1. As the ephippia (chitinous shells enclosing diapausing eggs) of pelagic crustaceans of the genus Daphnia have been occasionally reported to float at the water surface, we considered that this might be an adaptation promoting their passive dispersal. We investigated the mechanisms by which ephippia appear at the water surface.
2. While field surveys revealed that floating Daphnia ephippia are often numerous in various freshwater habitats, laboratory tests showed that newly-formed ephippia are not buoyant initially. Once transferred to the surface by ephippial female or some other external force, however, they may remain there due either to surface tension or gas absorption.
3. Video recordings showed that all ephippia at the water surface in laboratory vessels were shed there by ephippial females when moulting (despite the attendant risk of exposure to UV radiation). This implies that the moulting behaviour of female Daphnia may determine the fate of their dormant offspring, predetermining whether they remain in the natal environment (when the ephippium is released into the water column) or disperse (when it is deposited at the water surface).
4. Our findings reveal a potential mechanism underlying high dispersal capacity of the freshwater cladocerans inhabiting island-like aquatic habitats.
The production of dormant stages is a widespread adaptation among organisms inhabiting periodically deteriorating environments. The resistance of dormant stages to unfavourable conditions not only allows them to re-colonise the native habitat after conditions have recovered (dispersal in time), but may also facilitate their passive transport to new locations (dispersal in space) (Venable & Lawlor, 1980; Fryer, 1996; Gyllström & Hansson, 2004). For immobile (e.g. plants, sessile animals) or mobile organisms inhabiting isolated sites (e.g. parasites, animals on islands), the passive dispersal of dormant stages may be the only feasible way to colonise new habitats.
This seems also to be the case for pelagic crustaceans of the genus Daphnia, which are ubiquitous inhabitants of lakes and ponds (Hrbacek, 1987; Fernando et al., 1987) and are fast colonisers of new habitats (Louette & De Meester, 2005) despite lacking mechanisms for active dispersal. In their life cycle, they facultatively produce diapausing eggs enclosed in a chitinous shell, called an ephippium, formed from the dorsal part of the carapace and which enhances the resistance of diapausing eggs to unfavourable conditions. Another function of the ephippial shell that is less obvious is that it may facilitate the dispersal of diapausing eggs. It is widely appreciated that Daphnia owes its high dispersal capacity to diapausing eggs, which are passively transported by biotic (e.g. humans, Johnson et al., 2001; waterfowl and other amphibious animals, Figuerola & Green, 2002; Figuerola et al., 2005) or abiotic vectors (e.g. wind as suggested by Brendonck & Riddoch, 1999, or in outflows, Michels et al., 2001). However, additional specific features of ephippial shells (their hydrophobic nature or spiny surface structure), apart from providing mechanical protection, may also aid the passive dispersal of diapausing eggs.
Daphnia shed ephippia into the surrounding water during cyclical moulting. If the ephippium sinks to the bottom, diapausing eggs have to wait for favourable period in their natal habitat. Large numbers of ephippial eggs may be found in sediments (Carvalho & Wolf, 1989), where they may remain viable for years or even decades (Cáceres, 1998) ready to hatch upon the onset of suitable conditions. For reasons that remain unclear, some cladocerans glue ephippia to submerged plants (Brendonck & De Meester, 2003). Ephippia have also occasionally been reported to form vast accumulations of dormant propagules along surf lines at the water surface or on the shore (Fryer, 1996; Wetzel, 2001; Kerfoot et al., 2004). The common occurrence of ephippia at the water surface of freshwater habitats and their potentially significant role for Daphnia dispersal imply that this phenomenon may not be purely accidental but may have adaptive value. While the significant role of floating ephippia in Daphnia dispersal has been considered recently (Pietrzak & Ślusarczyk, 2006; Caceres et al., 2007), the mechanism by which they appear at the water surface has not yet been investigated.
Here we considered two potential mechanisms by which ephippia may appear at the water surface. Potentially, all ephippia might be shed in the water column and sink or float according to their relative densities determined by the content of additives such as gas or lipids. Alternatively, ephippia might be trapped in the surface film (despite their negative buoyancy) if deposited there by their mothers. To test these possibilities we first assessed the buoyancy of freshly deposited ephippia. Second, we investigated whether the females deposited their ephippia at the surface. We discuss the potential functions of the "intentional" deposition of ephippia at the water surface by females.
