Settlement Behavior of Zoospores of Ulva linza During Surface Selection Studied by Digital Holographic Microscopy
© The Author(s) 2012
Received: 22 March 2012
Accepted: 10 April 2012
Published: 3 May 2012
Settlement of the planktonic dispersal stages of marine organisms is the crucial step for the development of marine biofouling. Four-dimensional holographic tracking reveals the mechanism by which algal spores select surfaces suitable for colonization. Quantitative analysis of the three dimensional swimming trajectories of motile spores of a macroalga (Ulva linza) in the vicinity of surfaces functionalized with different chemistries reveals that their search strategy and swimming behavior is correlated to the number of settled spores found in spore settlement bioassays conducted over 45 min. The spore motility and exploration behavior can be classified into different motion patterns, with their relative occurrence changing with the surface chemistry. Based on the detailed motility analysis we derived a model for the surface selection and settlement process of Ulva zoospores.
A critical phase during the life cycle of sessile marine organisms is the planktonic dispersal stage, (larvae in the case of invertebrates such as barnacles and hydroids; motile spores in the case of many seaweeds), during which they settle and attach to a surface. Settlement is a selective process involving reversible contact with the substratum through exploratory behavior in response to a range of surface-associated signals. Having selected a suitable surface the attached larvae/spores then undergo metamorphosis to generate the adult organisms. When the immersed surface is man-made, the resulting colonization is a nuisance phenomenon generally referred to as ‘biofouling’, which causes major financial and environmental problems in various industries. Ships and leisure vessels, membrane filters, heat exchangers, underwater sensors and aquaculture systems are all subject to biofouling, with the consequence for ships of higher fuel consumption (and hence greenhouse gas and soot emissions) and demand for frequent cleaning .
Understanding the complex behavioral mechanisms involved in the recruitment of motile propagules to surfaces is fundamental to control the distribution, abundance and dynamics of organisms on hard substrata in the marine environment, and thus in the design of novel ‘antifouling’ coatings. The motile, quadriflagellated zoospore of Ulva linza with a spore body diameter of 4–5 µm has been extensively studied as a model biofouling organism. In order to complete the life cycle, zoospores must locate a surface and settle on it (i.e., permanently attach to it). Previous investigations by video microscopy (2) revealed that spores show complex swimming behaviors as they approach and make contact with surfaces. The attachment process appears to involve an initial, temporary, and reversible phase of varying duration, and is characterized by a rapid “top-like spinning”, although in certain circumstances spores may also settle without spinning. For example, video microscopy has recorded settlement i.e., permanent adhesion, of swimming spores against debris and previously adhered spores without any surface exploration or spinning (unpublished observations). Final commitment to irreversible and permanent adhesion involves the discharge of a preformed glycoprotein adhesive from cytoplasmic vesicles, as the cell contracts against the surface. The membrane of the sheaths surrounding the flagella are shed into the water, the axonemes are retracted into the settling spore [2, 3]. The attached spore secretes a cell wall and can then germinate to grow into the macroscopic, easily visible green seaweed.
2 Materials and Methods
2.1 Preparation of Surfaces
The surfaces used to seal the observation chamber and for the motility analysis were either acid washed glass (AWG) cover slips (Carl Roth GmbH) or cover slips coated with FOTS or polyethyleneglycoltriethoxysilane (PEG). FOTS silane was coated on glass cover slips through chemical vapor deposition (CVD) for 2 h at 80 °C . Subsequently samples were rinsed with ethanol (p.A.) and dried with N2 gas. Polyethylene glycol (PEG) triethoxysilane (PEG2000-urea) was synthesized according to previously published protocols . Prior to coating, glass cover slips were activated by piranha solution (H2SO4/H2O2 = 3:1). 0.25 mM PEG silane and 2.5 µM triethylamine were dissolved in dried toluene (p.A.). The activated glass cover slips were immersed in this solution and allowed to react for 48 h at 55 °C. Subsequently, the samples were rinsed with ethyl acetate (p.A.) and sonicated in ethyl acetate for 2 min and then rinsed again with ethyl acetate and methanol (p.A.). All surface coatings were characterized by ellipsometry, XPS, and contact angle goniometry. The AWG coverslips were prepared by immersion in 0.1 M HCl for 24 h, before washing extensively in deionised water and blowing dry in a stream of nitrogen. For the standard spore settlement assay, clean-room sealed Nexterion glass slides (Schott) were used as substrate. Slides were reacted with FOTS and PEG as supplied.
