Heterotrophy on ultraplankton communities is an important source of nitrogen for a spongerhodophyte symbiosis
1 School of Biological Sciences, Flinders University, Adelaide, South
Australia 5001, Australia
2 School of Biological Sciences (A08), University of Sydney, New South Wales
2006, Australia
3 School of Biological Sciences and Biotechnology, Murdoch University,
Murdoch, Western Australia 6150, Australia
* Author for correspondence (e-mail: apile{at}bio.usyd.edu.au)
Accepted 1 September 2003
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Summary |
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Key words: coral reef, heterotrophy, rhodophyte, sponge, symbiosis, ultraplankton, nitrogen
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Introduction |
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After carbon, nitrogen is the next most important nutritional requirement
for both autotrophs and heterotrophs, as it is essential for the synthesis of
amino acids. Marine autotrophs take up dissolved inorganic nitrogen (DIN) in
the forms of ammonium, nitrate or nitrite, whereas heterotrophs obtain their
nitrogen as particulate organic nitrogen (PON), as part of their diet, and in
some cases may also use dissolved organic nitrogen (DON). Cnidarian/algal
associations are amongst the most frequently studied symbiotic associations.
Cnidarian hosts are well-documented carnivores and/or omnivorous suspension
feeders, whose feeding supplies the nitrogen to maintain both partners of the
association (Cook et al., 1992;
Wang and Douglas, 1998
).
However, not all invertebrate hosts have the capacity to feed. The
vestimentiferan worms common to hydrothermal vents and cold seeps obtain
nitrogen by taking up DIN from the overlying water column
(Fisher, 1990
;
Childress and Fisher, 1992
).
Other cold seep and hydrothermal vent organisms such as bivalves can meet
their metabolic carbon demand through their chemoautotrophic symbionts while
the invertebrate host supplies a portion of the nitrogen by grazing on
ultraplankton (Pile and Young,
1999
).
Symbioses between sponges and algae are common on coral reefs. We have been
examining the nutritional relationship the symbiotic association between the
sponge Haliclona cymaeformis (Esper, 1794) (Demospongiae,
Haplosclerida; formerly Sigmadocia symbiotica) and the red macroalga
Ceratodictyon spongiosum (Zanardini, 1878) (Rhodymeniales,
Rhodophyta). For the HaliclonaCeratodictyon association most
or all of the carbon required by the alga is derived from photosynthesis, but
very little photosynthate is translocated to the sponge (less than 1.3% of
total photosynthetically fixed carbon; A. Grant, unpublished data). However,
nitrogen stable isotope values of +4.88±0.28 and
+2.33±0.18
for the sponge and alga, respectively, indicate that
nitrogen for both is most likely to be derived from heterotrophic sources
(Davy et al., 2002
). As sponges
are known to graze primarily on ultraplankton
(Pile et al., 1996
;
Pile, 1997
), it is the most
likely source of nitrogen to the association and carbon to the sponge.
Ultimately, the HaliclonaCeratodictyon association would need
to consume 0.275 mg N day-1 g-1 in order to maintain its
nitrogen balance/content (Davy et al.,
2002
). In the light of these findings we conducted a series of
feeding experiments to determine the natural diet of the sponge and its
feeding ecology, in order to ascertain if it is possible for the sponge to
consume enough PON to sustain the relationship. Since it is highly likely that
both food availability and water processing by the sponge would vary with
season and time of day, we used flow cytometry to measure feeding rates in
summer and winter, with time of day (day vs night) nested within
season.
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Materials and methods |
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Water samples for analysis of ultraplankton by flow cytometry were
collected with a Gilson (Middleton, WI, USA) pipettor from beakers that
contained either the association or seawater only (N=3). Triplicate 1
ml samples were collected at different points within the beaker at the
beginning of each trial and after 15 min. Water samples were preserved for
flow cytometry following standard protocols
(Pile et al., 1996),
transported to Flinders University on dry ice, and maintained at -80°C
until analysis. An additional 10 ml of water from each beaker was filtered
onto a 0.02 µm black polycarbonate filter and frozen for subsequent visual
confirmation of ultraplankton populations.
Treatment beakers contained one sponge branch, which was returned to the
lagoon at the end of the trial. At the conclusion of each experiment, each
sponge was blotted dry with a paper towel and weighed to the nearest 0.01 g.