Densities of ephippial females in the water column and ephippia floating at the water surface were determined in the central zone of 15 lakes and ponds in north-east Poland and in a single lake (Brome) in the south-east of Canada (see Table 1), at times when ephippia were being produced. The samples in Poland were taken in October (2005) and these from lake Brome in June (2007). In each lake, except the two shallowest ponds, three quantitative plankton samples were collected with a funnel shaped plankton net (of 14 cm wide circular opening, 150 m mesh size), that was towed from the bottom to the water surface. In all lakes and ponds three quantitative samples were collected with a neuston net (a modified plankton net of 15 cm wide and 20 cm high rectangular opening, 150 m mesh size) half-submerged during horizontal 100 m long tows. The samples were preserved in the field in 4% formaldehyde solution and analysed in the laboratory thereafter. Densities of ephippial females in the water column and floating ephippia at the water surface were estimated based on these samples.
Indirect test of physical forces that might keep ephippia at the water surface
This test was performed in 125 ml cylindrical glass vials (25 cm high, 2.5 cm wide) filled with Artificial Daphnia Medium (“ADaM”, see Klüttgen et al., 1994). The medium was supplied with the algae Scenedesmus obliquus Turp used as food at a concentration of 0.5 mg C L-1. Four Daphnia species, all originating from water bodies sampled in the field study, were tested separately. Three species originated from a shallow, temporary pond located in Warsaw, Poland (Waw, see Table 1): large bodied D. magna Strauss, medium size D. pulex de Geer and small daphnia from the D. longispina speciescomplex. The fourth species, medium sized D. pulicaria Forbes, originated from the permanent lake Brome in Canada. Daphnia pulex and D. pulicaria are closely related species and belong to the common species complex called D. pulex. Ephippial females found in the field were transferred to the laboratory and randomly assigned to experimental vials (12 females per vial) in four treatments, within 6 hours of their collection. The experiment was conducted under constant illumination (approximate intensity 2.5 µmol m-2 s-1) at 23°C, until all females had shed their ephippium (i.e. within 48 hours).
In the first “control” treatment conditions remained unmodified. In the second "uv" treatment Daphnia were exposed to constant UV radiation (generated by a 40 W Phillips Cleo UV fluorescent bulb, approximate intensity 2.5 µmol m-2 s-1) at the water surface. UV light was applied to discourage experimental females from staying close to the surface and prevent accidental shedding of ephippia there. In the third “net” treatment, the upper part of experimental vials was occluded with nylon mesh positioned 1 cm below the surface, in order to prevent any contact of ephippial females with the surface film. In the fourth “detergent” treatment surface tension of water in the open vials was reduced by adding to each vial 300 μg of non-toxic detergent, cetyl alcohol, commonly used in Daphnia cultures. In this experiment, if ephippia floated due to positive buoyancy, we should have observed them in the upper part of all experimental vials, regardless of the treatment. If they floated as a result of deposition at the water surface by the parent, we should have observed them at the surface in the open vials only (“control” and “UV”). Each treatment had seven replicates in case of D. longispina and D. magna and 11 replicates in case of D. pulex and D. pulicaria. Within 48 hours of their deposition, the buoyancy of ephippia found at the water surface was tested by pushing them below the surface film while ensuring that any attached air bubbles were dislodged.
Non-parametric tests were used for statistical analysis of the experimental results. Mann-Whitney test with Bonferroni correction was used for pairwise comparisons of the parameters tested. Kruskal-Wallis analysis with post-hoc Dunn's Multiple Comparison Test was used when more than two groups were compared simultaneously.
Direct test of the mechanismby whichephippia appear at the water surface
Daphnia of three common species groups: D. longispina, D. pulex and D. magna, all originating from the shallow temporary pond, were tested. The animals were cultured for several weeks prior to the experiment and ephippial production was stimulated by crowding.
This test, aiming at determining the way ephippia appear at the water surface, was conducted at 22oC in 60 cm tall and 7 cm wide aquaria filled with artificial water medium (ADaM) with the addition of food (the green alga Scenedesmus obliquus) at a concentration of 0.3 mg C L-1. Containers were exposed from above to UV radiation of the same source and intensity as in the previous tests. Daphnia were placed individually in 60cm long and 1cm wide glass tubes positioned vertically in the aquaria (four tubes in each aquarium). The top 10-cm water layer was continuously observed with a video recorder and the way ephippia appeared at the water surface was analysed thereafter from the moving pictures. Based on the proportion of the time spent at the water surface by experimental females in relation to the total time of the recording, we tested whether ephippial females deposited ephippia at the water surface accidentally or actively.
Floating ephippia were widespread and common. During our lake survey in autumn 2005 we recorded floating ephippia at various densities and intensities (in relation to density of ephippial females in the water column) in all (16) surveyed lakes and ponds (Table 1). The highest densities of floating ephippia were recorded in two contrastingly different habitats: a temporary Warsaw pond in autumn 2005 (1076 m-2) and in the permanent Lake Brome in early June 2007 (880 m-2), both of which were sources for experimental Daphnia populations. We did not test, however, the differences in densities of floating ephippia between lakes as they were likely to be determined by environmental factors not controlled in this study (e.g. wind, waves and rain action).
