-Román and Iglesias-Prieto, 2005). We speculate that this allows a safety margin in light exposure to be kept for the anemone's symbionts. Such behaviour was not seen in S. roscoffensis.

Doonan and Gooday (1982) reported a decreasing population of S. roscoffensis in summer months and speculated that this may be the result of high illumination levels, which can be up to 1200 µmol photons m⁻² s⁻¹ on summer days (Migné et al., 2004). An active regulation of the light exposure suggested by Serôdio et al. (2011) would avoid such a decline in the population. With respect the animal's inability to react to red light that we observed in this study, we think that the position of Doonan and Gooday (1982) is more likely, but further studies are necessary to determine whether there is a harmful effect of high illumination levels in summer months.

The specimens of S. roscoffensis were obtained from the Station Biologique de Roscoff (Roscoff, France) and cultured with artificial seawater provided by the zoological and botanical garden Wilhelma Stuttgart (Stuttgart, Germany). Cultures were kept in Petri dishes (Schott, Mainz, Germany) of different sizes in a climate chamber at 7°C and a low level of illumination for 12 h day⁻¹, starting at 08:00 h (PAR, 7.9 µmol photons m⁻² s⁻¹). For the experiments, the animals were transferred to single wells of 24-well plates, and acclimated in a climate chamber at 16°C for at least 1 day before measurements were made. The illumination was 16.7 µmol photons m⁻² s⁻¹ for 12 h day⁻¹, starting at 08:00 h. Despite the rather short time for acclimatization, the animals showed a normal behaviour, which was expected as large temperature differences of up to 10°C within a day are common in tidal environments (Morris and Taylor, 1983). All experiments were performed during standard laboratory work time from 09:00 h to 17:00 h.

Symbiotic algae were extracted from naturally deceased worms by pipette and transferred to 50 ml Erlenmeyer flasks (Schott, Mainz, Germany) with Seawater Medium (SWES) according the protocol (version 10.2008) of the SAG (Culture Collection of Algae, Göttingen, Germany). For chlorophyll fluorescence measurements of the algae, fine biofilms were grown in 9 cm Petri dishes on 1% agarose in SWES solution.

The intensities of the different light sources (PAR) were measured with a quantum sensor (QS, Delta-T Devices, Cambridge, UK). LEDs of different colours were used to provide different wavelengths: red (LED CQY 40 L, Osram, Munich, Germany; λ_max=660 nm, spectral line half-width Δλ_1/2=40 nm), green (LED CQY 72 L, Osram; λ_max=560 nm, Δλ_1/2=20 nm) and blue (LED L-53MBDL, Kingbright Electronic, Taipei, Taiwan; λ_max=466 nm, Δλ_1/2=60 nm). Each LED was run with a battery of its nominal voltage and controlled by a 1 kΩ potentiometer for adjusting an intensity of 5 PAR. LEDs were mounted in an interior reflector to provide a homogeneously illuminated field that allowed positioning with a well-defined border between illuminated and shaded parts of the arena (SMZ1089, Signal-Construct, Niefern, Germany). Because of scattered light, the average brightness in the shaded sector reaches 63% of the average brightness in the illuminated sector, which was a good compromise for getting enough light for video recording and a behavioural response of the animals. The response of the animals to different light intensities was tested in a light gradient. An adjustable light source from a binocular loupe (GSZ loupe, Zeiss, Jena, Germany) was fixed with an angle of 70 deg at a height of 20 cm, with a piece of aluminium foil on the upper half of the lamp for shading. The brightness in this gradient started at 24.92 PAR; peak value was 459.17 PAR within a Petri dish of 37 mm diameter.

The ETR of photosystem II of isolated algae was measured at varying actinic light intensities using a PAM 2000 fluorimeter with the default settings ‘run 9, actinic light intensity series’ (Heinz Walz, Effeltrich, Germany). The sensor had a distance of 1 cm to the surface of the algal biofilm.

Oxygen evolution by suspensions of algae isolated from the symbionts was measured with a Clark-type electrode (Type S1, Hansatech, King's Lynn, UK). The rate of photosynthesis following illumination by blue and red diodes was determined as the increase of the oxygen concentration in the suspension per dry algal biomass, measured after drying the algae for 2 days in a SpeedVac concentrator (Savant, Fisher Scientific, Schwerte, Germany). Five replicates (0.5 ml each) of algal suspension were tested at a photon flux density of 25 µmol photons m⁻² s⁻¹ (blue) and 38 µmol photons m⁻² s⁻¹ (red).

The behaviour of S. roscoffensis was recorded with a standard camcorder (DCR-PC 109 E, Sony, Tokyo, Japan) using the software Virtualdub v1.8.6 (www.virtualdub.org) (Fig. 3). The video frame rate for recording and analysis was one picture per second. The location coordinates of S. roscoffensis were evaluated as the allocation time in different sectors of the arena by the program BioMotionTrack D.S. (Shcherbakov et al., 2010) and stored in an Access (Microsoft, Redmond, USA) database. The response to the different wavelengths was tested for 900 s at an illumination of 5 PAR in Petri dishes with a diameter of 15.5 mm and a water depth of 5 mm seawater. The measurements within the light gradient were performed in Petri dishes of 37 mm diameter and 5 mm water depth for 1800 s. The behaviour in response to different wavelengths was tested in a series of experiments by comparing the length of stay within the illuminated and not illuminated half of the arena. In a second series, experiments were performed with sectors representing each of the three wavelengths plus a fourth not illuminated, shaded sector.

For testing the effect of light intensity, the blue light source was adjusted to a photon flux density of 5 µmol photons m⁻² s⁻¹, while the red LED (660 nm) was operated at 44 µmol photons m⁻² s⁻¹. The total irradiance for the blue LED was 1.3 mW m⁻² and for the red light source it was 7.6 mW m⁻². The conversion from photon flux density to radiation power was calculated for the dominating wavelength (Schröder and Treiber, 2007). During the behavioural experiments, the light sources were used in random order. To characterize motivation for active orientation behaviour, the animals' speed of motion was additionally calculated.

Statistical analysis of allocation time data was performed using either the Exact Wilcoxon signed rank test with continuity correction for pairwise analysis or an ANOVA on ranks. Statistical significance of differences in oxygen evolution was tested using a t-test. Significance was assumed for P<0.05 in all statistical tests. The data for ETR were fitted with Eqn 1. All statistics were performed with Sigmastat. Values are given as means±s.d.

Thanks are due to Thierry Comtet, Wilfried Thomas and Xavier Bailly from the Station Biologique de Roscoff, Roscoff, France, for the provision of the turbellarians. We would also like to thank the anonymous reviewers for their very useful comments.