Feeding on ultraplankton and dissolved organic carbon in coral
reefs: from the individual grazer to the community
By
Gitai Yahel
A thesis submitted in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
Submitted to the senate of the Hebrew
University of Jerusalem
2003
Feeding on Ultraplankton and Dissolved Organic Carbon in
Coral Reefs: from the Individual Grazer to the Community
By
Gitai Yahel
A thesis submitted in partial fulfillment of the requirements for the
degree of
Doctor of Philosophy
Submitted to the senate of the Hebrew University of Jerusalem
2003
This work was carried under the supervision of
Doctor Amatzia Genin
Department of Ecology, Systematics and Evolution
The Hebrew University of Jerusalem
Interuniversity Institute for Marine Science of Eilat
I
ACKNOWLEDGMENTS
I am grateful to my family, friends, and colleagues for their support and friendship
throughout the Ph.D. study.
Special thanks to my supervisor Dr. Amatzia Genin for his professional guidance,
enthusiasm, support, and friendship.
To my partner Ruthy and to the Weil, Perry, Reshef, Marom, Rappaport, and
Chukran families for the endless emotional, logistic, and financial support.
To R. Yahel, I. Ayalon, M. Ohevia, R. Wyeth & C. Holmes, B. Munkes, R. Motro,
S. Eckstein, E. Gotlibe, A. Brandes & G. Shoresh, Y. Shif, E&G Blumberg, D.
Weil, D. Chernov, K.B. Savidge, R. Andrews, and K. Rinker for there dedicated
help in the field and laboratory.
To the IUI staff for logistic support.
To B. Lazar, I. Brickner, M. Ilan, D. Lindell, A. Post, M. Kiflawi, E. Hadas, T.
Katz, R.P.M. Bak, D. Vaulot, T. Fenchel, K. L. Smith, Jr., L. Haury, G. C.Stephens,
and P. Jumars for useful comments and discussions.
Numerous people assisted to accomplish the studies depicted below. They are
acknowledged at the beginning of each chapter.
II
ABSTRACT
Benthic grazing on phytoplankton is a principal trophic pathway in shallow, temperate coastal
habitats. Traditionally, studies of benthic-pelagic coupling in coral reefs, where the planktonic
community is dominated by minute prokaryotic cells, have focused on zooplankton, rather than
the smaller phytoplankton and bacteria, as the principal source of prey. My goal was to study the
trophic role of ultra-phytoplankton (<0.01 mm), bacteria, and dissolved organic matter in the
coral reef ecosystem on scales ranging from the individual grazer to the whole community.
Mapping the spatial distribution of phytoplankton near coral reefs (Limnol. Oceanogr., 43,
551) revealed the occurrence of a 1-3 m layer depleted of phytoplankton. The depleted layer was
found over several coral reefs but not over sandy sites with similar bathymetry. The extent of the
depletion was dependent upon the hydrodynamic conditions. Using state-of-the-art underwater
technology, we were able to apply the "Control Volume" approach to measure in situ
phytoplankton grazing on a scale of a whole coral-reef community (Oceanography, 15, 90). The
results indicate that the import of carbon and nutrients via phytoplankton grazing is a major,
previously underestimated, trophic pathway in coral reefs.
By developing new methods for in situ measurements of feeding-rates (Limnol. Oceanogr.
Methods, 3, 46) I was able to measure the diet composition, prey preference patterns, and grazing
rates of 15 benthic suspension feeder taxa at the reef. Soft corals (Limnol. Oceanogr., 43, 354),
sponges (Limnol. Oceanogr., 48, 141), boring bivalves, and ascidians (Mar. Ecol. Progr. Ser.,
submitted) were found to be efficient phytoplankton grazers. Only sponges were efficient
bacteriovores. The yet un-described cryptic fauna inhabiting the outer surfaces of the so-called
"bare rocks" was found to be an important sink for phytoplankton (Coral Reefs, in press). The
preference for ultraplanktonic prey was similar for different grazers, exhibiting a general
selectivity for coccoid photosynthetic prokaryotes over both larger or similar-size eukaryotic
algae and smaller or similar- size non-photosynthetic bacteria. As this pattern does not maximize
carbon or energy gain, it is suggested that at the reef, where carbon is not a rare commodity,
suspension feeders have evolved to optimize the gain of other nutrients or to avoid harmful prey
taxa.
My new in situ technique allowed, for the first time, direct quantification of dissolved organic
matter removal by several active suspension feeders. The symbiont-bearing reef sponge Theonella
swinhoei removed up to 26% (12±8%, mean±1SD) of the total organic carbon (dissolved and
particulate) from the water it filtered during a single passage through its filtration system (Limnol.
Oceanogr., 48, 141). The amount of carbon gained by the sponge from the dissolved pool was an
order of magnitude greater than that gained from the total living cells it removed. Evidence for
removal of dissolved organic matter (DOM) was also recorded for a coral boring bivalve, a
solitary ascidian, and two other sponges. My findings suggest that DOM may be a major
component in the nutrition of metazoan and that the role of metazoans in DOM cycling may have
been grossly underestimated.
This study revealed the previously overlooked fundamental role of minute phytoplankton and
dissolved organic matter in the nutrition of benthic suspension feeders and benthic-pelagic
coupling in the complex coral reef community.
III
Table of Content
1.
Synopsis ................................................................................5
2.
Scientific Background ................................................................8
Fluxes of carbon, nutrients and phytoplankton in coral reefs ................8
Hydrodynamic control of mass transport.........................................9
Individual based grazing rates ................................................... 10
The role of DOC .................................................................... 12
Research goals ...................................................................... 13
3.
Phytoplankton distribution and grazing near coral reefs .................... 15
4.
In situ feeding and element removal in the coral-reef sponge
Theonella swinhoei: Bulk DOC is the major source for carbon ............. 28
5.
Selective filtration of ultraplankton by three tropical suspension feeders: a
sponge, an ascidian, and a bivalve .............................................. 38
6.
Phytoplankton grazing by epi- and in-fauna inhabiting exposed
rocks in coral reefs ................................................................. 69
7.
Discussion and conclusion ......................................................... 94
8.
General reference list For Introduction and Discussion sections ........... 98
9.
Appendix I In situ depletion of phytoplankton by an
azooxanthellate soft coral .......................................................107
10. Appendix II Intense benthic grazing on phytoplankton
in coral reefs revealed using the control volume approach ................111
11. Appendix III InEx” – an in situ method to measure rates
of element intake and excretion by active suspension feeders............119
.12 ‫ תוכן עניינים עברי‬.................................................................134
IV
Chap. 1. Synopsis
1.
Synopsis
This work was initiated about 8 years ago with the discovery that soft, asymbiotic corals
are apparently herbivores, ingesting and digesting minute, unicellular algae (phytoplankton) from
the flowing water (Fabricius, Benayahu, and Genin, Science, 268, 90, 1995). This finding was
rather surprising as corals were widely believed to be carnivores and phytoplankton grazing was
not considered significant in coral reef ecosystems. That discovery had triggered interest in the
ecological significance of phytoplankton as a nutritious source for soft corals and more generally
to the reef ecosystem. During the spring of 1994, when I was doing my bachelor studies in
biology, I approached Dr. Genin, seeking a subject for my BSc project. Within two days I found
myself in Eilat, diving deep (literally) into the beauty of the reef and the complexities involved
with underwater research. Soon after, I discovered that at the reef, no trivial answers were available
to even the simplest question, such as "what does that animal eat?" During my BSc project, we
discovered that the soft, asymbiotic coral Dendronephthya is able to remove significant amounts
of phytoplankton from the water passing through its branching colonies. The combination of this
field data with laboratory measurements of the rate of chlorophyll destruction within polyp's
lumen had allowed us to reevaluate Dendronephthya's feeding rates (Appendix I, Fabricius, Yahel,
and Genin, Limnology and Oceanography, 43, 354, 1998) which were previously grossly
underestimated.
Water samples collected in transects form the reef to the open sea indicated remarkable
phytoplankton depletion close to the reef bottom but not over a nearby sandy bottom site.
However, a significant unexplained variability existed between different sampling sessions. To
improve the accuracy of our phytoplankton measurements, and in order to gain more knowledge
of the planktonic community composition, we established a fruitful collaboration with Dr. D.
Marie and Prof. D. Vaulot from the CNRS institute in Rosscof, France, one of the worlds leading
groups in marine flow cytometry. After two more years, and much field work in Eilat and along
the western coast of the Sinai peninsula, we realize that it is mostly hydrodynamics (and not, for
example, shifts in grazers behavior or phytoplankton community) that controls the thickness of
the depleted layer over the reef (3, Yahel, Post, Fabricius, Marie, Vaulot, and Genin, Limnology
and Oceanography, 43, 551, 1998). By tracking water parcels flowing through the artificial
"perforated reef", which grows over old barbwires underneath the jetty of Eilat Oil Terminal, we
were able to estimate phytoplankton fluxes into that system as the product of water velocity and
phytoplankton depletion. Our estimates indicated that phytoplankton feeding can account for the
entire respiratory carbon demand of Dendronephthya and that phytoplanktonic carbon fluxes into the
"perforated reef" were comparable to the highest records of zooplankton fluxes into coral reefs.
Evaluating the role of phytoplankton as a nutritional source for the typical fringing reefs
of the Gulf of Aqaba, where soft, asymbiotic corals are sparse, was a much more complex task.
While phytoplankton depletion at the reef bottom was evident, the shear and turbulence created
by the rough micro-topography hindered simple rate measurements. To resolve this difficulty we
have decided to follow two complimentary approaches. One approach was holistic, i.e., we
wanted to establish a community scale methodology that will allow reliable quantification of
benthic-pelagic mass flux exchanges in the multifaceted environment of exposed reef slopes.
Soon, our preliminary flow measurements revealed, that on a meso-scale, the flow field over the
reef is even more complex, containing features such as vertical and cross-shore shears. We then
realized we needed the help of hydrodynamics engineers to properly design the community scale
methodology. This led to the establishment of a successful collaboration with Prof. J R. Kossef,
5
Chap. 1. Synopsis
and Prof. S. G. Monismith form the Dept. of Civil and Environmental Engineering, Stanford
University. A joint grant from the BSF allowed us to develop and deploy the "Control Volume"
experiments (Appendix II, Genin, Yahel, Reidenbach, Monismith, and Koseff. Oceanography 15,
90, 2002). Using a suit of underwater pump arrays, acoustic current meters, and current profilers,
we were able to achieve simultaneous, high resolution, measurements of both the flow and
phytoplankton concentration fields over some deep open sections of the reef slope community.
The entire setup was replicated twice in the summer 1999 and once again in the winter of 2001.
Our preliminary results from the 1999 experiments indicate that the overall grazing rate at the reef
was substantial, exceeding previous estimates by an order of magnitude. Import of carbon and
nutrients via this grazing is thus a major, previously underestimated, trophic pathway in coral
reefs. Independent estimates based on Lagrangian studies, as well as mesocosms experiments
yielded similar clearance estimates (Yahel, unpublished data). Buoyancy driven cross-shore flows
seems to be an important factors, replenishing the reef with fresh oceanic waters. Data analysis is
still in progress as over 2,000 flow cytometry samples and 3,000 chlorophyll samples were
collected in these experiments along with a high-resolution data set of physical data. At least three
more publications are currently under preparation by the participants of this study.
As a complimentary method, we used a "scaling up" approach, in order to extrapolate
phytoplankton-grazing rates of individual grazers to the reef scale. Surprisingly, a comprehensive
literature search has found no explicit report of a phytoplanktivorous reef dweller, let alone
reports on phytoplankton grazing rates. Moreover, the application of standard method used to
measure phytoplankton-grazing rates in temperate and boreal habitats (namely, putting the
animals in a laboratory vessel and measuring plankton depletion in the vessel) was clearly
inappropriate in the reef environments where many suspension feeders are cryptic and all have
tight symbiotic relationships. What we needed was a nondestructive method that would allow us
to survey, in situ, the feeding rates of different suspension feeders. The reference unit of these
feeding rates should have been also readily quantified at the reef (obviously, traditional biomass
reference unit are inappropriate for [e.g.] endolithic taxa). To that end I developed end evaluated a
technique termed “InEx”. InEx is based on the simultaneous, pair-wise collection and comparison
of the water Inhaled and Exhaled by the animal. Calculations of feeding (or excretion) rates are
obtained by multiplying the concentration difference by pumping rate. The latter is concurrently
measured by recording the movement of a dye front in a transparent tube positioned within the
ex-current jet (Appendix III, Yahel, Marie, and Genin, Limnol. Oceanogr. Methods, submitted).
Using InEx we collected over 500 separate measurements of filtration efficiency, pumping
rates, diet composition, and selectivity patterns in four sponge species, three tunicate species and
five bivalve species, some in the Gulf of Aqaba, and others, in the Indian Ocean. All species
studied were found to be phytoplanktivores with varying degree of efficiency and with a distinct
preferences pattern for each grazer taxa. In addition to phytoplankton, and non-photosynthetic
bacteria removals, measured with a flow cytometer, we have also analyzed inorganic nutrients,
respiratory gasses, and total and dissolved organic carbon in many of the InEx pairs. An
important, unexpected finding was the discovery of a previously undocumented pathway of
carbon flux in benthic habitats. DOC (dissolved organic carbon) appeared to be a major
component in the diet of several active suspension feeders at the reef belonging to three remote
phyla (Sponges, Tunicates, Mollusks). While we first documented this phenomena back in 1997
(using an un-calibrated Dohrmann TOC analyzer), it required the dedicated help of the world
leading authority in DOC analysis, Prof. J.H. Sharp of the Graduates College for Marine Sciences
in Delaware, and almost three years of painstaking work (with many excluded samples) to refine
the methodology and get a reliable, no equivocal, quantitative measurements of this DOC removal
(Chapter 4, Yahel, Sharp, Marie, Häse, and Genin, Limnol. Oceanogr. 48, 150, 2003). Another
6
Chap. 1. Synopsis
interesting and novel phenomenon revealed with InEx is the discovery that reef suspension
feeders (bivalves and tunicates) are highly selective in the planktonic bacteria they feed on. These
suspension feeders readily retain photosynthetic bacteria as well as non-photosynthetic bacteria
with high nucleic acid content but apparently reject other type of bacteria. This selectivity is
apparently size independent and thus should be based on a, yet unknown, cell recognition
mechanisms. Sponges in contrast are less selective but more efficient filter feeders. An in-depth
analysis of these phenomena for three representative species is presented in 5Chapter 5 (Yahel,
Marie, Eckstein, and Genin, Mar. Ecol. Progr. Ser., Submited).
Finally, phytoplankton-grazing estimates based on the individual based rate measurements
(InEx) and the actual abundance of active suspension feeders within the control volume area
yielded pumping rates estimates of <15% of the estimated total community grazing rate (Yahel et
al. in prep.). It was thus evident that the active suspension-feeders guild cannot be the sole, and
probably not even the major, sink for phytoplankton at the reef. Hydraulic phytoplankton
filtration through sand (Huettel and Rusch 2000) or reef framework (Haberstroh and Sansone
1999), as well as the activity of coelobite fauna, were all ruled out for Eilat reef slops. However,
laboratory experiments clearly demonstrate that the, yet un-described, cryptic fauna of reef rock
surfaces is a major sink for phytoplankton at coral reefs (Chapter 6, Yahel G., Zalogin T., Yahel
R. and Genin A., Coral Reefs, Submitted).
The studies presented here span over a six-year period. During that time, a few other
groups have published follow-up papers (e.g., Fabricius and Dommisse 2000; Richter et al. 2001)
or independent studies (e.g., Ribes et al. 1999; Roditi et al. 2000; van Duyl and Gast 2001)
related to aspects of phytoplankton grazing and DOC dynamics. In order to avoid confusion and
to maintain a clear flow of the scientific background and summary, the Introduction will review
the scientific background relevant to the time of the onset of the study and research-plan
formulation (1997). References for newer publications (after 1997) will be generally reserved for
the relevant chapters and to the concluding section, unless they pertain to recent reviews that
summarized older studies.
