Читать книгу Diatom Gliding Motility - Группа авторов - Страница 21

When examining drops hanging from a cover glass, the diatoms contained sink to the water-air interface. They can show movements there even without a solid substrate [1.26] [1.3]. Occasionally, living diatoms also float on the water surface of densely populated cultures. This was relatively common in Nitzschia sigmoidea and much rarer in Cymatopleura solea, as well as in certain species of the genera Cymbella, Rhopalodia and Pinnularia. Since only a small number of diatom species have been cultivated, it can be assumed that this phenomenon occurs in many other genera and species. These questions arise in particular:

 • How do the diatoms get to the water surface?

 • Why do the diatoms not sink immediately and how long do the diatoms remain on the water surface?

 • Can the diatoms survive on the water surface for a long time?

 • Which movement patterns and interactions between the diatoms occur and how can they be explained?

 • Do diatoms have an advantage from their ability to float and to move on the water surface?

It is anticipated that there will probably be no valid answers for all species, because the differences are considerable, especially with respect to their motility. The best observations to be interpreted are available in Nitzschia sigmoidea. For this reason, these are to be presented first. This is followed by comments on Pinnularia.

The cultivated Nitzschia sigmoidea came from various waters, a small reservoir (Aichstruter Stausee), its outflow (Lein) and a pond (in Stuttgart-Hohenheim). In a newly created culture, the phenomenon of floating diatoms is apparent after about 2 to 3 weeks, whereby the first isolated floating diatoms can often be found after only one week. In a fully developed culture, connected structures can form that cover a large part of the water surface. There are cultures in which the number of diatoms on the water surface is far greater than on the substrate. The diatoms are not located in a flake connected by EPS, which gets buoyancy by oxygen bubbles, something that occasionally occurs in this species at very high population density, but lie horizontally on the water surface. Whereas this species can occasionally be found in valve view when looking vertically at a solid substrate, on the water surface it is always found in girdle band view, apparently the equilibrium position. A possibility to rotate on the water surface around the apical axis does not seem to exist.

According to current knowledge, a buoyancy of the diatoms due to a lower specific weight than water can be excluded. The cultures were repeatedly carried a few meters to the microscopes for examination. In many cultures, one could observe that diatoms were whirled up and sedimented slowly. Apparently, even small water flows cause Nitzschia sigmoidea to detach from the substrate and to accumulate in the water. It can be assumed that diatoms also reach the water surface. As the diatoms obviously do not sink again, but show a remarkable floatability, they accumulate on the water surface. Further observations are required to substantiate this mechanism.

In the phase contrast, pronounced brightening is visible on most apices. Significant changes in brightness also occur in differential interference contrast (DIC) or differential interference contrast for plastic receptacles (PlasDIC). Figure 1.22 shows a typical appearance. Under the stereomicroscope you can see with an oblique view that the water surface is arched around the apices. There is a more or less distinct convex meniscus, which explains the appearance in phase contrast, DIC or PlasDIC. The diatom lies deep in the water (Figure 1.23).

The ends of the diatom have obviously hydrophobic properties, which lead to the deformation of the water surface and give it buoyancy due to the surface tension of the water, as is known from the water strider. In Figure 1.24 a diatom is sketched from a horizontal view. Wang et al. [1.33] have found that cleaned valves of Coscinodiscus sp. float on water surfaces. Here too, hydrophobicity is the cause. After examination of cleaned valves, the authors conclude that the hydrophobicity is based on the convex form and 40 nm sieve pores. I consider it an open question whether this explanation applies to floating living Nitzschia sigmoidea. Apart from the other structure of the valve, it can be assumed that living diatoms are surrounded by a layer of organic material. This could prevent the influence of the pores on one side and on the other side it could have hydrophobic properties itself. In this context, it should be mentioned that the cell lines of Nitzschia sigmoidea lost the ability to float after a few months and never regained it. At first the typical patterns of connected diatoms on the water surface became less regular and finally disappeared completely. The floating diatoms did not always lie in the same plane but frequently crossed, and occasionally they hung with only one end on the surface of the water. Later, the proportion of diatoms on the water surface decreased noticeably. An explanation of the origin of hydrophobia should also clarify this.

Figure 1.22 Nitzschia sigmoidea on the water surface viewed with PlasDIC.

Figure 1.23 Nitzschia sigmoidea with a stereomicroscope in oblique view.

Figure 1.24 Sketch of a Nitzschia sigmoidea on the water surface seen from the horizontal direction.

Nitzschia sigmoidea can survive many days on the water surface. This is probably made possible by the high proportion of wetted surface. The rapid increase of diatoms on the water surface and the sometimes high density of floating diatoms compared to benthic living diatoms suggest that they reproduce asexually on the water surface.