The sponges were then returned to the lagoon. The relationship between wet
mass and dry mass was quantified by linear regression analysis from paired
samples (Sokal and Rohlf,
1981). Fresh sponge branches (N=58) were collected and
their blotted wet mass determined. These branches were then dried at 60°C
and reweighed. The flux rate of association was then normalized to the
association dry mass to meet scientific conventions for the reporting of
fluxes.
Ultraplankton populations were quantified using a FACscan (Becton Dickson,
San Jose, California, USA) at Flinders University, South Australia following
the techniques of Marie et al.
(1997). Orange fluorescence
(from phycoerythrin), red fluorescence (from chlorophyll) and green
fluorescence (from DNA stained with SYBR Green) were collected through
band-pass interference filters at 650, 585 and 530 nm, respectively. The five
measured parameters, forward- and right-angle light scatter (FALS and RALS),
and orange, red and green fluorescence were recorded on 3-decade logarithmic
scales, sorted in list mode, and analysed with custom-designed software
(Vaulot, 1989
). Ultraplankton
populations were identified as general cell types of bacteria (Bac),
Prochlorococcus sp. (Pro), Synechococcus-type cyanobacteria
(Syn), and protozoans (Proto). Cell types were visually confirmed, and mean
cell diameter measured (N=50) using epifluorescence microscopy.
Depletion rates of ultraplankton were calculated assuming an exponential
growth and clearance of prey following the methods of Ribes et al.
(1998). In summary, prey
growth rate k (h-1) is computed as:
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A conservative estimate of daily carbon (C) and nitrogen (N) availability
was calculated empirically by converting the ingestion rate (I) to an
equivalent in g C and N. Computations assumed a 12 h:12 h light:dark cycle in
winter and a 14 h:10 h light:dark cycle in summer. For heterotrophic bacteria
we employed cell conversion factors of 20 fg C per cell with a C:N ratio of
3.5 (Wheeler and Kirchman,
1986). Available C and N from phytoplankton and protozoans was
determined as a function of biovolume, using epifluorescence microscopy
(Ribes et al., 1998
, and
references therein). We are aware of the limitations of such calculations and
sufficient data are presented so that if better cell-to-carbon and
cell-to-nitrogen conversions become available, fluxes can be recalculated.
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Results |
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Heterotrophic bacteria and two autotrophic prokaryotes, Prochlorococcus sp. and Synechococcus-type cyanobacteria, comprised over 90% the ultraplankton community in One Tree Lagoon regardless of season (Table 1). The only abundant eukaryotic organisms were a variety of heterotrophic ciliates that are grouped here as protozoans. No autotrophic eukaryotes were found during any of the sample periods. The C:N ratio of the ultraplankton community of 3.14 reflects the dominance of heterotrophic organisms found within the lagoon of One Tree Island (Table 1).
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Ultraplankton communities varied slightly with both the time of day and season. During the summer sampling period we recorded more than an order of magnitude increase in Synechococcus sp. at night. All other types of ultraplankton have variations of less than 20% both within and between seasons (Table 1).
The presence of the HaliclonaCeratodictyon association significantly reduced the growth rates of heterotrophic bacteria and protozoans. The net effect, combined day and night growth rates, of the HaliclonaCeratodictyon association on the growth rate of heterotrophic bacteria showed no difference by season. However, during winter the HaliclonaCeratodictyon association had a greater negative effect on growth during the day, while in summer there was a negative effect only at night (Table 2, Fig. 2). For protozoans, there was a seasonal difference, with more negative growth rates during the winter than summer and the same diel pattern as seen in heterotrophic bacteria was found (Table 2, Fig. 2). The HaliclonaCeratodictyon association did not appear to have a statistically significant effect on Prochlorococcus sp. and Synechococcus-type cyanobacteria (Table 2, Fig. 2).
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The HaliclonaCeratodictyon association removed more particulate material from the water column community in winter than summer (Table 3). Both the carbon and nitrogen fluxes were an order of magnitude higher in the winter than the summer. During the winter most of the carbon and nitrogen flux occurred during the night. During the summer there was only a slight difference between the day- and night-time fluxes (Table 3).
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Discussion |
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As in other Pacific coral reef water column communities, there was no
discernible seasonal variation in ultraplankton community structure
(Furnas and Mitchell, 1987;
Charpy, 1996
).