7
Chap. 2. Scientific background
2.
Scientific Background
Fluxes of carbon, nutrients and phytoplankton in coral reefs
An assessment of biogenic import and recycling of nutritious elements is essential for
understanding trophic dynamics in ecological communities. In coral reefs, as in many other open
systems, heterotrophic animals utilize carbon from two sources (reviewed by Hatcher 1997):
allochthonous, that is, carbon they import into the ecosystem from outside (e.g., benthic predation
on pelagic zooplankton), and autochthonous, the consumption of carbon originated from members
of the local community (e.g., fish feeding on coral polyps or snails grazing on benthic algae). As
coral reefs typically flourish in clear oligotrophic waters, a fundamental question that motivated
ample research in this field is how the reef can be so much more productive than the surrounding
waters. In coral reefs, high primary production by zooxanthellate cnidarians and algae, together
with an occurrence of high abundance and most diverse assemblies of closely interacting species,
has led earlier researchers to suggest a tight recycling of autochthonous carbon and nutrients
(Odum and Odum 1955; reviewed by D’Elia and Wiebe 1990). Reports on nutrient leaks (D'Elia
1977; Crossland 1983; Erez 1990; Korpel 1991; Hatcher 1997) required explanations that involve
efficient mechanisms of net nutrient import from the ambient waters.
Zooplankton was considered the major source
Indeed, import mechanisms by which inorganic nutrients are brought into the reef,
primarily by autotrophic uptake from the dissolved pool, were recognized (Atkinson 1992;
Atkinson and Bilger 1992; Bilger and Atkinson 1992; Baird and Atkinson 1997). Heterotrophic
import of particulate carbon and nutrients was also thought to be significant, mainly in the form of
zooplankton predation by corals, fish and other planktivores (e.g., Tranter and George 1972;
Glynn 1973; Hamner et al. 1988; reviewed by Erez 1990). Such heterotrophic sources of “new”
nitrogen (sensu Dugdale and Goering 1967), for example, should compensate, at least partly (Erez
1990), for the advection and biogenic losses of nutrients, particularly in situations where physical
oceanographic sources (upwelling, mixing) are not significant. The potential of small living
water-born organisms such as protozoans, phytoplankton and bacteria, as a source for
heterotrophic import of allochthonous carbon and nutrients into coral reef ecosystems, was
acknowledged by some authors (Linley and Koop 1986; Sorokin 1973; Ayukai 1995). I have
noticed, however, an apparent gap in the reference to the latter mechanism (import from the living
pool), related to a traditional "bias" among coral-reef investigators to selectively address the role
of zooplankton, rather than phytoplankton, as an allochthonous source for carbon and nutrient in
this ecosystem. In that respect, this bias fundamentally contrasts the situation in temperate coastal
habitats, such as estuaries and bays, where phytoplankton is thought to be the key link in benthicpelagic couplings (Fréchette and Bourget 1985; Loo and Rosenberg 1989; Asmus and Asmus
1991; Chardy and Dauvin 1992; Graf 1992; Yamamuro and Koike 1994) and in extreme cases, to
be controlled by benthic grazing (Cloern 1982 1991; Fréchette et al. 1989; Hily 1991).
Phytoplankton was overlooked
A historical perspective may shed some light on the reason for the apparent scarcity of
studies of phytoplankton grazing at the coral reef. Three decades ago, Glynn (1973a), in a seminal
study that for years set the stage for our understanding planktivory in coral reefs, suggested that
8
Chap. 2. Scientific background
the import of carbon to the reef via zooplankton predation surpasses that of phytoplankton
removal by an order of magnitude. A set of important zooplankton-predation studies then
followed (reviewed by Sebens 1997). While evaluating the findings of Glynn (1973a) and other
earlier studies, one should be aware that in those days, neither epi-fluorescence microscopes nor
flow cytometers were available. Glynn, for example, indeed tested for phytoplankton removal, but
did so only for cells caught with his 76µm mesh, we know now that such large cells constitute an
insignificant fraction of the total phytoplankton biomass in the typically warm oligotrophic
oceans which surround most of the world's coral reefs (Furnas and Mitchel 1987; Furnas et al.
1990; Chisholm 1992; see also Yahel et al. 1998).
Theoretically, phytoplankton grazing should be important
Given the presence of numerous taxa that are notorious phytoplankton grazers in
temperate habitats, including bivalves, sponges, ascidians and polychaetes (Jørgensen 1966), it
was somewhat surprising that the contribution of phytoplankton grazing has been overlooked in
community-wide, as well as taxon-specific investigations, of the coral reef. Theoretical
perspectives related to the topographical roughness of reef bottom (Baird and Atkinson 1997) and
the occurrence of high slenderness ratio (height over width) in numerous reef animals, suggest an
excellent adaptation for feeding on small-suspended particles such as phytoplankton (Abelson et
al. 1993). It was not until very recently, that evidence started mounting suggesting that
phytoplankton grazing may indeed play an important role in coral reefs trophic pathways. For
example, a study done at A. Genin's laboratory (Fabricius et al. 1995a,b) found that efficient
phytoplankton grazing by soft corals can fully support their extremely fast growth and Ayukai
(1995) documented a strong depletion of picoplankton (<2µm) near some reefs in Australia.
Hydrodynamic control of mass transport
A major objective of this thesis was to test the hypothesis that phytoplankton is a major
allochthonous source of carbon and nutrients at the coral reef ecosystem. However, the supply,
renewal, and possibly removal rates of plankton at the reef are dependent on, and possibly
controlled by the flow (e.g., Fréchette and Bourget 1985; Fréchette et al. 1989; Monismith et al.
1990; Shimeta and Jumars 1991; Atkinson and Bilger 1992; Koseff et al. 1993; Carlson et al.
1994; Lenihan et al 1996; Sebens 1997). To adequately test the above hypothesis, a thorough
characterization of the hydrodynamics is required. In fact, ill characterization of the flow patterns
near the reef is probably the major shortcoming of past community-level studies on planktivory
(reviewed by Hatcher 1997). Near-bottom shear and mixing greatly affect the hydrodynamics
near corals (Denny 1988), making little value of primitive measurements, such as those where the
passage time of fluorescein-dyed waters is measured (e.g., Glynn 1973b; Hamner et al. 1988),
unless advanced "blob"-monitoring techniques are applied (Koehl et al. 1993, 1997). Even then,
the use of the data to infer mass transfer is quite limited. Detailed measurements of currents in
coral reefs are generally limited to either large-scale investigations or single-point measurements
(e.g., Roberts et al. 1975; Wolanski and Pickard 1983; Sebens and Done 1992; Helmuth and
Sebens 1993; Sebens 1997), and are typically lacking a thorough coverage of the complex
hydrodynamics near the bottom. Flow measurements appropriate for benthic studies must be
performed in multiple strata, a task rarely done in the coral reef (see Shashar et al.,1996, for
limited measurements of flow indices in the benthic boundary layer). Adequate in situ flow and
turbulence measurements in this hydrodynamically complex zone are a major challenge of
benthic ecology (Butman et al. 1994). In the case of benthic environments, flow patterns are
9
Chap. 2. Scientific background
complicated due to the interactions of several key factors operating on several scales, from the
meso-scale flow field (e.g., wind-driven cross-shore circulation) to small-scale boundary-layer
processes over rough topography (e.g., mixing and turbulence) (see Denny 1988; Koehl et al.
1993; Vogel 1994; Shashar et al. 1996 and references therein). A major challenge for coastal
hydrodynamics is to integrate the small- and meso-scale flow dynamics, together with biogenic
processes, to derive the overall mass exchanges between the benthic communities and adjacent
sea.
Individual based grazing rates
Almost no studies have been devoted to phytoplanktivory in coral reefs. In fact, little
research was devoted to the nutritional ecology of any non-anthozoan suspension feeders in coral
reefs. A notable exception was the pioneering work of H.M. Reiswig (see below) on the
nutritional ecology of some Caribbean's giant reef sponges back in the early sixties.
Unfortunately, there was hardly any follow-up to Reiswig outstanding works (see, Wilkinson
1978; Pile 1997). The only studies of the nutrition of actively pumping reef suspension feeders
were of the commercially reared giant clams (Klumpp et al. 1992; Klumpp and Lucas, 1994;
Klumpp and Griffiths, 1994; Hawkins and Klumpp, 1995) and pearl oysters (reviewed by Loret et
al. 2000). The paucity of research in these fields is most likely due to the remoteness of reefs from
the traditional centers of modern marine biology, and to a lesser extent the lack of proper
methodology for the study of the ultraplankton dominating oligotrophic waters. In a sharp
contrast to coral reefs, almost any aspect of the nutritional ecology and physiology of temperate
and boreal actively pumping suspension feeders (most notably bivalves) has been intensively
studied since the onset of modern marine biology (reviewed by Jørgensen 1966; Wildish and
Kristmanson 1997). Surprisingly, as reviewed below, very few of those methods are suitable for
in situ studies whereas, the application of in vitro methods was clearly inappropriate in the reef
environments where many suspension feeders are cryptic and all have tight symbiotic
relationships.
Laboratory studies
The vast majority of active suspension feeders’ nutritional ecology research was
conducted in closed laboratory vessels (Ribes et al., 2000; Riisgård, 2001a). The methodology
involved is reviewed in Jørgensen (1966), Wildish and Kristmanson (1997), and Riisgård (2001a).
Briefly, most laboratory studies as well as some field studies (e.g., Frost and Elias 1985;
Hopkinson et al. 1991; Ribes et al. 1998, 1999) adopted indirect techniques (see Jørgensen 1966)
where removal/excretion rates are deduced from temporal concentration shifts (of colored beads,
radioactive label, food items, or excretion products) in the vessels containing the experimental
organisms (but see, Møhlenberg and Riisgård 1979). A major drawback of these indirect methods
is the inability to differentiate between pumping rate and retention efficiency. Instead, both
parameters are indistinguishably combined and reported as “clearance rate”. As a result, clearance
rate based estimates of diet composition, selectivity, and feeding rates may be biased by
interactions between food concentration and pumping rate (Newell et al. 1989; Ward and
MacDonald 1996; Kryger and Riisgård 1988; Riisgård 2001a). In many experimental setups, the
lack of simple means to estimate pumping rates lead to the use of unduly small vessels where refiltration and accumulation of waste products cause further problems (Møhlenberg and Riisgård
1979; Riisgård, 2001a). Wildish and Kristmanson (1997, page 49) discuss other drawbacks
associated with the indirect method.
10
Chap. 2. Scientific background
Biodeposition, gut content, and fecal pellet analysis were all used for the study of active
suspension feeders’ nutritional ecology in both field and laboratory conditions. The major
limitation of these techniques is that the differential digestion rates and extent of food component
in the digestive system of the studied specimen are unknown (review by Iglesias et al. 1998).
Wright and Stephens (1978) suggested a direct method for in vitro study of active
suspension feeders’ intakes and excretion that involve minimal interference. This method applies
direct sampling of the exhaled water after a single passage across the animal filtration apparatus.
Unfortunately, it has not gained much attention.
Indeed, in vitro experiments were highly effective in promoting our understanding of
active suspension feeders' nutritional ecology. Nevertheless, due to the numerous, welldocumented laboratory effects (e.g., Vogel 1974, 1977; Møhlenberg and Riisgård 1979; Famme
and Riisgård 1986, Riisgård 2001a), inference from laboratory obtained rates to actual behavior at
the field is limited (Møhlenberg and Riisgård 1979; Wildish and Kristmanson 1997; Ribes et al.
2000; Cranford 2001). Moreover, expensive and complicated experimental systems are required
for a proper laboratory simulation of even the most basic environmental factors (e.g., water flows,
O'Riordan et al. 1993; Nowell and Jumars, 1987), while accurate experimental reconstruction of
real world situations such as (e.g.) the complex web of symbiotic relationships maintained by
most coral reef active suspension feeders is unlikely.
In situ methods
H.M. Reiswig, in his comprehensive fieldwork on the ecology of Caribbean sponges
(1971a, 1971b, 1974) pioneered the use of direct in situ techniques for the study of active
suspension feeders diet composition, metabolic performance and water transport rates. His direct
methods were based on comparison of the content of the water inhaled and exhaled by the
sponges and therefore, were free of most of the methodological problems associated with both
indirect and in vitro experiments. Combined with pumping rate estimates obtained by hot wire
thermistors or hand held flow meters, Reiswig’s data allowed calculation of the sponge’s actual
grazing rates. Unfortunately, the methodology used by Reiswig for his giant Caribbean sponges is
not suitable for many smaller sponges (Wilkinson 1978) and even less so, for more sensitive
organisms such as bivalves and ascidians (Yahel, unpublished data). Water collection by syringes
from small or medium size ex-current jets (Reiswig 1974, 1985; Wilkinson 1978; Pile et al. 1996,
1997) offers no control over the risk of “contaminating” the exhaled water sample by sucking in
ambient water. Additionally, it is unclear how a 1 ml sample drawn by a syringe from a 10 mm
bivalve can be ensured to contain solely exhaled water. Indeed, retention efficiencies reported by
Pile and Young (1999) are suspiciously low, especially for their smaller size classes (<50 mm),
suggesting such contamination might have occurred (see also Reiswig 1985; Pile 1997).
Hopkinson et al. (1991) used bell jars in-situ to study the metabolism and nutrient fluxes
of benthic organisms. More recently, Ribes at al. (1998, 1999, 2000) introduced a re-circulating
bell jar system. While this indirect technique uses natural seawater under natural temperature and
illumination, it is still afflicted with most of the other drawbacks of its analogous pulse-chase
laboratory method as discussed above. The bell jar method is also quite demanding in terms of the
amount of equipment and work required per specimen sampled (Ribes et al. 2000).
11
Chap. 2. Scientific background
Pumping rates
A considerable effort was made throughout the last century to the study of water transport
rate by various active suspension feeders’ taxa (review by Jørgensen 1966; Famme and Riisgård
1986; Joens et al. 1992; Wildish and Kristmanson 1997; Riisgård 2001a). Methodology ranged
from the straightforward quantification of dyed water movement within a tube inserted into a
sponge osculum (Parker 1914; Gerrodette and Flechsig 1979) via the constant-level tank method
where the ex-haled water is channeled into a measuring flask (Jørgensen 1966 and ref therein) to
complex mechano-optic devices (e.g., Jones et al. 1992; Famme and Riisgård 1986 ; Riisgård
2001a). Tank head pressure (e.g., Jørgensen et al. 1990; Riisgård et al. 1993), temperature
differences between the animal interior and its environment (Defossez et al. 1997), algal clearance
rate (Møhlenberg and Riisgård 1979; Petersen and Riisgård 1992; Riisgård et al. 1993), and
microscopic observations (Turon et al. 1997) were also used. Many workers used jet speed and
aperture area products to estimate water-transport rate, applying different techniques to measure
these parameters (e.g., Fiala-Medioni 1978; Jones et al. 1982). Clearance-rate based estimates of
pumping rates assume either 100% or at least an invariable retention efficiency, but this
assumption has rarely been tested (e.g., Riisgård et al. 1993). Jet speed based estimates assumed a
known distribution of water velocities across the ex-current aperture (Charriaud 1982). While this
latter assumption may be valid for some ascidians and bivalves, it does not hold for many sponge
species (e.g., Mycale fistulifera, Subarites clavatus) in which large exhalent canals are merged
from different directions just below the osculum opening. Experiments and observations with
dyed water revealed that ex-current jets in those sponges are frequently composed of several
independent jets (Yahel, personal observations).