There is an attractive interaction between hydrophobic bodies on the water surface. When floating hydrophobic bodies move towards each other due to this force, energy is released into the environment. The system strives for a state of minimal energy. Lycopodium spores scattered on a water surface, for example, bond together and form two-dimensional structures with local order. In this process, restructuring takes place in which contacts are broken up and are closed at other locations until a local minimum of energy and thus a stable equilibrium is reached. The global minimum will only be reached in the case of very few particles. Wang et al. [1.33] report on the formation of regular structures at Coscinodiscus sp. on the surface of a water droplet. This self-assembled monolayer is a consequence of the short-range attractive interaction between the hydrophobic frustules.

In Nitzschia sigmoidea, the attractive force acts at the ends of the diatoms. Figure 1.25 shows two diatoms in the valvar plain with the resulting water surface. The water surface has a lower energy than with two separate diatoms according to Figure 1.24. When several diatoms drift with hydrophobic apices on the water surface, patterns are formed in which the ends preferably stick together. This results in locally star-shaped and polygonal structures. Not all diatoms have a pronounced hydrophobic apex. It is enough to stay on the water surface, but the attractive interaction is barely recognizable in the movement. There are systematic differences in the strength of the hydrophobicity in diatoms from different localities that affect the patterns. A low interaction leads to loose arrangements (Figure 1.22), a strong interaction to patterns where the apices are close together (Figure 1.26). It will probably not be possible to give an analytical representation of the attractive force. A simplified modeling consists in the replacement of the diatom by two rotationally symmetric hydrophobic particles, which are connected by a rod having the length of the diatom. For the experimental determination of the attractive interaction one can examine the motion of diatoms, which move toward each other, or the movement of a hydrophobic particle like spores of Lycopodium in the vicinity of a diatom. Under the plausible assumption that the inertia force can be neglected in the equation of motion, and Stokes’ law holds, the velocity is proportional to the force. In Figure 1.27 the speed of a diatom, which is proportional to the force, is plotted versus the distance between two approaching diatoms. It was determined from a video recorded at 30 fps. This is not sufficient to measure the high speeds shortly before the collision. The fluctuations in velocity at longer distances are probably due to the different drift velocities of the diatoms. This image is primarily intended to illustrate the method.

Figure 1.25 Sketch of two adjacent Nitzschia sigmoidea on the water surface seen from the horizontal direction.

Figure 1.26 Very regular structure of a diatom cluster on the water surface (dark field).

A remarkable aspect is the dynamics of movement. Different forms of movement occur, which indicate different mechanisms. Single floating diatoms are able to move slowly in the direction of the apical axis. This is similar to the movement on substrate, but hardly any longer distances are covered. Adhering lumps of EPS are typically not visible. Since no axenic cultures are used, bacteria can usually be seen on the water surface in phase contrast, making water movement observable. One recognizes changing currents of the water along the raphes, which run opposite to the movement of the diatoms. Apparently, the activity of the raphes can couple to a liquid medium. I do not want to exclude the possibility that the bacteria have an influence on the movement by mechanically coupling to the raphes, but in view of their small size and their lack of mutual contact, I consider their influence to be small. Theories of motility that require adhesion to a solid substrate cannot adequately explain this phenomenon. The movement of diatoms in hanging drops at the interface to air was interpreted by Nultsch in 1957 on the basis of the since disproved cytoplasmic streaming theory. Wavelike movements of microfibrils, as suggested by Bertrand [1.4] in 2008 (translation in Chapter 12 of this book), could cause the observed water transport along the raphe. They strengthen this hypothesis. I could not observe this form of movement with certainty in clusters of several connected diatoms, possibly because they contribute little to movement compared to the other forces acting there.

Figure 1.27 Relative speed of two diatoms plotted versus their distance.

It is obvious in Nitzschia sigmoidea cultures with high population density that the diatoms secrete EPS. These excretions can accumulate and lead to the formation of flakes of living diatoms. Sometimes it can be noticed that more or less large lumps of EPS adhere to individual diatoms on the water surface. When two diatoms come into contact through the mediation of an EPS lump, the transport of this lump along the raphes of both diatoms leads to a fast and remarkable movement. As there is no adhesion to a solid substrate, the two diatoms “dance” around each other. This movement pattern often occurs without the EPS lumps being recognizable.