Indirect test of physical forces keeping ephippia at the water surface
We found different proportions of ephippia floating in the upper part of experimental vials in different treatments and for different species (Fig. 1). All ephippia were found at the bottom in the ”net” and “detergent” treatments, where access to the surface film was denied or surface tension reduced. In the control treatment, D. longispina, D. pulicaria and D. pulex left more ephippia at the water surface than D. magna (post-hoc Dunn's Multiple Comparison Tests, p<0.05, after a significant difference between species was found using a Kruskal-Wallis test, F3,36=18, p<0.0005). The first three species, however, did not differ between each other in this parameter. Exposure to UV significantly reduced the proportion of ephippia deposited at the surface compared to the control treatment in three of four tested species: D. pulex (U1,11=6, p<0.0001), D. pulicaria (U1,11=20.5, p<0.01) and D. magna (U1,7=3.5, p<0.005) (pairwise comparisons in Mann-Whitney tests with Bonferroni correction).
Most of the ephippia that were found at the water surface in the four species tested were negatively buoyant and sunk when pushed below the water surface (Fig.1). They were clearly kept at the water surface by surface tension forces. Some of the floating ephippia did not sink even when pushed below the water surface and remained at the water surface thanks to gas bubbles lodged either inside the double wall of the ephippial “shell”, or between the two parts of the shell. The highest proportion of positively buoyant ephippia were recorded in D. longispina (over 20%), and no positively buoyant ephippia were found in D. magna.
Direct test of the mechanismof ephippia appearance at the water surface
Video recordings of ephippial females exposed to the surface threat of UV radiation revealed that all ephippia found floating had been actively deposited at the water surface by the mother while moulting. Some of the ephippial females (different proportions in different species) approached the surface, broke the surface film, shed the ephippium at the water surface within a few tens of seconds and moved down promptly thereafter (Fig. 2). The highest proportion of experimental females depositing ephippia at the water surface was observed, as in previous tests, in D. longispina (31 out of 32 individuals). Fewer females of D. pulex left ephippia at the surface (39 out of 73 individuals, χ2=18.9, df=1, p<0.0001), while none of D. magna (out of 82 individuals) approached the surface nor left ephippia there during this trial. Females that left ephippia on the bottom (117 of all 187 tested animals) either did not approach the surface while moulting (98%) or did not manage to leave the ephippium there (2%).
The brief appearance of ephippial females at the water surface compared to total observation time (with the relative time ratio 1:243, in D. longispina and 1:1100 in D. pulex) indicates active deposition of ephippia at the surface film in two of three species of Daphnia tested.
Although reports of the success with passive dispersal and establishment in a new favourable location seem relatively limited (De Meester et al., 2002; Bohonak & Jenkins, 2003), many aquatic organisms have propagules apparently adapted for dispersal (Bilton et al., 2001). Ephippial eggs can resist unfavourable conditions for long periods, making them ideal for passive dispersal between aquatic “islands” scattered across an inhospitable terrestrial landscape (Fryer, 1996; Gyllström & Hansson, 2004). Such passive dispersal of dormant stages may potentially be promoted in various ways. However, since most vectors of overland dispersal operate at the surface of freshwaters, deposition of propagules at the surface might enhance their chances of travelling to new locations (Pietrzak & Ślusarczyk, 2006). Indeed, Davison (1969) was the first to report the release of ephippia by pelagic Daphnia at the water surface, though this anecdotal observation has since been overlooked, the potential adaptive value going unrecognised for all this time. Our research is now able to confirm Davison’s (1969) observations, and to challenge the view that the appearance of ephippia at the water surface is a mere accident.
Our field survey showed the widespread occurrence of floating ephippia at times when they were being produced, even if it could not identify mechanism by which they appeared at the water surface. While the highest density of floating ephippia was observed in a shallow pond, it is not known if this reflects a higher intensity of ephippia deposition at the water surface or simply better protection from the wind, which would carry ephippia toward the shore in more exposed lakes.