In situ measurements of active suspension feeders pumping rates rely mostly on hot wire
or hot film thermistors (Reiswig 1974; Vogel 1974, 1977; Fiala-Medioni 1978; Savarese et al.
1997). These instruments are sufficiently small and allow a continuous record of ex-current speed,
but have a tendency to drift and require constant and tedious calibrations (LaBarbera and Vogel
1976; Charriaud 1982; Wildish and Kristamson 1997). As hot film probes measure only excurrent jet speed, continuous information of aperture area and ambient flow are also required.
When such information is available, pumping rates can be recorded for prolonged periods.
Instantaneous pumping rates were estimated by Reiswig (1974) for the giant sponges he
investigated using a hand held flow meter, whereas Savarese et al. (1997) used dye injection to
the inhalant canals and subsequent measurements of the dye jet trajectories within the ambient
flow field.
The role of DOC
The dissolved organic pool is operationally defined as the organic carbon passing through
a fine filter, typically GF/F (Benner 2002; Carlson 2002) although a plethora of ranges (0.001 µm
to 1.0 µm) is found in the literature (McCarthy et al. 1996). The vast majority (>97%) of the
organic carbon pool in seawater resides in the dissolved phase (Benner 2002). Only a small
fraction of the dissolved pool is labile, the rest is thought to be refractory and unavailable for
utilization by marine organisms (reviewed by Carlson 2002). While the methodology for accurate
DOC determination was still evolving when my thesis was initiated, the notation that DOC
concentrations are 10 to 100 fold higher than those accounted by both living and dead organic
particles (POC) was already generally accepted (Sharp 1997). Even in extreme cases, such as
those reported for the Antarctic by Martin and Fitzwater (1992), DOC still constitutes some 75%
12
Chap. 2. Scientific background
of total organic carbon (POC+DOC). Potential sources for DOC in coral reefs are numerous:
Algae excrete high proportions of their photosynthetically-assimilated carbon as DOC (e.g.,
Mague et al. 1980; Zlotnik and Dubinsky 1989). “Sloppy” feeding and grazing of benthic algae
and animals would also cause DOM leakage from broken prey (Thomas 1997). Moreover, corals
directly excrete substantial amounts of DOM (Muscatine et al. 1984), sometimes as free amino
acids (Schlichter and Liebezeit 1991) or mucus (Richman et al. 1975; Crossland et al. 1980).
Indeed, a substantial DOM increase was reported by Crossland and Barnes (1983) over a reef flat
in Lizard Island (Great Barrier Reef).
The paucity of in situ tests of the nutritious role of DOC is likely related to
methodological problems, which have long inflicted DOC measurements in the marine realm
(Peltzer and Brewer 1993; Suzuki 1993; Sharp 1997; reviewed in Sharp 2002). This gap in our
knowledge goes way beyond coral reefs to benthic communities in general. A decade ago,
Sugimura and Suzuki (1988) introduced a new technique for the oxidation of organic compounds
in seawater commonly referred as HTCO. Despite Suzuki’s later criticism of his earlier technique
(Suzuki 1993), the HTCO methodology has since been improved and is now acknowledged as the
most reliable method for DOC determination, with reported preciseness of ±1-2% (Carlson 1994;
reviewed by Sharp 1997, Sharp 2002).
In a sharp contrast to the well-established role of DOC in the so-called “microbial loop” of
pelagic food webs (e.g. Azam et al. 1983, 1994; Fenchel 1988), the role of DOC in the nutrition
of benthic animals is still unresolved (Wright and Manahan 1989; Thomas 1997). Indeed, a role
for DOC in the nutrition of aquatic metazoans has been repeatedly proposed since the turn of the
century (Pütter 1909) and was recently suggested to explain the observed discrepancies between
the supply and demand of carbon in benthic suspension feeders (Reiswig 1981, 1985) and deepsea soft-bottom communities (Smith et al. 1989). However, except for bacteria (Azam et al.
1983, 1994) and some invertebrate larvae (Wright and Manahan 1989), direct evidence of bulk
utilization of DOC in the ocean is lacking. Indeed, numerous laboratory experiments have
demonstrated the ability of soft-bodied metazoans to take up specific components from the
dissolved pool (e.g., Stephens and Schinske 1961; Johannes et al. 1969; Jørgensen 1976; Wright
and Manahan 1989; Thomas 1997) including sponges (Jaeckle 1995). We are not aware of direct
evidence for in situ, uptake of bulk DOC by benthic metazoans.
Research goals
In coral reefs, neither the heterotrophic import of organic matter, nor its recycling, are well
understood (Hatcher 1997). Specifically, two major pathways have been overlooked in coral reef
ecology: benthic grazing on small (<8µ) phyto- and bacterio-plankton and consumption of
dissolved organic carbon (DOC) by benthic invertebrates. Addressing these two pathways and
their correspondence to hydrodynamic conditions were the principal goals of this thesis.
To address these questions we combined biological, physical and chemical investigations
to study the distribution and removal of ultraplankton and DOC by benthic suspension feeders in
coral reefs. Dictated by logistic limitations, the fieldwork focused on the reef slope, which is the
most unexplored (Hatcher 1997), largest and biologically most diverse part of coral reefs in the
Red-Sea (Loya 1972).
Specifically, my major objectives were:
(1) To characterize the distribution of phytoplankton in two-dimensions horizontally, from the
reef slope to the open water, and vertically, from the bottom-water interface to the sea
surface.
13
Chap. 2. Scientific background
(2) To quantify in situ the rates of ultraplankton grazing and DOC removal by individual
benthic suspension feeders and to compare individual-based removal rates of DOC vs.
living POC (ultraplankton).
(3) To combine simultaneous measurements of the physical field (near-shore circulation,
benthic boundary flow and mixing) and biological processes (removal rates) over defined
sections of the reef community, together with in situ manipulation experiments, in order to
quantify in situ phytoplankton removal rates by the coral-reef community.
(4) To compare the contribution of the mass fluxes mediated by the benthic grazers studied to
overall reef community mediated fluxes.
Due to space limitations, only four chapters are included in the thesis. Three other chapters are
included as external appendix.
14
3.
Phytoplankton distribution and grazing near coral reefs
By
Yahel G., A.F. Post, K.E. Fabricius, D. Marie, D. Vaulot, and A. Genin
1998
Limnology and Oceanography 43, 551-563
15
LIMNOLOGY
AND
June 1998
OCEANOGRAPHY
Lmnol
Oceanogr.. 43(4), 1998, 551-563
0 1998, by the Amencan Society of Limnology
and Oceanography,
Phytoplankton distribution
Volume
43
Number
4
Inc
and grazing near coral reefs
Gitai Yahel
H. Steinitz
Department
Marine Biology Laboratory,
Interuniversity
Institute of Eilat, PO. Box 469, 88103 Eilat, Israel;
of Evolution,
Systematics and Ecology, Hebrew University
of Jerusalem, Jerusalem, Israel
and
Anton F. Post
H. Steinitz
Department
Katharina
Australian
Marine Biology Laboratory,
of Microbial
and Molecular
Interuniversity
Institute of Eilat; and
Ecology, The Hebrew University
of Jerusalem
Fabricius
Institute
of Marine
Science,
PMB
3, Townsville,
Queensland
4810, Australia
Dominique Marie and Daniel Vaulot
Station
Biologique,
CNRS;
INSU
et Universitk
Pierre et Marie
Curie,
BP 74, 29682 Roscoff
Cx, France
Amatzia Genin
H. Steinitz
Department
Marine Biology Laboratory,
Inter-university
Institute of Eilat; and
of Evolution,
Systematics and Ecology, Hebrew University
of Jerusalem
Abstract
Depletion of phytoplankton cells and pigments over coral reefs was studied in the Gulf of Aqaba, Red Sea,
during 1994-1996. Phytoplankton abundance and chlorophyll (Chl) a concentrations were 1565% lower near the
reefs than in the adjacent open waters. The decrease in chlorophyll near the reef was typically associated with an
increase in the concentration of its degradation products, the pheopigments. The steepest slope of these cross-shore
gradients occurred within l-3 m above bottom. More than 50% of the variation in the extent of the chlorophyll
gradients, but not of pheopigments, could be explained by the advection of water during 2 h preceding the transect
and by the concentration of Chl a in the open water. No cross-shore gradients were observed at a sandy-bottom
site without reef. Eukaryotic phytoplankton (<5 pm) contributed >70% of the total depleted carbon near the reef
during winter, while the cyanobacterium Synechococcus (1 pm) contributed the largest share in summer. The proportions of different taxa in depleted fractions were similar to those in ambient waters, indicating no size selectivity.
Direct measurements of phytoplankton removal rates were made in water passing through a unique 5-m-long
perforated reef, dominated by herbivorous soft corals. The waters downstream of that reef were strongly depleted
of phytoplankton (10 to >36%, or 32 to >lOO ng Chl a liter-‘). When converted to carbon fluxes, these rates
greatly exceeded reported values of carbon input to coral reefs via zooplankton predation. Phytoplankton grazing
is an important component of benthic-pelagic coupling in coral reefs.
Acknowledgments
We are indebted to Ruthy Yahel, Yaron Shiff, Dan Weil, Inball Ayalon, Debbi Lindell, Eitan and Galy Bluemberg, Alon and Anat Bookai,
Roberto Erlich, Itzik Lherer and Amr Attia for their professional and most dedicated help with the field and laboratory work. We thank
Marcel Veldhuis for his invaluable help with the flow cytometry analysis of the 1995 samples. Itzchak Brickner kindly helped with species
identifications in the benthic survey. We thank the Oil Terminal company Katza for a permit to work at the oil jetty and the Israel Nature
Reserves Authority for a permit to work at the closed section of the Eilat Nature Reserve. We are grateful to the Interuniversity Institute
of Eilat for logistic support. Comments made by R. Yahel, Russell Wyeth, and two anonymous reviewers significantly improved the
manuscript.
This study was partly supported by the “Red-Sea program,” a joint German, Egyptian, Palestinian, and Israeli program funded by the
German Ministry of Science, Technology and Education (BMBF). The flow cytometry analysis in Roscoff was funded by the European
Community contract MAS2-CT95-00 16.
551
552
Yahel et al.
Benthic grazing on phytoplankton is a principal trophic
pathway in shallow, temperate coastal habitats (Asmus and
Asmus 1991; Koseff et al. 1993; Yamamuro and Koike
1994). In some bays and estuaries, phytoplankton density in
the water column is controlled by benthic grazing (Cloern
1982; Frechette and Bourget 1987; Cloern and Alpine 1991;
Frechette et al. 1989; Hily 1991). Dominant grazers in such
situations appear to be bivalves, ascidians, and polychaetes
(Monismith et al. 1990; Riisgkd et al. 1996).
Traditionally, studies of benthic-pelagic coupling in tropical coral reefs, unlike those in shallow temperate habitats,
have focused on zooplankton rather than phytoplankton as
the principal source of prey (Tranter and George 1969;
Glynn 1973; Johannes and Gerber 1974; Hamner et al. 1988;
Erez 1990). Indeed, numerous coral-reef inhabitants, including the corals themselves, feed on zooplankton (Sebens
1997). The concept that phytoplankton is not a principal
food source in coral reefs probably originated from an early
comparison between zooplankton and phytoplankton removal rates in a Caribbean coral reef (Glynn 1973), which suggested that zooplankton are by far the most important source
of heterotrophic carbon in this ecosystem. Recently, however, Ayukai (1995) observed a substantial retention of picophytoplankton at the Great Barrier Reef, Australia, and Fabricius et al. (1995a,6) discovered phytoplankton feeding by
some common soft-coral species that had previously been
considered exclusive zooplanktivores.
In fact, strong phytoplankton grazing is to be expected at
the reef, as numerous members of the coral-reef community
are known to feed on particles within the size range of phytoplankton. Such taxa include bivalves (Klumpp et al. 1992;
Lesser et al. 1992), gastropods (Lesser et al. 1992), sponges
(Reiswig 1971, 1974; Pile et al. 1996, 1997), ascidians (Petersen and Riisgard 1992), crinoids (Rutman and Fishelson
1969), foraminiferans (Cedhagen 1988), polychaetes (Vedel
and Riisgard 1993), and soft corals (Fabricius et al.
1995a,b). Conceptual considerations also support expectations for intense phytoplanktivory at the reef, as phytoplankton biomass in coral reefs commonly exceeds that of zooplankton by an order of magnitude (Roman et al. 1990; R.
Yahel pers. comm.). Furthermore, the bottom topography in
coral reefs is typically rough, with numerous living and nonliving objects having a high “slenderness ratio” (height over
width), which is an optimal morphology for feeding on small
suspended particles (Abelson et al. 1993). Boring mussels
(Lithophaga spp.), for example, growing in protruding corals, are less likely to experience refiltration of exhaled water
than their counterparts in flat soft bottoms (O’Riordan et al.
1995). Direct evidence showing that the above taxa feed on
phytoplankton in coral-reef communities are nevertheless
scarce (Rutman and Fishelson 1969; Klumpp et al. 1992;
Fabricius et al. 1995a,b; Pile 1997).
The goals of this study were to characterize the spatial
and temporal patterns of phytoplankton distribution near
fringing coral reefs and to quantify the rates of phytoplankton removal at a “perforated” reef, where the flow of water
through the reef could be measured. Our study focused on
small (<8 pm) phytoplankton, termed “ultraphytoplankton”
(e.g. Lindell and Post 1995). The trophic contribution of ultraphytoplankton to the reef community, in terms of carbon
0
10
20
3okm
-
29’00’N
-
ZS’OO’N
-
Gulf of
Aqaba
-
RED
I
34’30’E
SEA
I
3900
Fig. 1. The study sites in the Gulf of Aqaba (Eilat). Arrows on
the left point out coastal sites (E, Eiiat; H, Hibik; R, Ras Abu Galurn); letters within full circles indicate the locations of the oceanographic stations near the center axis of the gulf (A, B, M, E and
9.
influx from the ambient waters, is compared with fluxes reported for other heterotrophic pathways.
Materials
and methods
Study sites-The study was carried out in the Gulf of Aqaba (Eilat), Red Sea, between February 1994 and May 1997.
The cross-shore distribution of phytoplankton was studied at
three sites with fringing reefs and a single control site with
a sandy bottom without a reef. The three reef sites (Fig. 1)
were at the Coral-Beach Nature Reserve in Eilat (29”30’N,
34”56’E), Ras Abu Galum (28”36’N, 34”33’E), and Hibik
(28”52’N, 34”38’E). The sandy site was near Taba (29”28’N,
34”55’E), -1.5 km south of the nature reserve in Eilat. Measurements of phytoplankton grazing rates were carried out
at an artificial reef (hereafter “perforated reef”) located at
Phytoplankton
lagoon 7
c
grazing in coral reefs
reef flat
Distance
from shore (m)
Fig. 2. A schematic plot of the cross-shore topography (dark
area) and the sampling points (A) across the reef in Eilat. Note that
the sampling points were all at 5-m depth.
the southern jetty of the Oil Terminal in Eilat, -1 km north
of the nature reserve.