2.2 Preparation of zoospore suspension
Fertile plants of Ulva linza were collected from the seashore at Llantwit Major, South Wales, UK (51°40′N; 3°48′W) in June 2008, 5 days before full moon. Plants were stored (maximum 48 h) at 4 °C until zoospores were released. Zoospores were released from fertile tips into filtered (0.22 µm) artificial seawater (ASW: Tropic Marin). The spore suspension was filtered into a beaker through 3 layers of nylon mesh (100, 50 and 20 µm) to remove debris. The beaker containing the spore suspension was plunged into ice, which concentrates the spores (spores swim towards the bottom of the beaker), which were pipetted into another beaker. This procedure was repeated and then the spore suspension was filtered through 2 layers of nylon mesh (20 µm pore size). The spore suspension was kept on a magnetic stirrer and the absorbance at 660 nm measured. The spore suspension was diluted with filtered (0.22 µm) ASW to a final concentration of 1.3 × 104 spores/ml for holography, or 1.0 × 106 spores/ml for standard settlement assays. As only one holographic setup was available, the spores used for the experiments were released at different times. For the FOTS surfaces spores were released 7 h after collection from the seashore, for PEG 21 h and for AWG 30.5 h. One dataset obtained on each of the respective surfaces has been analysed. While the release time after collection did not have a major influence on the observed motion patterns, we noted that the mean velocity of the spores decreased over time.
For the standard assay, 10 ml of freshly released spores were added to individual compartments of sterile Quadriperm dishes each containing a test surface. Three replicates of each test sample were immersed simultaneously. The slides were incubated in darkness for 45 min and then washed gently with ASW to remove unsettled i.e., motile, spores. The three replicates were used to determine the number of settled (attached) spores. Spores were fixed in 2.5 % glutaraldehyde in ASW, washed in deionized water and dried. Spore counts were taken using a Kontron 3000 image analysis system attached to a Zeiss epifluorescence microscope. Spores were visualized by autofluorescence of chlorophyll and counts were recorded for 30 fields of view on each replicate slide as described by Callow et al. .
2.3 Digital In-Line Holographic Microscopy
An in-line holographic microscope as developed by the Kreuzer group [16, 22, 25] was used, consisting of a light source, a pinhole, a wet cell and a detector, all arranged on the same optical axis. For illumination a diode pumped solid state laser providing continuous wave (cw) light with a wavelength of 532 nm with a power of 30 mW was used (IMM Messtechnologie, model GLML4C1-30). A commercial (National Apertures) pinhole with a diameter of 500 nm was used to generate the divergent wave front. The detector was an OEM CCD module (Lumenera Lu160M) with a resolution of 1,392 × 1,040 pixels (pixel size 6.45 µm) and a frame rate of 15.4 Hz located 16 mm behind the pinhole. The wet cell had a volume of 50 µl and was made out of Teflon® and sealed with the surfaces of interest. From the recorded holograms, the wavefront was back-propagated through the complete volume via a Kirchhoff-Helmholtz transformation . From such reconstructions, different projections in real space were calculated and spore trajectories were extracted as described previously . For each of the three surfaces, holographic movies at three different time points (immediately and up to 13 min after injections) with a typical duration of 40 s were analyzed. The full data set consisted of 414 spore trajectories containing a total of 39,650 spore positions and correspondingly velocity vectors.
The result of a standard spore settlement assay (after 45 min exposure to a zoospore solution)  is shown in Fig. 3c. In line with previous studies, FOTS showed the highest amount of settlement after 45 min and glass (AWG) an intermediate coverage of zoospores, while the PEG coated surface had no settled spores [6, 26]. These results positively correlate with the deceleration of the spores Fig. 3b, i.e., the most ‘attractive’ surface for settlement showed the greatest spore deceleration.
Figure 4c–e shows the occurrence of the different motion patterns depending on the surface chemistry as they occurred in the volume extending 200 µm away from the surface. For the distributions only the three earliest time points immediately after injection were investigated (≈60 trajectories with a total of ≈5,500 data points). The probability of observing the different motion patterns differed between the surfaces. Gyration was detected as the dominant pattern on PEG and on AWG (Fig. 4c, d), which means that spores explored the surfaces and established temporary surface contacts. However, on PEG the probability of observing a hit and run event was nearly twice as high (44 %) compared to AWG (23 %), indicating that the PEG surfaces were less attractive to the spores. The situation was different on FOTS and spores exploring the surface showed predominantly the hit and stick behavior (Fig. 4e). A hit and stick pattern never occurred on PEG and AWG. The high probability of observing a hit and stick pattern indicated that the pristine and hydrophobic fluorinated surface attracted spores.
In summary, insight into the differences in spore settlement density on three different surfaces has been provided by DIHM. Quantitative analysis of DIHM data revealed that the mechanism by which spores select a surface for settlement involves deceleration, followed by a number of different surface probing behaviors, the nature and duration of which vary with the attractiveness of the surface for settlement and permanent adhesion. The spinning phase in particular could serve as a sensing mechanism by which spores probe the surface and predict its ability to bind the permanent adhesive. The exploration behavior of the spores correlates with the final spore coverage after a conventional 45 min settlement assay on the respective surface.
This work has been funded by the EU 6th Framework Integrated Project “AMBIO”, the Office of Naval Research (Grant N00014-08-1-1116) and the DFG projects RO 2497/7-1 and RO 2524/2-1. We gratefully acknowledge the stimulating discussions with Hans Jürgen Kreuzer and Bodo Rosenhahn. We thank Celine Rüdiger and Sebastian Weiße for help with data analysis.
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