Equally, our laboratory tests refuted the possibility raised in other studies (Caceres at al., 2007) that Daphnia ephippia are positively buoyant. Had that been the case, floating ephippia would have been found in all treatments in the first experiment (Fig.1). Rather, we found floating ephippia only in those experimental vials offering females unrestricted access to the surface. This implies that ephippia were not initially buoyant, yet found their way to the surface through maternal behaviour or some other mechanism. Video recordings confirmed these speculations, demonstrating that a proportion of the ephippial females (varying from species to species) release ephippia directly at the water surface while moulting. Moreover, our data show that this is not accidental. Daphnia seems to exploit the surface tension to keep non-buoyant ephippia at the air-water interface. The hydrophobic structure of the ephippial shell may facilitate its binding to the water surface. Under natural conditions, the persistence of ephippia in the surface film is unlikely to be assured by these weak forces easily counteracted by external agents like wind or rain. In addition, natural substances accumulating in the surface film may reduce surface tension significantly (Goldacre, 1949) and affect the proportions of floating ephippia like the detergent in our study did (Fig. 1).
Although freshly–released ephippia are apparently non-buoyant, some may become buoyant having come into contact with the water surface. Our trials revealed the presence of gas bubbles, either between shells or within the double wall of the buoyant ephippia. Since bubbles in ephippia were only reported from vials offering experimental females unrestricted access to the air-water interface, they probably comprised atmospheric air that shells captured after contact with the surface. Air absorption by ephippial shells might be of adaptive value , if it kept ephippia at the water surface and furthered dispersal.
The release of ephippia at the water surface puts both parental females and ephippial eggs at increased risk, for instance of enhanced exposure to UV radiation or predation by waterfowl (Gardarsson & Einarsson, 2002) or fish (Mellors, 1975). Moreover, we observed females becoming trapped by surface tension at the water surface as they released ephippia. While active individuals are indeed vulnerable at the surface, ephippial eggs are found to be highly resistant to many of the risks associated with the surface. The ephippial eggs of Daphnia are believed to tolerate high levels of UV radiation, drying and freezing, and they may even survive consumption by predators if not mechanically damaged (Mellors, 1975). The fact that Daphnia mothers are willing to run the risk of depositing of ephippia at the water surface suggests that the phenomenon has an adaptive function. Furthermore, the risks may be diminished in nature if the females shed ephippia at night (what is indicated by our preliminary unpublished data).
The smallest species, D. longispina, deposited the greatest proportion of ephippia at the surface among the four species studied. Moreover, this was the only species that left ephippia at the surface regardless of whether it was or was not being exposed to the surface threat of UV radiation (Fig. 1). Finally, D. longispina left the highest proportion of positively buoyant ephippia. The reasons for these species differences are unclear.
While ephippia deposited at the water surface may be the most likely to disperse, it is not clear whether dispersal operates mainly within or between aquatic habitats. There could be adaptive value in either case. Though risky, dispersal between environments may avoid unfavourable conditions, facilitates the colonisation of vacant habitats (Levin et al., 1984) and reduces kin competition (Hamilton & May, 1977). On the other hand, deposition of ephippia at the water surface may facilitate passive transport by currents to the shallows of the natal habitat. This may help avoid the sedimentation of ephippia in the profundal of deep lakes, where reliable hatching cues like light or temperature changes may be absent (Cáceres, 1998). Moreover, in temporary ponds the deposition of ephippia in the shallows might promote hatching only after the pond is completely refilled, thus assuring persistence of the aquatic habitat, the mechanism known in fairy shrimps inhabiting ephemeral pools (Hildrew, 1985). The ultimate reasons for the phenomenon will only be determined by further investigation, however.
The study was supported by grant from Polish Ministry of Science and Higher Education (2P 04F 069 27). We thank Tomasz Grabowski for help in field samples collection and analysis and Joanna Pijanowska and Piotr Dawidowicz for comments on the manuscript.
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Table 1. Mean density of floating ephippia and ephippial females in the water column at the centre of lakes and ponds in Poland in autumn 2005. The only Canadian lake (Brome) was sampled in late spring 2007. L - D. longispina, P - D. pulex, M - D. magna. na – data not available
Density of floating ephippia [m-2]
Density of ephippial females in the water column [m-2]
M, P, L
Fig 1. Mean ratio of floating ephippia vs. ephippia recorded at the bottom (standard error indicated by black error bars) in four Daphnia species, in four types of experimental vials: "control" - open at the top; "UV" - open at the top and exposed to UV radiation; "net" - with net installed below the water surface; and "detergent" - open at the top, with detergent added. White error bars indicate standard error for proportion of positively buoyant ephippia among the ephippia found at the surface. Horizontal lines above the bars indicate that treatments in each species tested for the proportion of floating ephippia were not significantly different (post-hoc Dunn's Multiple Comparison Tests, p<0.05).
Fig. 2. Timing of appearance at the surface of experimental Daphnia before/after shedding ephippium (indicated by 0 on the X axis) when exposed to UV radiation.
ephippia found at the bottom of the vial
D. pulex ig. 1.
ephippia found in the upper part of the vial; positively buoyant
ephippia found in the upper part of the vial; negatively buoyant