The fringing coral reefs at the three sites were similar with
regard to their topography and community structure. They
all had a wide (30-50 m) shallow (l-2-m depth) lagoon,
located shoreward of a lo-30-m-wide reef flat. The flat ended at its seaward edge at a steep vertical wall (2-5 m in
height), seaward from which a rich gradually sloping forereef was found, extending some hundreds of meters seaward
of the flat (Fig. 2). Our sampling work was carried out over
the slope and extended seaward into deeper waters (30 m).
The reef community was described by Fishelson (1970) and
Benyahu and Loya (1977, and references therein). In short,
these reefs were dominated by hermatypic corals, many of
them bearing numerous herbivorous endolithic filter-feeders
(Hutchings 1983; Loya 1981). Other common taxa included
hydrozoan corals (Millepora dichotoma), soft corals, anemones, sponges, tunicates, and polychaetes. Soft corals were
rare at the fringing reefs.
The perforated reef at the Oil Terminal has grown on the
jetty’s pilings and their surrounding barbed wire, constructed
some 25 years ago. The local community was described by
Goren and Benayahu (1992). The section studied (6-15-m
depth) was dominated by the herbivorous soft corals Dendronephthya hemprichi and Scleronephthya corymbosa. A
gradual shift of dominance, from D. hemprichi to S. corymbosa, took place during the course of our study. Other
common invertebrates at this reef included a gorgonian coral
(Acabaria sp.), sponges, ascidians, stony corals, and actinians. Clearly, both the topography and community structure
at the perforated reef were different from those at the abovereef slopes. Nevertheless, there are numerous reefs in the
Red Sea (and elsewhere) where dominant Dendronephthya
colonies form similar structures on steeply sloping walls and
overhangs (Benayahu 1985). The water in the perforated reef
flowed through the benthic community rather than above it
(as in the case of the fringing reef). This flow pattern allowed
direct measurements of water passage times, which were required to estimate rates of phytoplankton grazing by the benthic community (see below).
The slope at the sandy sites was similar to that of the reef
slope, except that it had no reef and the bottom was partly
553
covered with sea-grasses (mainly Halophila stipulacea). A
few coral-bearing boulders were found at the sandy site, but
the total abundance of corals and their associated benthic
community was several orders of magnitude lower than at
the reef slope.
Oceanographic and meteorological conditions in the Gulf
of Aqaba were described by Reiss and Hottinger (1984) and
Genin et al. (1994, 1995). The currents at our study sites
were weak to moderate (<20 cm s ‘) and primarily longshore. Semidiurnal reversals of flow direction occurred during summer-fall, while fluctuations of lower frequencies
dominated in winter-spring (Genin et al. 1994; Genin et al.
unpubl. data). The cross-shore components, although being
much weaker (< 10%) than the long-shore components, commonly formed a well-defined coastal circulation consisting
of a weak onshore flow in the upper lo-20 m, slow downwelling near the coast, and a return, offshore flow above
bottom (Genin et al. unpubl. data). Sea conditions at all sites
were relatively calm (wave height <<l m), except for rare
southerly winter storms.
Cross-shore transects-Intense
benthic grazing on phytoplankton is expected to produce a prey-depleted layer
above the bottom (Cloern 1982; Frechette et al. 1989) possibly forming a gradient of decreasing phytoplankton concentrations from the open waters toward the reef. If strong
grazing is unique to the coral-reef community, such crossshore gradients are expected to be more pronounced at the
reef than at the sandy sites. To test these expectations, water
samples were taken at seven points along a cross-shore transect (Fig. 2), starting at a 5-m bottom depth and extending
seaward to a bottom depth of 30 m (hereafter “open water”).
The sampled water was filtered through a loo-pm mesh to
remove large zooplankton and stored in a darkened ice box
until processing in the laboratory. All samples were taken at
the same depth (5 m); thus, the shorewardmost sample was
taken a few centimeters above the bottom, while the seawardmost sample was taken 25 m above bottom (mab), with
intermediate samples taken at 1, 2, 3, 5, and 10 mab. Transect lengths varied between 150 to 250 m, depending on the
topographic slope. Two such transects, 500-800 m apart
(hereafter north and south transects), were sampled at each
study site during each visit. Except for a single visit (see
below), all 57 transects were carried out during the day (Table 1).
The study sites in Eilat were visited 10 times, while the
more remote sites along the Gulf of Aqaba were visited
once, during a cruise aboard the RV University I in August
1996. In order to examine diurnal changes in cross-shore
patterns, nocturnal transects preceded the daytime transects
at the two reef sites in Eilat in February 1996, while changes
between consecutive days were examined through a replication of the complete sampling protocol for 4 and 3 consecutive days in February and June 1994, respectively (Table
1).
Lagrangian sampling-This
part of the work was based
on consecutive sampling of the same water parcel as it
moved above, or through, the reef. Two modes of Lagrangian sampling were used: (1) repetitive sampling of a water
554
Yahel et al.
Table 1. Dates and times (hour of day) of the cross-shore transects carried out at the Eilat sites. Two transects >500 m apart were
used at each site, one at the northern (N) and the other at the southem (S) section of the reef.
Coral reef
Visit
No.
Month
Day
1
Feb 94
2
3
4
6
7
8
9
13
14
15
28
23
18
23
11
9
16
1
4
6
2
3
4
5
6
7
8
9
10
Jun 94
Aug 94
Dee 94
Feb 95
Jun 95
Aug 95
Dee 95
Feb 96
Aug 96
N
Sandy bottom
S
N
S
1200
1000
1230
1000
1400
1430
1215
1015,
0940
0950
0930
0815,
1525
1130
0940,
0540,
1115
1500
1045
1030
0930
1515
0930,
0900
1030
0845
0845,
1610
1215
0850,
0400,
1215
1515
1110
1100
1200
1140
1200
1030
0900
1345
1435
1000
1431
1120
1100
1125
1005
0945
1430
1335
1130
0840
Nrjte: Transects were also carried out at Ras Abu Galum on 4 August 1996
(1120 and 1235 h at N and S sections of the reef, respectively) and at
Hibik on 6 August 1996 (1215 and 1350 h at N and S sections of the
reef).
parcel marked with a drifter drogue as it moved above the
reef slope (hereafter “the drogue experiment”), and (2) sampling a water parcel prior to (upstream) and after (downstream) it passed through the perforated reef at the Oil Terminal (hereafter “the perforated-reef experiment”). The goal
of the drogue experiment was to test for phytoplankton depletion by grazers dwelling in the water column (e.g. herbivorous zooplankton). The goal of the perforated-reef experiment was to quantify the rates of phytoplankton grazing
by the benthic community at that reef.
The drogue experiment was carried out at two sites: Ras
Abu Galum and Hibik. A drifter (Davis et al. 1982) was
drogued at 5-m depth above a bottom of 6-8-m depth and
monitored from a nearby skiff for 30-90 min. Water samples
were taken next to the drogue by divers every 5 min.
The perforated-reef experiment protocol was as follows.
Prior to sampling, fluorescein dye was released upstream of
the reef and the time it took the dyed water to pass through
the reef was measured (passage time). Due to mixing and
consequent dispersal of the dye during its passage through
the reef, the visual estimate of passage time was based on
the bulk of the dyed blob, and should therefore be considered
an approximate measure. Sampling started 15 min after the
fluorescein dye cleared the reef and lasted 30-40 min afterwards. Each sampling set consisted of 8-11 pairs. Within
each pair the downstream sample was taken after the upstream sample, delayed by the predetermined passage time.
Grazing rates by the perforated-reef community were calculated for each pair as the difference in phytoplankton
abundance between the upstream and downstream samples
divided by the relevant passage time.
Sample processing-Concentrations
of chlorophyll a and
pheopigments were measured with a fluorometer (Model
AU-lo, Turner Designs) by using the acidification method
(Parsons et al. 1985) after filtering 280 ml of seawater on a
Whatman GF/F filter followed by 24 h dark extraction in
90% acetone at 4°C. To allow direct comparisons between
the molecular concentrations of the parent chlorophyll and
its degradation products, pheopigment concentrations are reported using chlorophyll equivalent units as in Head and
Home (1993). Starting in February 1996, Chl a was measured both as indicated above and by the nonacidification
method (Welschmeyer 1994) using a Turner Designs TD-700
fluorometer. A reliability test of our chlorophyll measurements indicated a precision of t5.1% (n = 90) within replicates.
Flow cytometry was used to process a subset of the above
samples in order to estimate the concentrations of two dominant autotrophic groups in our waters, Synechococcus and
pica-eukaryotes. Prochlorococcus, the third major autotrophic group in the Red Sea (Lindell and Post 1995), has a
very weak chlorophyll fluorescence near the surface, especially in summer. It could not be detected during most of
this study with the flow cytometers used, except in February
1996 (see below). Taxonomic discrimination was made on
the basis of cell-side scatter and forward scatter (a proxy of
cell size), orange fluorescence of phycoerythrin, and red fluorescence of chlorophyll (Partensky et al. 1996). Aliquots of
1.5 ml were withdrawn from the same water samples used
for pigment analysis and preserved with 0.2% buffered paraformaldehyde (June 1995, February 1996) or with 1% paraformaldehyde and 0.05% glutaraldehyde (August 1996).
Samples were frozen in liquid nitrogen and stored at -70°C
until processing. Samples of the cross-shore transects and
the perforated reef experiment from June 1995 were analyzed on a Coulter EPICS XL flow cytometer equipped with
a 488-nm argon laser. Data processing was performed using
the EPICS XL workstation (vers. 1.5, 1993, Coulter). The
cross-shore transect samples from February and August
1996 were analyzed with a FACSort (Becton-Dickinson) using the standard setup. All cellular parameters were normalized to the values measured for 0.95-pm YG Polysciences beads. Data processing was performed using the
Windows software CYTOWIN (Vaulot 1989). Cell numbers
were converted to carbon following Campbell et al. (1994).
Size and biomass of phytoplankton in the open watersPigment measurements based on water filtered on GF/F filters included a large range of cell sizes (up to 100 pm). The
flow cytometer measurements, on the other hand, were confined to ultraphytoplankton cells, i.e. smaller than 8 and 5
pm for the 1995 and 1996 samples, respectively. To evaluate
the relative contribution of ultraphytoplankton
to total extracted Chl a, we used data collected in the Gulf of Aqaba
during five oceanographic cruises that partly overlapped the
period of our field sampling near the coast (June 1994-August 1995). A total of 24 water samples were collected at
0-20-m depths at five oceanographic stations along the Gulf
Phytoplankton
grazing in coral reefs
of Aqaba (Fig. 1). Each sample (500 ml) was passed through
a lOO-pm mesh to remove large zooplankton and then split
into two equal (250 ml) aliquots. One aliquot was filtered
directly on a GF/F filter, while the other was prefiltered
through a 8-Frn nylon mesh to remove larger cells and then
filtered on a GF/F filter.
Abundance of benthic suspension feeders at the reef-The
coral-reef community at our study sites in Eilat was surveyed in order to estimate the densities of the main suspension feeders that are either known to feed on phytoplankton
or belong to taxonomic groups that include species that feed
on phytoplankton in other habitats. The taxa recorded included sponges, bivalves, ascidians, and tunicates but did not
include bryozoans and polychaetes. Three benthic transects,
one at each of the 4-, 5-, and 6-m isobaths, were made near
each of the 0 mab points of the above-described north and
south cross-shore transects. Each benthic transect consisted
of 11 0.5 X 0.5-m quadrats, positioned 0.5 m apart by scuba
divers. A total of 65 quadrats were surveyed. Visual counts
of the above taxa were made in each quadrat.
Current measurements-Because
currents can affect both
the feeding rate of suspension-feeders (Lenihan et al. 1996)
and the mixing of depleted and affluent layers, 36 of the 38
cross-shore transects were complemented with current measurements. An electromagnetic current meter (model S4,
InterOcean) was deployed on the slope in the vicinity of the
transects (<500 m distant). The current meter was attached
on a mooring line usually at 12-m depth, except from February and June 1994 visits where the instrument depth was
5 m. The current meter was set to record a l-min average
vector every 10 min for a duration of at least 2 h (but usually
several days) prior to sampling. The recorded data were used
to calculate the average advection vector for the 1, 2, 4, and
6 h preceding the sampling time of each cross-shore transect.
These vectors, together with the magnitude of their crossand long-shore components, their scalar values, as well as
the instantaneous current speed during the transect sampling,
were examined for possible correspondence with the strength
of the respective cross-shore chlorophyll and pheopigment
gradients (see below).
Statistical analysis--The
significance levels of crossshore trends were tested using the “Page test” for ordered
alternatives (Siegel and Castellan 1988). This nonparametric
test is a modified version of the Kruskal-Wallis
one-way
ANOVA of ranked data. Hence, H, for chlorophyll was an
occurrence of a gradient of decreasing concentrations from
the open water (25 mab) toward the reef (0 mab), whereas
H, for pheopigment was the occurrence of an opposite gradient.
A multiple linear regression analysis was used to examine
the relationships between the various flow parameters (see
above) and the strength of the cross-shore gradient. This
strength was operationally defined as the difference, in percent, between the pigment concentration at the reef (0 mab)
and that in the open waters (25 mab). A complete residuals
analysis was performed to validate the robustness of the resulting model.
555
A comparison of the square-root-transformed Chl : pheopigment ratio at the different sampling sites in Eilat was
done using a repeated-measures ANOVA. The open-water
(25 mab) and shorewardmost (0 mab) values within a transect were considered repeated measures and their statistical
effect was crossed with the site effect (coral reef vs. sand
site). Because the interaction between site and transects was
highly significant (see results), a Scheffe post hoc test was
used to examine the difference between the reef and the
sandy site and between the 0 and 25 mab points within each
of the two sites. Homogeneity of variance and normality
were verified by Co&ran and Kolmogorov-Smirnov
tests,
respectively.
The occurrence of significant phytoplankton depletion in
waters passing through the perforated reef was tested by the
Wilcoxon signed-ranks test on pairs of upstream and downstream samples. The overall difference was tested using the
mean of the normalized t statistics of the Wilcoxon test
(W,, of Frechette and Bourget 1985).
Except for the manual calculations of the Page test, statistical analyses were done using STATISTICA for Windows
(vers. 5.0, 1995, StatSoft).
Results
Cross-shore patterns-A
significant gradient (P < 0.01,
Page test) of decreasing Chl a concentrations from the open
waters to the reef was observed during 6 of the 10 visits to
the study sites in Eilat, while an opposite gradient, consisting
of a reef-ward increase in pheopigment concentrations, was
observed during seven visits (Table 2). Opposite gradients
(increasing chlorophyll or decreasing pheopigment toward
the reef) were not observed. The gradients of chlorophyll
and pheopigment at Hibik and Ras Abu Galum were similar
to those in the reef of Eilat, but the chlorophyll gradient at
the former site was not significant (Fig. 3). The steepest
sections of the gradients usually occurred within 10 m from
the fore-reef, l-3 mab. Chl a concentrations in this depleted
layer were lo-69% (21-115 ng liter ~I) lower than those in
the offshore waters (25 mab). An average cross-shore transect (Fig. 4) based on all 38 reef transects, exhibited a clear
18.6% (SE 3.1%) decrease in Chl a and 73.1% increase (SE
17.0%) in pheopigments from the open water toward the
reef. A decrease in the ratio of Chl a to pheopigment from
the open water toward the reef was observed in all but a
single visit (December 1994), with values near the reef being
2-4 times higher than in the offshore region (Table 3).