Synechococcus was the only genus to show a diel variation, with
greater abundance at night than during the day
(Table 1). There could be two
sources for this increase. First, the doubling of Synechococcus
cells, which occurs synchronously at night in natural populations
(Campbell and Carpenter, 1986
;
Jacquet et al., 1998
), will
result in new cells. This characteristic of ultraplankton communities means
that for a certain window of time, there is an increase in the amount of both
C and N available for capture by suspension feeders. Second, there may be a
diel migration of benthic cyanobacteria into the water column, which will
significantly increase the number of cells. It remains to be seen if
invertebrate grazers can take advantage pulsed increases in food availability
by changing their grazing activities.
The HaliclonaCeratodictyon association is capable of
retaining heterotrophic bacteria and protozoans but is apparently unable to
retain Prochlorococcus and Synechococcus
(Fig. 2). While this may
suggest that H. cymiformis grazes selectively on heterotrophic
organisms, this is highly unlikely as sponges have no known mechanism for
particle selection. Rather, it is more likely that the low abundance of
Prochlorococcus and Synechococcus in One Tree Island Lagoon
as compared to other coral reefs (Ayukai,
1995; Charpy, 1996
;
Charpy and Blanchot, 1998
)
prevents their capture by the choanocytes as seen in Caribbean sponges
(Pile, 1999
). Regardless of
the mechanism, the natural diet of H. cymiformis is nitrogen-rich
with a C:N ratio ranging from 3.3 to 4
(Table 1).
Surprisingly the HaliclonaCeratodictyon association removed
nearly an order of magnitude more PON and POC from the water column in winter
than summer (Table 3). As there
was no discernible difference between the ultraplankton communities, the
greater flux is probably due to an increase in the amount of water processed
by the sponge. It has been demonstrated in a temperate sponge that water
processing and temperature are positively correlated
(Riisgård et al., 1993).
H. cymiformis, however, appears to be increasing water processing
rates as the temperature decreases from summer to winter. This result, while
unexpected, may be linked to the unique association between the sponge and
algal partners. It may be that, in summer, higher rates of photosynthesis
(Trautman, 1996
) supply more
oxygen and organic carbon to the sponge partner than in winter, allowing it to
process less water and still meet its metabolic needs.
Grazing on ultraplankton appears to be an excellent source of carbon and
nitrogen to the sponge partner in the association. Unlike most symbioses with
either photo- or chemotrophic partners
(Muscatine et al., 1984;
Fisher, 1990
;
Childress and Fisher, 1992
), in
this association there appears to be little transfer of organic carbon from
the autotrophic partner to the heterotrophic partner (A. Grant, unpublished
data). Grazing by the heterotrophic partner is required as we have no evidence
of significant nutritional support to it from the algal partner. This lack of
nutrient transfer may be a result of the extracellular nature of the
association; in associations in which significant transfer of nutrients has
been reported from algal symbionts, the algae have been intracellular,
facilitating the exchange of nutrients
(Trautman and Hinde,
2001
).
We conclude that nitrogen obtained from grazing on ultraplankton supports
the nitrogen metabolism of both partners in this spongealgal
association. The sponge can retain enough PON to meet the projected nitrogen
budget of the association. The sponge partner would need to take up 0.275 mg N
g-1 dry mass day-1 to meet the nutritional needs of both
partners (Davy et al., 2002).
Thus, uptake in summer is sufficient to maintain the association, while in
winter the fluxes are an order of magnitude greater than required to supply
the association's nitrogen requirements for maintenance. The surplus of
nitrogen obtained during the winter could only be allocated to growth if the
association's other metabolic requirements were met through the overall
greater uptake of particulate material.
Research on the nutritional interactions in many invertebrate/autotrophic
symbiotic associations has focused on the flux of carbon between the partners.
However, recent advances in understanding the feeding ecology of invertebrate
partners other than the Cnidarians are revealing that grazing on ultraplankton
is an important nutritional source of nitrogen for these associations
(Pile and Young, 1999).
Ultraplankton is the most common food source in the world's oceans
(Stockner and Antia, 1986
;
Stockner, 1988
;
Sherr and Sherr, 1991
) and its
role in structuring benthic communities is only beginning to be elucidated
(Gili and Coma, 1998
). The
role that grazing on ultraplankton plays in maintaining complex biological
communities where symbiotic associations are prevalent, such as coral reefs
and hydrothermal vents, remains to be explored.
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Acknowledgments |
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