Diatoms that have come together in structures such as those shown in Figure 1.22 and Figure 1.26 show a peculiar dynamic. The attractive interaction of the hydrophobic apices is of great importance here. If only this attractive interaction existed, a pattern would be formed on the water surface, whereby the movement would come to rest as soon as a local minimum of energy is reached. However, the activity along the raphe and in the area of the apical pore field leads to continuous changes of the pattern. According to the simple model of two hydrophobic particles in the distance of the apices one expects structures with few unbound ends. For example, when there are three diatoms in a cluster, energetically favorable configurations in which all ends have bonds to other diatoms should prevail (Figure 1.28). A chain of three diatoms should close to a triangle because of the attractive interaction of the free ends. But the diatoms are not passive floating objects, but show active movement. They release energy and thus temporarily produce patterns with higher energy. There are elementary processes which are observable locally in the entire structure pattern. The end of one or more parallel diatoms can be moved by another diatom along its raphe (Figure 1.29). At the beginning of this movement, an existing connection at an apex of the moved diatom can be broken. Energy is released locally from the diatom into the structure. Angular changes at connected apices (Figure 1.29b) are very frequent. Diatoms that adhere with the apex on a substrate are capable of performing tilting movements. Probably it is the same mechanism as observed on the water surface. It is by no means certain that all motion sequences can be explained thereby, because the simultaneously occurring changes in this many-body system are very complex. One striking feature is the separation of connections at the apex without subsequent transport along a raphe. It could be due to opposing forces within the cluster. The static attractive force of the apices, the active movement along the raphe, the active change of the angle between two connected diatoms and possibly other mechanisms can generate a very large number of patterns and sequences in a system of several diatoms (Figure 1.30). These processes are fundamentally different from those on a solid substrate.

In nature, the currents in the water are also likely to whirl up diatoms that do not have a high adhesion to the ground. They may occasionally reach the water surface. The ability to stay there for a longer period of time could lead to dispersal by drifting, i.e., hydrochory.

Figure 1.28 Energetically favorable patterns of three diatoms on the water surface: all diatoms parallel (a) and diatoms form a triangle (b).

Figure 1.29 Frequently observed movement patterns: movement along the raphe (a) and angular changes at connected apices (b).

Figure 1.30 Image sequence showing the temporal development of seven connected diatoms. The time between the first and last image is 170 seconds.

Figure 1.31 Pinnularia gentilis.

I am not convinced of this mechanism and without evidence it is nothing but speculation. A benefit from the mobility at the surface is not evident. Structures of interconnected Nitzschia sigmoidea, as described above, require a very calm water surface in addition to a high population density. Under light winds and waves, these fragile structures will certainly not be able to form or preserve themselves. I consider these patterns and their dynamics to be an artifact that only occurs in cultures, but it allows an insight into the motility of this species.

The observations of Craticula cuspidata, Cymbella spp., Rhopalodia spp. and Pinnularia spp. on the water surface differ in several aspects from those of Nitzschia sigmoidea. In the following I will restrict myself to comments on Pinnularia gentilis (Figure 1.31) from a small pond in Stuttgart-Hohenheim. At the time of observation, the diatoms were already six months in culture and had a typical length of 200 μm. When Pinnularia cultures are prepared, a fast sedimentation of inserted diatoms is usually observed. In the case of diatoms from these cultures, it is noticeable that many diatoms are stirred up when the Petri dish is carried to the microscope and settle relatively slowly on the substrate. Immediately after swirling up the diatoms, one regularly finds a few to several tens of diatoms on the water surface. Already in the first minutes many of the diatoms sink to the ground. Others remain on the surface for hours and only a few for days. In all observed cases, the sinking starts with the diatom taking a position perpendicular to the water surface. Often it remains on the water surface in this orientation for a while. Either the diatom drops to the ground in this orientation or it rotates around the transapical axis or pervalvarous axis as it sinks. It was not possible to recognize which axis it is. During one observation, a diatom returned from the vertical position back to the horizontal position on the surface, which requires an energy supply by the diatom.

Pinnularia floating horizontally on the surface are almost completely enclosed by water. A deformation of the water surface is not visible in phase contrast. There is also no formation of regular patterns due to an attractive interaction. Nevertheless, I consider a slight hydrophobicity to be possible.

Bacteria can often be found on the water. If these form a coherent turf, this probably has a significant influence on the movement of the diatoms. Also, with these observations the water surface showed only a low bacterial density, so that I consider its influence negligible.

When looking vertically at the water surface, the diatoms appear on the water surface in both valve view and girdle band view, with the valve view dominating. Occasionally there is a 90° rotation around the apical axis and thus a change between the two views. Presumably an activity of the raphe in the area of the helictoglossa is responsible for it. As on substrate, Pinnularia in valve view have a high mobility and cover longer distances, while in girdle band view back and forth movements are carried out. The movement is very similar to that on a solid substrate. Occasionally Pinnularia in valve view show rotations around the pervalvarous axis, which cannot be found on substrate. The adhesion to the substrate will probably prevent such rotations. In practice, this movement is often accompanied by a drift movement. In this context, it should be noted that the collective movement of two Pinnularia also appeared. I suspect the coupling by adhering EPS lumps.

The observations of Pinnularia gentilis, which move actively on the water surface, whereby the driving raphe is completely below the water surface, again support Bertrand’s [1.4] hypothesis of a wavelike movement of microfibrils.