The repetitive sampling in February 1996 revealed no diel
changes in cross-shore patterns of chlorophyll (Fig. 5A),
while the daily replications in February and June 1994 exhibited a persistence of the cross-shore patterns over periods
of 3-5 d (Fig. 5B,C).
No cross-shore gradients were observed in summers 1994
and 1995, when the ambient concentrations of Chl a were
low (although a gradient was observed in similar conditions
during summer 1996; cf. Figs. 3 and 5C) and in December
1994. The occurrence of significant gradients of chlorophyll
did not correspond with the occurrence of significant pheopigment gradients (Table 2). Of all the flow parameters ex-
556
Yahel et al.
Table 2. Average (SD) concentrations of chlorophyll a, chlorophyll : pheopigment (Chl : Pheo.) ratios, and percentage difference of the
concentration of pigments between the open water and the coast at the coral reef and the sand site in Eilat. “Open water” and “coast”
indicate the distantmost (25 mab) and the shorewatdmost (0 mab) sampling points in each cross-shoretransect, respectively. Concentration
difference is calculated by subtracting coast values from those in the open water. P values are for the Page test (testing for the occurrence
of a cross-shore gradient), where H, was a shoreward decreasefor chlorophyll and an increase for those in the pheopigment. For the sand
site, nonsignificant P values were also observed for the opposite H, (a shoreward increase of chlorophyll) (N, number of transects; ns, no
significant gradient; *, P 5 0.05; **, P 5 0.01; ***, P 5 0.001).
Chl a (pg liter-l)
Site
Month
n
Open water
Reef
Feb 94
Jun 94
Aug 94
Dee 94
Feb 95
Jun 95
Aug 95
Dee 95
Feb 96
Aug 96
4
8
2
2
4
2
2
4
4
2
0.212(0.017)
0.148(0.019)
O.lZS(O.003)
0.522(0.019)
0.284(0.017)
0.208(0.011)
Feb 94
Jun 94
Aug 94
Dee 94
Feb 95
Jun 95
Aug 95
3
6
2
2
2
2
2
Sand
Coast
Chl : Pheo. ratio
Open water
Coast
0.165(0.040)
1.90(0.20)
0.147(0.021)
0.123(0.006)
3.24(0.79)
3.66(0.04)
2.38(0.29)
1.42(0.36)
2.06(0.60)
1.36(0.25)
2.50(0.65)
0.508(0.000)
0.231(0.032)
0.134(0.021)
1.63(0.28)
5.38(1.31)
0.087(0.006)
0.092(0.012)
4.34(0.90)
0.295(0.010)
0.206(0.011)
0.161(0.026)
0.199(0.019)
0.171(0.062)
1.99(0.36)
0.189(0.000)
0.124(0.036)
0.170(0.048)
0.216(0.090)
0.137(0.045)
0.099(0.003)
0.502(0.009)
0.293(0.002)
0.227(0.039)
0.495(0.000)
0.334(0.057)
0.178(0.000)
0.178(0.000)
O.OSS(O.003)
0.082(0.008)
2.77(0.38)
4.88(1.36)
2.14(0.00)
3.33(0.42)
3.86(0.75)
2.49(0.03)
1.78(0.06)
3.35(0.00)
3.49(2.19)
amined (see methods), the net advection of water during the
2 h preceding the transect had the highest (negative) correlation value with the strength of the cross-shore gradients of
Chl a, but not with the strength of pheopigment gradients
(rp = -0.456, P < 0.05 for chlorophyll and r[, = 0.269, P
> 0.15 for pheopigment). Temporal variations in the strength
of the cross-shore chlorophyll gradients at the reef could be
best explained by the above advection term and the concentration of Chl a in the ambient (offshore) waters (through
multiple linear regression) as follows: Chlorophyll depletion
(%) = 0.239 - 2.412A + 0.972C (multiple-adjusted R2 =
0.502, F2.28= 16.106, P < 0.0001) where A is the advection
distance (m) during the 2 h preceding the transect sampling
and C is the chlorophyll concentration (pug liter’) at the 25
mab sampling point of the transect.
No cross-shore gradients were observed at the sandy site
(P > 0.1, Page test, n = 19), where the concentrations of
both Chl a and pheopigment near the shore were either similar or higher than those found in the open water (Table 2,
Fig. 4). Marginally significant higher concentrations of chlorophyll were found at the nearshore sampling points compared with the open water (Fig. 4). The Chl: pheopigment
ratios at 0 mab at the sandy site were similar to those found
at 25 mab, or 2-4 times higher than the values found at the
coral reef on the same sampling dates (Table 3, Fig. 6).
More than 80% of the total Chl a measured with GF/F
filters in the upper waters (0-20-m depth) in the Gulf of
Aqaba was contributed by ultraphytoplankton (cell size <8
pm; Table 4). Direct cell counts with a flow cytometer
showed that the observed cross-shore gradients in chlorophyll reflected a decrease in both Synechococcus and eukaryotes (Fig. 7). Numerically, Synechococcus constituted
1.23(0.27)
1.04(0.33)
1.68(0.09)
1.21(0.24)
1.84(0.45)
1.34(0.44)
1.97(0.09)
3.08(0.35)
3.51(1.65)
2.38(0.12)
2.08(0.24)
3.71(1.63)
3.87(0.05)
Chl difference
%
-27(19)
- W)
-2(7)
-3(4)
-19(12)
-36(14)
5(7)
-32(7)
-22(7)
-47(24)
l(18)
lO(10)
43(63)
-1m
14(19)
O(O)
43(21)
P
***
ns
ns
ns
***
***
ns
***
**
***
ns
ns
ns
ns
ns
ns
ns
Pheo. difference
%
8(15)
70(76)
71(67)
-3(33)
lO(26)
235(47)
170(59)
14(21)
7(66)
147(147)
25(28)
20(17)
57(26)
36)
-3(l)
31(O)
22(60)
P
*
***
**
ns
ns
***
*
**
ns
**
ns
ns
ns
ns
ns
ns
ns
77-94% of the removed phytoplankton cells. In winter, however, eukaryotes contributed >71% to the carbon depletion
near the reef while in summer their share was <36%. Prochlorococcus was present but could not be enumerated with
confidence in all but a single transect, where its decreasing
trend was similar to that of the other two groups of ultraphytoplankton (data not shown).
Water parcels-Chl
a and pheopigment concentrations remained unchanged along the 25-70 min (800-50 m) tracks
of the five drifter drogues released in this study, including
situations with weak (< 1.5 cm s’) and strong (>20 cm s ‘)
currents. Pigments concentrations were similar to those
found at l-3 mab in corresponding cross-shore transects.
Conversely, water parcels passing through the perforated
reef were significantly depleted of Chl a (Wilcoxon sign
ranks test, P < O.Ol), with a mean removal efficiency of
21.5% (SE 3.0%), equivalent to a mean removal of 61 (SE
9) ng Chl a per liter of water passing through the 5-m-long
section of the reef (Table 5). Considering a cross section of
1 m2 (5 m3) and a passage time of 40-80 s, the removal
rates ranged from 2.29 to 8.67 pg Chl a s I. Surprisingly,
the concentrations of pheopigment did not significantly
change during the passage through the perforated reef (Table
5).
Counts of Synechococcus and eukaryotes in 10 pairs of
samples taken in June 1995 indicated that the removal of
these two groups was nonselective and well represented by
values of Chl a removal (Table 6; Spearman rank correlation
between removal of ultraphytoplankton
and chlorophyll, r,
= 0.833, n = 8).
The most abundant suspension-feeders in the coral reef of
Phytoplankton
557
grazing in coral ree$y
sand
***
t
TM1
1
-25
:Ii
**
0.0 II
0
5
10
15
20
-El-
-50
25
0
5
Height above bottom (m)
Fig. 3. The cross-shore profiles of chlorophyll a (solid symbols)
and pheopigments (open symbols) in August 1996 measured in transects across the coral reefs of Eilat (A), Hibik (B), and Ras Abu
Galum (C). Triangles are used for northern transects, circles for the
southern transect at each site (ns, nonsignificant; **, P 5 0.01; ***,
P 5 0.001).
Eilat were the coral-boring mussels (Lithophaga spp.),
sponges (including members of the genera Mycale and
Cliona), benthic tunicates, and ascidians (Table 7). Octocorals were rare, and none was included in our quadrats. The
densities of each of the 15 taxa surveyed did not differ significantly between the north and south sections of the reef
in Eilat (Mann-Whitney
U-test, n = 32, 33, P > 0.1).
Discussion
Phytoplankton was effectively removed by the benthic
community at the coral reefs. A phytoplankton-depleted layer, -3 m in thickness, was commonly found above the reef
slope. At the perforated reef (an arborescent community),
-20% of the phytoplankton was removed during the -lmin passage of water through a 5-m-long section of the reef.
In terms of biomass, the phytoplankton deficiency and removal reported in this study refers mostly to small cells (<8
w).
Pheopigment
10
15
20
25
Height above bottom (m)
Fig. 4. The average difference in pigment concentration between each sampling point and the open water (25 mab) at the same
transect, calculated for all the transects carried out at the sand site
(upper panel, n = 19) and at three coral reefs (lower panel, n =
38). Solid symbols indicate chlorophyll a; open symbols indicate
pheopigment; error bars indicate 95% confidence interval.
Table 3. The average (SE) contribution (%) of pheopigment to
the total pigments (Chl a + pheopigment) measured at the openwater and the shorewardmost (coast) points (25 and 0 mab, respectively) for all cross-shore transects made during our study. ANOVA
of the square root-transformed pheopigment to chlorophyll ratios
showed a highly significant interaction between the two study sites
and within the two extreme sampling points of each transect (F,,,,
= 12.43, P < 0.0001). P values are for a Scheffe post hoc test,
which measured the difference between the open water and shoreward samples (in the rows) and between the coral reef and the sandy
site (in the columns) (ns, nonsignificant; ***, P < 0.001).
n
Coral reef
Sand bottom
P
Open water,
% (SE)
Coast,
% (SE)
34
26.3(1.3)
40.7( 1.4)
16
26.1(1.3)
ns
26.7(1.3)
***
P
***
ns
Yahel et al.
558
A
0.2 --
0.1
I
I
I
I
II
i
q
10
20
30
40
Percent pheopigment
50
60
70
at 0 mab
Fig. 6. Differences in the concentration of chlorophyll a between the open water (25 mab) and the shorewardmost (0 mab)
points vs. percentage of pheopigment at the 0 mab point for all
cross-shore transects. The percentage of pheopigment was the concentration of pheopigment divided by the total measured pigments
(Chl a + pheopigment).
0.1 I
0
5
10
15
20
25
Height above bottom (m)
Fig. 5. Short-term variations in the cross-shore distribution of
chlorophyll a along the coral reef of Eilat. A. Comparison between
a nocturnal (filled symbols) and diurnal (open symbols) transects in
February 1996. Triangles are used for the northern transects, circles
for the southern transect. B. Replications performed daily during 4
consecutive days at the southern transect in February 1994. C. Replications performed daily during 3 consecutive days at the southern
transect in June 1994.
The role of benthic grazing in the observed phytoplankton
removal was obvious at the perforated reef. Because this reef
was inhabited by massive colonies of soft corals reported as
phytoplankton grazers (Fabricius et al. 1995a,b, 1998), high
rates of phytoplankton
removal
were expected.
However,
the
occurrence of a well-defined depleted layer over the fringing
reef slopes was surprising, as soft corals at those reefs were
rare and the local communities were dominated by hermatypic corals, which are not known to be phytoplanktivorous.
The confinement of the depleted layer to the nearest l-3 m
above bottom, together with the lack of grazing at the sandy
site, indicates that the key grazers responsible for the observed depletion were inherent members of the benthic-reef
community. The reason for the slight increase of phytoplankton concentration near the sand bottom is not yet known.
Bottom-associated, yet water-borne grazers (e.g. demersal
zooplankton), some of which are phytoplanktivores (Roman
et al. 1990), could also contribute to phytoplankton depletion
near the reef. However, their contribution was expected to
be most conspicuous during night, when the waters overlying coral reefs are replete with demersal plankton (Alldredge
and Ring 1977, 1985; R. Yahel unpubl. obs.). The lack of
diel changes in chlorophyll gradients across the reef (Fig.
5A) suggested an insignificant contribution of the grazing by
demersal plankton. This conclusion is further corroborated
(for daytime) by the absence of phytoplankton depletion in
the drogue experiments-the
drogue was suspended l-3
mab so that the water sampled along its path had not been
Table 4. The average (SD) concentration of chlorophyll a retained on GF/F filters and the contribution (%) of ultraphytoplankton to those chlorophyll values in seawater collected at 0-20-m
depth at the five oceanographic stations in the Gulf of Aqaba (see
Fig. 1). Ultraphytoplankton is defined as cells passing through a 8pm mesh (n, number of replications).
Cruise
date
Jun 94
Mar95
May 95
Jun 95
Aug 95
Total
Station
n
Chlorophyll
(pg liter ‘)
A, B, M, F
A
A, B, M
A, B,M, E S
A
7
2
6
7
2
24
0.131(0.067)
0.870(0.052)
0.069(0.036)
0.046(0.015)
0.055(0.007)
0.160(0.239)
Ultraphytoplankton,
% (SD)
87.2(27.6)
83.3(4.7)
91.1(22.9)
85.4(9.1)
81.7(2.4)
86.9(18.5)
110
100
!
Phytoplankton
grazing in coral reej%
80
s
g)
70
8
6
60
s.
3
2
z
I
0
I
1 0
June 1995
1
0
110
I
I
,
Eukaryotes
100
90
80
70
60
0
0
I
1
with the benthos, except via turbulent mixing
(Shashar et al. 1996).
The taxa responsible for the depletion of phytoplankton
at the reef were most likely the coral-boring Lithophaga
spp., sponges, and ascidians. We are currently studying in
situ feeding by individual specimens, and find efficient
(>60%) grazing rates on eukaryotic phytoplankton, Prochlorococcus, and Synechococcus by the bivalves Lithophaga
simplex, Lithophaga purpurea, the sponges Cliona mussae,
Mycale jistulifera, Subarites clavtus, and the ascidians Halocynthia gangelion and Didemnum candidum (G. Yahel unpubl. data). Meroz and Ilan (1995) reported densities of the
sponge M. jistulifera near our site in Eilat similar to those
observed in our survey (Table 7). Although the densities
reported in Table 7 are high, for some infauna species those
values should be regarded as gross underestimates. The
cryptic nature of many suspension-feeders, some of which
are dwelling within corals and rocks, precluded accurate visual counts of all the specimens present in a quadrat (Hutchings 1983). For example, in the case of the boring mussel
Lithophaga lessepsiana in branches of the coral Stylophora
pistillata, visual counts made as above were -10 times lower than complete counts made after the corals were brought
to the laboratory, broken apart, and fully inspected (G. Yahel
pers. obs.). Because the information on densities of suspension feeders was not a principal objective of this study, we
chose to avoid damaging corals during our survey.
Whereas phytoplankton removal was observed at the perforated reef during all visits, a cross-shore chlorophyll gradient at the slope reef was observed in most, but not all,
in contact
Synechococcus
90
g
559
n
0
I
I
I
I
,
I
0
5
10
15
20
25
Height above bottom (m)
Fig. 7. Cross-shore distribution of ultraphytoplankton based on
cell counts in the coral reef of Eilat from three different seasons.
Owing to substantial seasonal differences in the absolute densities
of these taxa, the data are presented as a percentage relative to the
density in an open-water (25 mab) sample in a transect. Absolute
densities ranged from 800 to 8,000 cells ml-’ for eukaryotic phytoplankton and 8,000 to 30,000 cells ml-’ for Synechococcus. Each
point represents a seasonal average from both northern and southern
transects. Full lines indicate averages over all seasons.
transects. The variations
in the cross-shore
gradients
can best
be explained by the linear regression model (see results),
suggesting significant contributions by both biology (positive correlation with the ambient concentration of chlorophyll) and physics (negative correlation with water advection). The biological term was related to the fact that three
of the four visits with no chlorophyll gradients occurred in
summer (Table 2), a season characterized by low Chl a concentrations
(Genin
et al. 1995) and a dominance
of small
prokaryotic taxa (Lindell and Post 1995). Thus, this parameter in the above model may, in fact, reflect some seasonality, either in phytoplankton,
their grazers, or even in some
Table 5. Average (SE) changes in the concentrations of chlorophyll a and pheopigments after passing through a 5-m-long section of
the perforated reef. Passage time is a bulk measure of the flow speed through that section. Negative and positive values indicate a decrease
and increase in the pigment concentrations, respectively. P values are for a pairwise Wilcoxon sign rank test of the difference in pigments
concentrations between the upstream and downstream samples (ns, no significant difference; *, P < 0.05; **, P < 0.01; n, number of
sample pairs).
Change in pheopigments
Change in Chl a
Passage
time (s)
Dee 94
Feb 95
Jun 95
Aug 95
Dee 95
Nov 96
Mean
60
60
80
70
40
60
62
n
9
%
8
8
-23.1(7.7)
-23.4(16.4)
-65(47)
-79(26)
-48(17)
-32(12)
11
-21.9(8.1)
-37(13)
9
-36.3(2.2)
-104(g)
56
-21.5(30)
-61(9)
11
-10.1(8.8)
Absolute
(ng liter-‘)
- 15.5(5.0)
P
ns
**
**
*
**
**
**
%
7.7(14.1)
-5.0(3.3)
35.9(21.4)
7(7.3)
-20.4(5.0)
-1.3(1.9)
1.2(3.9)
Absolute
(ng liter ‘)
318)
-12(7)
28(18)
W)
-68(17)
-xv
-13(6)
P
ns
ns
ns
ns
*
ns
ns
560
Yahel et al.
Table 6. Average (SE) densities of Synechococcus and eukaryotic phytoplankton and the corresponding concentrations of chlorophyll u
in the upstream samples (ambient value) and the decrease in those values after the passage of the water through the perforated reef in the
June 1995 experiment. Cell numbers, measured with a flow cytometer, were converted to carbon values as in Campbell et al. (1994). A
third group of ultraphytoplankton, the Prochlorococcus, was abundant at that time but for technical reasons could not be counted. Thus,
values of carbon removal do not cover the total contribution of ultrphytoplankton (n, number of pairs; *, P 5 0.05; **, P 5 0.01).
Synechococcus (no. ml ml)
Picoeukaryotes (no. ml- I)
Chlorophyll (pg liter-l)
n
Ambient value
Decrease
10
10
8
6,533(327)
935( 16)
0.209(0.006)
1,760(596)
260(28)
0.048(0.017)
seasonal abiotic factor(s), rather than the chlorophyll
concentration as such. Note, however, that a lack of gradient
was also observed in December
1994, when the ambient
chlorophyll
concentration
was high (Table 2). This irregular
observation could be related to the exceptionally
rough conditions caused by a strong southerly storm that occurred a
day before our visit. This storm caused massive resuspension
of sediment and could have mixed the phytoplankton-depleted waters near the bottom with effluent waters aloft. The
physical term in the above model likely reflected a similar
effect, namely, an enhanced water mixing and the obstruction of a defined depleted layer. Our model, however, is
based on single-point
measurements of currents, sometimes
made a few hundred meters away from the location of a
transect. A much more comprehensive
investigation
of the
current regime should be carried out in order to better explain the effect of water physics on temporal variations in
the distribution
of phytoplankton
near the reef.
The observed phytoplankton
depletion
encompassed
a
wide range of cell sizes (0.5-5 pm> with no apparent selec-
Fraction
removed (%)
Carbon removed
(pg liter I)
2%‘)
0.44(0.05)
0.55(0.06)
28(3)
23(8)
P
**
**
*
tivity either at the fringing reef, where a diverse guild of
active suspension-feeders
was found, or at the perforated
reef, where the benthic community
was dominated by passive filter-feeders
(soft corals). It is yet unknown whether
this nonselectivity,
documented here on the community
level, reflected a similarly
nonselective
feeding by individual
phytoplanktivores.
The depletion of cells in the range of 1 pm in size suggests that heterotrophic
bacteria may also be readily eaten
at the reef. Because heterotrophic
bacteria are extremely
abundant in tropical waters, with a carbon pool similar in
magnitude to that of phytoplankton
(Campbell et al. 1994),
their trophic contribution
to the coral reef deserves careful
attention. Ayukai’s
(1995) recent observation
(see Table 8)
showed that the depletion of heterotrophic
bacteria downstream of his study reefs in Australia were similar to those
of phytoplankton.
A third group of small cells that may turn
out to be an important source of carbon for the reef community, but has so far been overlooked,
is Prochlorococcus.
In the Gulf of Aqaba this is the dominant phytoplanktonic
Table 7. Average (SE) and maximum densities of suspension-feeders that are likely phytoplankton grazers (see Discussion) in the coral
reef of Eilat (4-6-m depth). Densities are reported per quadrat size (0.25 mZ). Percent occurrence refers to the number of quadrats (of a
total of 65) in which at least one specimen was found. For the boring mussel Lithophaga, the name of the host coral is given. The bottom
three rows list the average (SE) percent cover of living organisms (mostly stony corals), bare rocks (rocky substrate with no visible
invertebrates), and sand.
Avg density
Taxon
Lithophaga simplex (in Goniastrea spp.)
Lithophaga purpurea (in Montipora erythruea)
L. purpurea (in Cyphastrea spp.)
Lithophaga lessepsiana (in Stylophora spp.)
Lithophagu spp. in other corals
Mycale jistulifera (red sponge)
Cliona spp. (boring sponges)
Unrecognized blue sponge
Other sponges
Halocynthia gangelion (solitary ascidian)
Other solitary tunicates
Didemnum candidum (colonial ascidian)
Other colonial tunicates
Tridacna spp. (Giant Clam)
Pedum sp. (medium-size bivalve)
% live cover
% bare rock
% sand
Max density
No. 0.25 mm*
1.06(0.47)
1.23(0.65)
1.80(0.41)
l.OO(O.59)
0.32(0.20)
O.lZ(O.05)
0.38(0.09)
0.94(0.15)
0.3 l(O.09)
O.OS(O.03)
0.46(0.11)
0.12(0.06)
0.06(0.04)
0.03(0.02)
0.25(0.10)
40(3)
‘WV
12(3)
% occurrence
19
40
14
37
11
2
3
5
3
1
3
3
2
1
5
100
95
100
15.4
13.8
30.8
10.8
7.7
9.2
24.6
50.8
18.5
7.7
26.2
9.2
4.6
3.1
12.3
Phytoplankton
grazing in coral reej%
561
Table 8. Comparison of reported carbon contributions to coral reefs via grazing on different types of planktonic organisms. All values
were calculated similarly to ours, based on a comparison of particle concentrations upstream and downstream of a coral reef. Our measurements and the original values of Ayukai (1995) and Hamner et al. (1988) were converted to g C m z yr I. A conservative conversion
factor of 1 : 30 for the Chl : C ratio was used (Ayukai 1995) although our measurements suggested a factor >60 for our study sites (Yahel
unpubl. data).
Organisms
Phytoplankton
Bacteria
Protozoa
Microzooplankton
Zooplankton
group during summer-fall (Lindell and Post 1995). Both our
single transect that included Prochlorococcus
(February
1996) and the few transects that included heterotrophic bacteria counts (August 1996) showed that the depletion values
for both groups were similar to that of ultraphytoplankton.
Pile (1997) and our recent study on individual grazing (G.
Yahel unpubl. data) indicated high grazing on heterotrophic
bacteria and Prochlorococcus by sponges and Lithophaga
SPP.
Reference
Flux (g C me2 yr- ‘)
4.2-20.2
413.6 (<lo0 pm, D. hemprichi thicket)
719.1 (<lo0 pm, perforated reef)
3.9 (diatoms >68 pm)
9.2-10.2
1.4-6.3
0.3 l-O.52
116.8 (>330 or 200 pm, night only)
61.8 (>68 pm)
197.1 (>60 pm)
2.8 (>250 pm, only at day, owing to predation by reef fishes)
A significant elevation of pheopigment concentrations was
observed at the fringing reefs. In general, phytoplankton
grazing increases the concentrations of pheopigments (e.g.
Shuman and Lorenzen 1975) and such an increase at the
Great Barrier Reef was attributed to grazing by reef-associated zooplankton (Roman et al. 1990). In our study, higher
values of chlorophyll depletion near the reef were usually
associated with lower values of the Chl : pheopigments ratio
at the 0 mab samples (Fig. 6), suggesting that phytoplankton
grazing was (at least partly) responsible for the elevated levels of pheopigments at the reef. However, the gradients of
pheopigments across the reef did not always coincide with
decreasing gradients of chlorophyll. In 5 of our 10 visits to
the reef, a significant gradient of chlorophyll co-occurred
with a nonsignificant trend of pheopigments or vice versa
(Table 2). Note that in all but one visit the Chl : pheopigment
ratio was lower near the reef than in the open waters (Table
2). A likely cause for this partial decoupling between phytoplankton chlorophyll and pheopigments is the occurrence
of an additional source of pheopigments at the reef, namely,
grazing on benthic algae by fish, echinoids, and gastropods.
In particular, herbivorous fish, discharging their feces aloft,
could have been an additional source for suspended pheopigments in the water column over the reef.
The lack of pheopigment increase downstream of the perforated reef is not well understood. Soft corals may transform the ingested chlorophyll
into nonfluorescent compounds or, as with other benthic anthozoans (Sebens and
Koehl 1984), they may egest packed material that rapidly
sinks to the bottom. A similar lack of pheopigment increase
was observed in our past experiments with the soft coral D.
Ayukai 1995
Fabricius et al. 1998
This study
Glynn 1973
Ayukai 1995
Ayukai 1995
Ayukai 199 1
Tranter and George 1969
Glynn 1973
Johannes and Gerber 1974
Hamner et al. 1988
hemprichi (Fabricius et al. 1998), a dominant taxon at the
perforated reef.
Traditionally, investigators of planktivory in coral reefs
referred to zooplankton rather than phytoplankton as the
principal source of prey (Table 8). Historical perspective,
together with the fact that stony corals are exclusively zooplanktivorous, may explain why phytoplankton has been
overlooked. Glynn (1973), in a seminal study that for years
set the stage for our understanding of planktivory in coral
reefs, suggested that the import of carbon to the reef via
zooplankton predation surpasses that of phytoplankton removal by more than an order of magnitude. Glynn’s estimates of phytoplankton removal by the reef were 3.9 g C
mm2 yr ‘, more than two orders of magnitude lower than
those reported here for the perforated reef. An evaluation of
such earlier studies, however, should consider the fact that
at the time of Glynn’s study neither epifluorescence microscopy nor flow cytometers were available for marine ecologists. Not being aware of the great importance of small cells
in warm-water oceans (Furnas and Mitchell 1987; Chisholm
1992), Glynn (1973) used a 68-pm mesh net to sample phytoplankton, totally missing ultraphytoplankton.
The occurrence of a concentration boundary layer (Monismith et al. 1990) depleted of phytoplankton is a well-documented characteristic of mussel and polychaete beds in the
temperate bays and estuaries (Frechette and Bourget 1985,
1987; Frechette et al. 1989; Asmus and Asmus 1991; Riisgard et al. 1996). Our findings, together with Ayukai’s
(1995) report on the strong depletion of Synechococcus hundreds of meters downstream of two Australian reefs, suggest
that benthic grazing on ultraphytoplankton may be a ubiquitous characteristic of coral reefs as well. Future studies of
carbon and nutrient fluxes in coral-reef communities should
consider phytoplanktivory
an important allochthonous
source of food to the reef community.
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Received: 20 February 1997
Accepted: 1 October 1997
Amended: 26 October 1997
Chap. 6. Phytoplankton grazing in reef rocks
4.
In situ feeding and element removal in the coral-reef sponge
Theonella swinhoei: Bulk DOC is the major source for carbon
By
Yahel G., J.H. Sharp, D. Marie, C. Häse, and A. Genin
2002
Limnology and Oceanography 48, 141-159
28
Limnol. Oceanogr., 48(1), 2003, 141–149
q 2003, by the American Society of Limnology and Oceanography, Inc.
In situ feeding and element removal in the symbiont-bearing sponge Theonella
swinhoei: Bulk DOC is the major source for carbon
Gitai Yahel 1
Interuniversity Institute for Marine Sciences of Eilat and The Hebrew University of Jerusalem, P.O. Box 469, Eilat 88103,
Israel
Jonathan H. Sharp
Graduate College of Marine Studies, University of Delaware, Lewes, Delaware 19958
Dominique Marie
Station Biologique, CNRS, INSU et Université Pierre et Marie Curie, BP 74, Roscoff Cx 29682, France
Clivia Häse 2
Interuniversity Institute for Marine Sciences of Eilat and The Hebrew University of Jerusalem, P.O. Box 469, Eilat 88103,
Israel; Center of Tropical Marine Research, Bremen, Germany
Amatzia Genin
Interuniversity Institute for Marine Sciences of Eilat and The Hebrew University of Jerusalem, P.O. Box 469, Eilat 88103,
Israel
Abstract
The vast majority of organic matter in the world ocean is found in the dissolved pool. However, no evidence has
been demonstrated for direct uptake of bulk dissolved organic matter (DOM) by organisms other than bacteria and
some invertebrate larvae. The total organic carbon (TOC) is 10–30% higher in coral reefs than in adjacent open
waters. The dissolved organic carbon (DOC) accounts for .90% of the TOC. Using a new in situ technique for
clean sampling of the seawater inhaled and exhaled by benthic suspension feeders, we measured directly the removal
of DOC in the symbiont-bearing reef sponge Theonella swinhoei. The sponge removed up to 26% (mean 6 SD:
12% 6 8%) of the TOC (dissolved and particulate) from the water it filtered during a single passage through its
filtration system. Most of the carbon gained by the sponge was from the dissolved pool (10 6 7 mmol C L21), an
order of magnitude greater than the carbon gained from the total living cells (phytoplankton and bacteria) the sponge
removed (2 6 1 mmol C L21). In T. swinhoei, over two-thirds of the sponge biomass consists of symbiotic bacteria,
which likely play an important role in DOC uptake. Our findings indicate that the role of DOC in metazoan nutrition
and the role of metazoans in DOC cycling may have been grossly underestimated.
The total organic carbon (TOC) in the ocean is divided
into two major compartments: particulate (POC) and dis-
solved (DOC). The dissolved pool is operationally defined
as the organic carbon passing through a fine filter, typically
GF/F (Benner 2002; Carlson 2002). The particulate fraction
consists of larger living organisms and detrital particles. The
vast majority (.97%) of the organic carbon pool in seawater
resides in the dissolved phase (Benner 2002). Only a small
fraction of the dissolved pool is labile; the rest is thought to
be refractory and unavailable for utilization by marine organisms (Carlson 2002).
A role for DOC in the nutrition of aquatic metazoans has
been repeatedly proposed since the turn of the last century
(Pütter 1909; Jørgensen 1976). DOC was recently suggested
to explain the observed discrepancies between the supply
and demand of carbon in benthic suspension feeders (Reiswig 1974, 1981; Gili and Coma 1998) and deep-sea softbottom communities (Smith and Kaufmann 1999). However,
except for larvae of soft-bodied invertebrates (Wright and
Manahan 1989), direct evidence of metazoan utilization of
bulk DOC in aquatic habitats is lacking. Thus, in sharp contrast to the well-established role of DOC in the microbial
loop of pelagic food webs (Carlson 2002), the role of DOC
Corresponding author ([email protected]).
Present address: German Center for Aerospace Research, Oberpfaffenhofen, PF 1116, D-82230 Wessling, Germany.
1
2
Acknowledgments
We thank R. Yahel, I. Ayalon, M. Ohevia, R. and C. Wyeth, B.
Munkes, R. Motro, S. Eckstein, A. and G. Brandes, Y. Shif, D. Weil,
A. Inditzky, N. Cohen, E. Hazan, T. Cohen, L. Gutman, I. Meallem,
K. B. Savidge, R. Andrews, D. E. Canfield, and K. Rinker for help
in the field and laboratory, the Interuniversity Institute staff for logistic support, B. Lazar, M. Ilan, D. Lindell, A. Post, M. Kiflawi,
R. P. M. Bak, D. Vaulot, K. L. Smith, Jr., L. Haury, and G. C.
Stephens for comments on earlier versions, M. Birkicht (Center of
Tropical Marine Research) for the POC analysis, and the Rieger
Foundation and E. and A. Reshef for supporting G.Y. This study
was funded by grants from the U.S.-Israel Binational Science Foundations, the Israel Science Foundation, and the German Ministry of
Science, Technology and Education through the Red Sea Program.
The flow cytometry analysis was funded by the French program
PROSOPE and the European program PROMOLEC. The DOC
analysis was partially funded by U.S. National Science Foundation.
141
142
Yahel et al.
in the nutrition of benthic animals is still unresolved (Wright
and Manahan 1989; Thomas 1997).
Numerous laboratory experiments have confirmed the
ability of soft-bodied metazoans, including sponges (Jaeckle
1995), to take up specific components from the dissolved
pool (reviewed by Johannes et al. 1969; Jørgensen 1976;
Stephens 1988; Wright and Manahan 1989; Thomas 1997;
Ambariyanto and Hoegh Guldberg 1999; Roditi et al. 2000).
However, the uptake of components other than dissolved
glucose and free amino acids has received little attention
(Wright and Manahan 1989; Thomas 1997). Because glucose
and amino acids are found in trace concentrations in natural
aquatic habitats, their removal is generally considered energetically insignificant (e.g., Ambariyanto and Hoegh Guldberg 1999). The very few laboratory studies that included
naturally occurring DOC produced contradictory conclusions (e.g., Alber & Valiela 1995). Reiswig’s (1985) attempt
to quantify DOC feeding in situ with boreal glass-sponges
was limited by a small data set and a variance too high to
draw clear conclusions. More recently, Ribes et al. (1998,
1999) used recirculating bell jars to measure in situ the diet
of a Mediterranean sponge and ascidian. Bulk DOC removal
was not observed in either case. We are not aware of any
direct evidence for in situ uptake of bulk DOC by benthic
metazoans.
One reason for the paucity of direct empirical data for the
nutritional role of DOC in benthic communities was the lack
of easy, accurate DOC analyses (Sharp 2002). In the 1990s,
careful reevaluation of methods and utilization of high-temperature combustion methods have resulted in much easier
DOC analyses (Sharp 2002; Sharp et al. 2002). Routine oceanic DOC analyses can now be performed with reproducibility on the 2% level (Sharp 2002).
Coral reefs sustain high levels of recycling of autochthonous carbon and nutrients, driven by the high productivity
of algae and zooxanthellate cnidarians and the unusually
high abundance of closely interacting species. Sources for
DOC in coral reefs are numerous (reviewed by van Duyl
and Gast 2001). Algae excrete high proportions of their photosynthetically assimilated carbon as DOC. ‘‘Sloppy’’ feeding and grazing of benthic algae and animals should cause
DOC leakage from broken prey (Thomas 1997), but most
significant is probably the excretion of DOC by corals, frequently as mucus and free amino acids (Ferrier-Pages et al.
1998). Recently, Van Duyl and Gast (2001) ascribed the 10–
15% DOC elevation they measured near the corals to a labile
fraction excreted by the corals. They suggested that in reef
crevices benthic suspension feeders may consume considerable amounts of this DOC.
Theonella swinhoei (class Demospongiae, order Lithistida,
family Theonellidae) is a common sponge in coral reefs
throughout the Indo-West Pacific. The sponge typically occurs in clusters. The body material is dense, and the barrelshape body has a single osculum at its apex. In the Gulf of
Aqaba, clusters are typically small (usually fewer than six
specimens per cluster), and specimens are usually 5–15 cm
in length and 3–8 cm in diameter. Similar to other dense
tropical sponges (e.g., Verongula sp. [Reiswig 1971] and
Verongia fistularis [Reiswig 1981]), T. swinhoei is actually
a consortium of several symbiotic populations (Bewley et al.
1996; Magnino et al. 1999). Prokaryotic symbionts include
unicellular heterotrophic bacteria (mean concentration, 20%
of the sponge tissue), the unicellular, phototrophic cyanobacterium Aphanocapsa feldmanni (mean concentration,
15% of the sponge tissue), and filamentous bacterial symbionts (mean concentration, 40% of the sponge tissue).
These filamentous bacterial symbionts contain a specific antifungal peptide (theopalauamide) and were recently proposed as a new species Entotheonella palauensis of the dProteobacteria (Schmidt et al. 2000). In addition, large
populations (.50 individuals ml 21) of symbiotic polychaetes
(Syllidae) typically inhabit the water channels of these
sponges (Magnino et al. 1999).
In the present study, we focused on the first step in the
utilization of DOC, its removal from the ambient water. Removal rates of TOC, DOC, and living phytoplankton and
bacteria (hereinafter LvPOC) by the sponge were measured
in situ based on the difference in the concentrations of these
components between the water inhaled and the water exhaled
by the sponge.
Materials and methods
The InEx technique (Yahel et al. unpubl.) was used to
sample in situ the water inhaled and exhaled by the sponge.
A scuba diver sampled the inhaled water by slowly (;0.5
ml s21) withdrawing water into a 32-ml open-ended glass
tube attached at its proximal end to a syringe while holding
its open (intake) end 3–5 mm from the sponge ostia (inhaling
apertures). A sample of exhaled water was taken simultaneously using an identical tube held within the exhalent jet
with the tube’s intake end positioned ;2 mm above the
sponge’s osculum (exhaling aperture), using the excurrent jet
to flush and then fill the tube. Filling time (mean 6 SD: 135
6 43 s) was determined individually for each pair so that it
would last 150% of the time it took the exhalant jet to flush
clear an identical tube prefilled with fluorescein dye (measured a few minutes prior to each sampling). Sampling duration was about 100 times longer than the few seconds it
took the water to pass through the sponge. Thus, each InEx
pair represented an integration of approximately 2 min of
sponge activity (Yahel et al. unpubl. data).
All samples were collected by scuba divers at the Coral
Reef Nature Reserve of Eilat, Israel. For a description of the
study site, see Yahel et al. (1998, 2002). Two sampling sessions were conducted: the first in 1998 at the height of the
summer stratification (10 specimens) and the second in 2000
soon after the termination of spring bloom (20 specimens).
The length and diameter of each sponge was measured to
the nearest 0.5 cm and converted into volume assuming a
cylindrical shape. The osculum diameter was measured to
the nearest millimeter. The average length of the specimens
was 9 6 2 cm, the external diameter was 5 6 2 cm, and the
osculum diameter ranged from 6 to 15 mm (Table 1).
Sample handling was performed within 2 h after each dive
in a clean, open-air glass hood constructed at the seaward
end of a local pier. Only precombusted glassware was used
throughout the experiment, and a new set of pipettes was
used for each sample. A 1-ml aliquot was withdrawn from
Bulk DOC removal by a sponge
143
Table 1. Sampling times, cluster dimensions, and description of the individual specimens studied. Sampling sessions were undertaken
at midday (1000–1200 h).
Cluster
Specimen
Sampling date
and time
ID
No.
specimens
Depth
(m)
15 Sep 98 1045
A
9
5
27 Sep 98 1100
B
4
8
28 Sep 98 1020
C
D
1
4
8
4
02 Oct 98 1030
E
10
13
11 Jul 00 0930†
F
4
5
G
4
5
F
4
5
G
4
5
H
I
3
3
10
10
J
K
8
2
10
14
L
2
14
M
N
O
P
Q
2
2
1
8
2
14
14
6
8
18
R
S
2
2
463
1–10
13
10
964
4–18
12 Jul 00 1000†
24 Jul 00 1000
(17 Dec 00 O2,
18 Dec 00 NH4)
25 Jul 00 1000
26 Jul 00 1000
27 Jul 00 1000
Average 6 SD
Range
ID
A1
A2
A3
B1
B2
C1
D1
D2
E1
E2
F1
F2
G1
G2
F1
F2
G1
G2
H
I1
I2
J
K1
K2
L1
L2
M
N
O
P
Q1
Q2
R
S
Length
(cm)
Diameter
(cm)
8
7
9
10
10
7
7
6
12
11
10
5
12
10
10
5
12
10
10
10
10
6
8
7
6
6
8
10
7
12
10
5
10
10
962
5–12
5
6
3
5
5
5
2
3
4
5
5
4
7
4
5
4
7
4
6
8
8
6
4
6
4
3
6
3
6
8
5
3
5
5
562
2–8
Analyses*
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS, TOC
DFS, FACS
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS
FACS, TOC-Ex,DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS
FACS
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC-In
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
FACS, TOC, DOC
* DFS, due-front speed; FACS, flow cytometry; TOC, total organic carbon; DOC, dissolved organic carbon; Ex, exhaled; In, inhaled.
† Specimens F1–G2 were sampled on both 11 and 12 July 2000.
each InEx sample, preserved in 0.1% glutaraldehyde solution, and frozen in liquid nitrogen for subsequent flow cytometry analysis.
To avoid the risk of contamination associated with filtering the small InEx (32 ml) samples (Yoro 1999), the water
was not filtered during the 1998 experiment; therefore, the
parameter measured was TOC. The water was acidified directly in the InEx sampler using 100 ml Merck Suprapure
30% HCl, and two 5-ml aliquots were transferred to precombusted 10-ml glass ampoules. Ampoules were immediately
heat sealed and stored at 2808C for subsequent TOC analysis at the laboratory of J.H.S. using high-temperature combustion oxidation with a Shimadzu 5000 analyzer. At the
beginning of each day of TOC analysis, instrument performance was tested by running a set of reference DOC samples
distributed globally by J.H.S. to verify accuracy (Sharp et
al. 2002). The error associated with TOC measurements was
1–2 mmol C L21 (coefficients of variation of repeated injection were ,2%) with an average difference of TOC in duplicate ampoules of 4.0 mM C. To be conservative, a single
InEx pair from 1998 with extreme TOC removal, exceeding
2 SD from the mean, was considered an outlier and omitted
from further analysis.
Auxiliary POC and chlorophyll measurements were undertaken concurrently with each of the 1998 InEx sampling
dives (Table 1). The samples were taken at the study site by
a scuba diver at approximately the same height above bottom
as the animals, using a prewashed, horizontally held Niskin
bottle. In the laboratory, 4 L were filtered on a precombusted
GF/F filter. Carbonate particles were removed from the filters
using acidification in HCl fumes for 24 h at 328C. POC was
then measured using a NA2100 CHN analyzer (FISONS)
with an absolute detection limit of 0.16 mmol C. Ambient
(inhaled) DOC for the 1998 samples was therefore calculated
144
Yahel et al.
by subtracting the ambient POC from the measured TOC
(DOCIn 5 TOCIn 2 POCAmbient). A conservative estimate of
exhaled DOC was calculated by assuming a removal of all
POC except the exhaled living cells (DOCEx 5 TOCEx 2
LvPOCEx).
In 2000, DOC was measured directly. A set of InEx pairs
was collected from the sponge as described above and analyzed with and without filtration of the water. To minimize
the risk of contamination, a carbon-free filtration device with
minimal internal volume was fabricated from a Millipore
stainless-steel syringe filter holder (13 mm) equipped with a
custom-made stainless-steel gasket. The filter holders, assembled with GF/F glass fiber filters (two filters per holder),
were fitted to glass syringes, and the entire apparatus was
wrapped in aluminum foil and baked for 2 h in a muffle
furnace at 4508C. A separate filtration assembly was used
for each sample. Sampling commenced as describe above,
the syringe was filled with the sampled water, 10 ml was
used to rinse the filter, and duplicate 5-ml samples were filtered directly into the ampoules (DOC samples). The filter
assembly was removed, and nonfiltered water was injected
into a second set of ampoules (TOC samples), resulting in
eight samples for each InEx pair (two replicates of each In
and Ex, filtered and unfiltered). Ampoules were heat sealed
immediately after acidification (18 ml, Merck Suprapure
30% HCl) and stored at 2808C for later DOC/TOC analysis.
The DOC/TOC analyses were done with duplicate ampoules, and the number of samples required analytical runs
on many days. A strict quality control was used so that precision for the analyses of all the samples could be calculated.
DOC reference materials prepared by J.H.S. (Sharp et al.
2002) were run on each analysis day. For the 11 sample runs
in 2000, the reference blank gave an average of 20.1 6 1.5
mmol C L21, and the deep-ocean reference gave a value of
46.2 6 2.3 mmol C L21. Two of the InEx pairs obtained
were considered contaminated based on duplicate inconsistency (.7 mmol C L21) and were therefore omitted from the
data set. The average difference between duplicate ampoules
of the filtered samples was 2.7 mmol C L21 (n 5 41). Therefore, we consider the precision of our DOC analysis based
on sample preparation and analysis to be on the order of 63
mmol C L21.
A flow cytometer (FACSort, Becton Dickinson) was used
for total counts of phytoplankton and bacteria up to 8 mm,
as described by Marie et al. (1997). The carbon content in
the live cells counted by the flow cytometer (LvPOC) was
estimated using published conversion factors (Campbell et
al. 1994).
Changes in the concentration of oxygen between inhaled
and exhaled water were measured for three specimens (H,
I1, and J; Table 1) at dawn, midday, and dusk of 17 December 2000. InEx pairs were collected as described above using
a 4-ml sampler, and oxygen concentrations were measured
upon retrieval using a calibrated microelectrode (Revsbech
1989). Ammonia concentrations were measured during the
following day for four specimens (H, I1, J, and K1; Table
1) using the fluorometric method of Holmes et al. (1999).
Instantaneous water transport rate through the osculum
was measured a few minutes after InEx sampling using the
dye-front speed (DFS) technique. This measurement was
based on three to five video recordings of the movement of
a dye in a transparent tube located within the excurrent jet.
The diameter of each tube was similar or slightly larger than
that of the sponge’s osculum; thus, speed measurements
were converted to rates of water transport by multiplying the
tube’s cross section by the DFS. This approach was corroborated through a series of laboratory measurements in a
flume using artificial oscula with known flow rates (Yahel
et al. unpubl.). A current meter (MicroADV, 16 MHz Acoustical Doppler Velocitometer, SonTek) was used to continuously measure the excurrent velocity for four specimens for
1–5 d each (Motro et al. unpubl.). The continuous
MicroADV measurements were made in September 1999
and January 2000 at a water depth of ;5 m at the same
study site.
Results
The TOC concentration at the reef’s benthic boundary layer can be estimated by averaging the water inhaled by the
sponges. In 1998, the average TOC in the water inhaled by
the sponges was 79 6 7 mmol C L21. After subtracting the
ambient measured POC content, this leaves an average DOC
of 72 mmol C L21, accounting for 91% of the TOC. In 2000,
the ambient TOC concentration was slightly higher and more
variable (87 6 12 mmol C L21), but the DOC content (directly measured as 81 6 11 mmol C L21) accounted for
essentially the same proportion (93%) of the TOC as it did
in 1998. The LvPOC values (calculated from the flow cytometry measurements) were also higher and more variable
in 2000 than in 1998 (2.5 6 1.3 mmol C L21 and 1.9 6 0.2
mmol C L21, respectively). However, the difference between
the two sampling periods was not significant (Mann–Whitney U-test, P 5 0.10 and 0.08 for LvPOC and TOC, respectively). Ambient POC concentration (6.3 6 5.9 mmol C
L21) calculated from the TOC minus DOC measurements of
2000 was similar but more variable than the directly measured POC in 1998 (7.2 6 1.9 mmol C L21). Sampling in
1998 was performed at the height of the water-stratification
season, whereas sampling in 2000 was performed close to
the end of the spring bloom.
Most of the T. swinhoei we sampled removed TOC (Fig.
1), with an average removal of 12% 6 7% of the inhaled
TOC (Wilcoxon matched pairs test, T 5 6.0, n 5 29, P ,
0.0001). Overall, the sponge removed about 10 mmol C from
every liter it pumped (Table 2). Of the total 29 paired InEx
TOC samples, 24 showed removals exceeding the 3 mmol
C L21 estimated analytical precision, 1 showed an increase
of 3.9 mmol C L21, and 4 showed no significant difference.
The direct measurements of DOC also indicated a highly
significant removal (Wilcoxon T 5 10.0, n 5 20, P ,
0.0001). Of the 20 paired InEx DOC samples from 2000, 14
showed removals, 1 showed an increase of 3.7 mmol C L21,
and 5 showed no significant difference. Our direct measurements in 2000 indicated that the DOC accounted for .90%
of the TOC removed (Table 2, Fig. 2). In 1998, the sponges
removed an average of 10.7 mmol C L21 TOC (Figs. 1, 2;
Table 2). The average particulate concentration was 7.2
mmol C L21; therefore, at least 33% of the removed TOC
Bulk DOC removal by a sponge
Fig. 1. The removal of organic carbon by T. swinhoei in a single
passage of the water via its filtration system measured at the coral
reef of Eilat, Israel. Solid and open symbols represent the inhaled
and exhaled concentrations, respectively; TOC and DOC concentrations are averages of duplicate sample ampoules. The magnitude
of the organic carbon removal is indicated by the length of the
vertical lines connecting each pair of measurements. Circles represent the TOC measurements made in 1998 and 2000. Triangles represent direct DOC measurements made in 2000. Specimen IDs are
indicated on the abscissa (see Table 1).
must have been from the dissolved pool. An indication of
DOC removal (cases where TOC removal exceeded the maximal ambient POC level) was evident in seven of the nine
InEx pairs sampled in 1998. Thus, considerable DOC removal was demonstrated in the 1998 sampling and by the
direct measurements of DOC removal in 2000 (Fig. 1).
An increase of POC in the exhaled water was observed
in about one-third of the samples despite the sponge’s efficient removal of nearly all the living cells. The flow cytometry analysis confirmed that most of the excreted POC was
nonliving detrital particles. The POC removal (1.3 6 6.7
mmol C L21, median 5 2.3 mmol C L21) calculated as TOC
removed minus DOC removed was similar to the values of
LvPOC removal (2.1 6 1.0 mmol C L21) calculated from
the flow cytometer measurements.
T. swinhoei removed live phytoplankton and bacteria in
all cases. Removal efficiency was remarkably high (average
85–95%, Fig. 3) except for eukaryotic cells in 1998 (30%
6 11%). In 2000, however, eukaryotic cells were removed
as efficiently as were other types of phytoplankton. Over the
wide range of ambient concentrations encountered, the number of cells removed increased linearly with increasing cell
145
Fig. 2. Average TOC and DOC concentrations in the inhaled
and exhaled water. P values (Wilcoxon matched pairs test) indicate
the significance level of the difference in the concentration of the
respective carbon pools between the inhaled and exhaled samples
in n pairs of InEx samples. Error bars 5 SE.
abundance, with no indication for a threshold or for saturation (Fig. 3). This pattern is similar to that reported for other
sponges (e.g., Reiswig 1971; Pile et al. 1996; Ribes et al.
1999). However, in comparison to the TOC removal, the
amount of LvPOC removed (maximum: 4.6 mmol C L21)
was always small (Table 2, Fig. 4).
The amount of molecular oxygen removed from the water
passing through T. swinhoei (9 mM O2) was equivalent to
the amount of organic carbon removed (10 mmol C L21,
Table 2). This removal peaked at dawn (7% of the inhaled
oxygen) and was minimal at midday (2%), when oxygen
production by the photosynthetic symbionts was expected to
be maximal (Fig. 5; Beer and Ilan 1998). Ammonium was
also efficiently removed by the four specimens sampled
(62% 6 7%), resulting in a 204 6 49 nmol N reduction
from each liter of seawater pumped by the sponge.
The DFS measurements indicated an average excurrent
speed of 7.6 6 2.3 cm s21, corresponding to a water transport
rate of 230 6 69 ml min21. Specific water transport (normalized to the volume of the sponge) was inversely correlated with sponge size (r 5 20.87). Large sponges (specimen volume .100 ml) pumped ,2 ml min21 (ml sponge)21,
small sponges (,50 ml) pumped .4.5 ml min21 (ml
Table 2. The removal of organic carbon and its components by the sponge Theonella swinhoei as measured in 1998 and 2000. Averages
(6SD) were calculated for each year and for the grand total (n 5 number of InEx samples).
Component removed
1998 (n59)
2000 (n520)
Total (n529)
Total organic carbon (TOC, mmol C L21)
Dissolved organic carbon (DOC, mmol C L21)
Living plankton (LvPOC, mmol C L21)
% LvPOC of TOC removed
% DOC of TOC removed
1167
665*
1.460.2
13
40*
1067
1068
2.461.1
24
97
1067
* A conservative estimate of DOC removal was used by considering the POC to be fully (100%) removed by the animals.
2.161.0
20
146
Yahel et al.
Fig. 3. The removal of ultraplankton (,8 mm) plotted against ambient (inhaled) concentrations.
The diagonal line (x 5 y) represents 100% removal. Data are from the same InEx pairs that were
used for the TOC/DOC analysis (opens symbols 5 1998; solid symbols 5 2000).
sponge)21, with an overall mean pumping rate of 2.6 6 1.8
ml min21 (ml sponge)21. The average excurrent speeds measured continuously with the MicroADV (7.8 6 3.5 cm s21,
n 5 4 sponges) was in excellent agreement with the above
discrete DFS measurements. Based on this agreement, the
DFS measurements were used to calculate hourly integrated
fluxes for matching InEx pairs (Table 3).
Discussion
DOC accounts for most of the carbon removed by the
tropical symbiont-bearing sponge T. swinhoei from the water
it pumps. This sponge is also an efficient filter feeder, removing most of the living cells from the water it pumps.
The few oxygen measurements made indicated a respiratory
demand of the same order of magnitude as the TOC/DOC
removal (Fig. 5, Table 2). These findings confirm earlier suggestions by Reiswig (1974, 1981) that DOC uptake can reconcile the .70% discrepancy between the particulate gain
and respiratory demand of several tropical sponges. Because
T. swinhoei harbors several prokaryotes and worms, it is still
unknown whether and to what extent the sponge participates
in the uptake and utilization of the removed DOC.
The composition of dissolved organic matter in waters
overlying coral reefs is poorly understood (van Duyl and
Gast 2001), rendering speculative any discussion of the nature of the material removed from the water passing through
T. swinhoei. The DOC content in the water exhaled by the
sponge (average of 71 6 10 mmol C L21, Fig. 1) was reduced
to a level similar to the ambient DOC in the adjacent opensea surface (68 6 8 mmol C L21, 0–20 m depth; Häse et al.
unpubl. data). However, the exhaled values were always
much higher than the concentration of the refractory DOC
in the deep ocean (45 mmol C L21; Benner 2002). The exhalent level is similar to that reported from the surface waters of oligotrophic oceans, presumably representing the
semilabile DOC (Carlson 2002). We therefore suggest that
T. swinhoei removes primarily the labile fraction (cf. Van
Duyl and Gast 2001). Potential sources for labile DOC in
coral reefs are numerous. Such sources are expected to vary
over both time and space. Unlike the comparatively uniform
distribution of DOC in the ocean interior (Benner 2002 and
references therein), DOC in the coral reef was much more
variable (Fig. 1).
The typical low surface-to-volume ratio of metazoans is
thought to limit the uptake of dissolved elements from the
water, to a point where mass feeding on DOC by metazoans
is considered unlikely (Siebers 1982). Sponges, on the other
hand, have evolved to be functionally adapted for bulk DOC
removal by actively pumping water through complex filtration systems with immense internal surface areas.
At present we have no information on the mechanisms
involved in the observed DOC removal. A possible role of
symbiotic bacteria was suggested by Reiswig (1971, 1981).
Bulk DOC removal by a sponge
147
Fig. 4. The average contribution of different pools (stacked bars
on the left) to the TOC (gray bars at the right) gained by the sponge
T. swinhoei in 1998 and 2000. The unfilled region denoted by a
question mark in the 1998 bar is the unresolved organic matter
(UnPOC) from a conservative estimate of DOC considering remaining TOC removed as particulate detritus. Horizontal arrows
indicate the corresponding ambient POC levels and thus represent
an upper bound for POC removal by the sponge. Pro, Prochlorococcus; Syn, Synechococcus; Euk, eukaryotic algae ,8 mm; Bact,
nonphotosynthetic bacteria. Error bars 5 95% confidence interval
for the TOC removal.
Fig. 5. The concentration of oxygen in the water inhaled and
its removal by three specimens of T. swinhoei at dawn, noon, and
dusk in a single day (17 December 2000). Data were obtained by
the InEx method. The bar at the top indicates dark (solid) and light
(open) time. Error bars 5 SE.
His findings indicated that the discrepancy between respiratory demand and particulate feeding occurred in sponges
with large populations of endosymbiotic bacteria but not in
asymbiotic sponges. Another indirect indication of the importance of symbiotic bacteria is the typical proximity of the
endosymbiotic bacteria to the choanocyte cambers of some
sponges (Wilkinson 1978; Webster and Hill 2001). Fast incorporation of labeled amino acids by the symbiotic bacteria
of the sponge Chondrosia reniformis further strengthens the
assertion that bacteria are involved (Wilkinson and Garrone
1980). However, ultrastructural studies of T. swinhoei (Bewley et al. 1996; Magnino et al. 1999) and other sponges
(Reiswig 1981) indicated that the bacteria are not found
along the surface of the water canals but rather further inside
in the sponge’s mesohyl behind the pinacoderm. It is reasonable to assume that the removal of dissolved matter dur-
Table 3.
volume.
Comparison of published specific metabolic activity of several sponges (mean 6 SD or range) normalized to 1 ml sponge
Sponge
Theonella swinhoei
Osculum
diameter
(mm)
6–15*
Excurrent Pumping rate Carbon gain
O2 removal O2 removal rate
efficiency (nmol O2 min21
jet speed
(ml min21
(nmol C (ml
21
21
21
21
(%)
(cm s ) (ml sponge) ) sponge) min )
(ml sponge)21)
7.662.3
2.661.8
462
23.4613.0
26.0618.2
Mycale lingua
Halichondria panicea
Haliclona urceolus
Dysidea avara
Therea muricata
Mycale sp.
Tethya crypta
Verongula sp.
Verongia fistularis
1267
0.15
20–100*
.100
108
33
1469.7
5.6
7.960.7
12.862.6
17.360.6
10.160.5
* Range.
† LvPOC, particulate organic carbon of living cells.
‡ DOC, dissolved organic carbon.
2.761.1
2.561.7
1.060.8
3.0–3.6*
14.4
10.8
5.1
7.460.4
0.6–9.7*
27.8
15.5
10.5
9.161.3
Source
This work (LvPOC)†
4.262.6
1.2
1.1
5.6
5.3
34
9
50
79
This work (DOC)‡
Pile et al. 1996
Riisgård et al. 1993
Riisgård et al. 1993
Ribes et al. 1999
Witte et al. 1997
Reiswig 1974
Reiswig 1973
Reiswig 1974
Reiswig 1981
148
Yahel et al.
ing the brief (,3 s) passage of the water via the sponge
filtration system would require the removing organs/organisms to be in direct contact with the flowing water. Nevertheless, the initial uptake and transport of dissolved substances in the sponge must occur across the pinacoderm cells
or via the microvilli of the choanocytes. In other sponges,
bacteria are also found in intracellular vacuoles (Reiswig
1981 and references therein) or concentrated in specialized
bacteriocytes (Ilan and Abelson 1985). Thus, dissolved substances must first be transported across at least two cell
membranes before they can come in contact with the symbiotic bacteria. The ability of sponge cells to transport and
assimilate dissolved elements was shown for asymbiotic larvae of the sponge Tedania ignis by Jaeckle (1995). These
morphological and physiological studies suggest that T.
swinhoei may have a direct role in the uptake and transport
of DOC from the water it pumps.
T. swinhoei excretes organic carbon, as do all heterotrophs, in dissolved, particulate, or both forms (Reiswig
1971; Ribes et al. 1999). The InEx technique allowed measurement of the net flux of each element. Therefore, unlike
living cells (counted with the flow cytometer) our TOC/DOC
measurements did not discriminate between removal and excretion of detrital particles. When measured directly, net
DOC removal accounted for almost all the TOC removed
(Table 2). However, the flow cytometer measurements
showed that consistently ;20% of the TOC removed was
LvPOC (Table 2). How is it then possible for the DOC to
account for almost all the TOC removed? Moreover, a net
removal of detrital particles (total POC minus LvPOC) was
evident in 9 of 19 samples. This apparent discrepancy (Fig.
4) is most likely a consequence of excretion by the sponge.
Furthermore, a relatively large error was inherent in the way
we estimated detrital particles by subtracting two large quantities one from another: TOCremoved 2 (DOCremoved 1 LvPOCremoved ). On average, the removal of POC by the sponge
(1.3 6 6.7 mmol C L21, median 2.3 mmol C L21) was similar
in magnitude to LvPOC removal (Fig. 4), suggesting that the
detrital removal is either small or totally masked by excretion. Thus, the role of nonliving particles in the nutrition of
T. swinhoei remains unresolved. In contrast, detritus encompasses .90% of the carbon ingested by the temperate ascidian Halocynthia papillosa (Ribes et al. 1998).
Specific pumping rates of T. swinhoei were in the lower
range of those reported for other (larger) tropical sponges
but resemble those of temperate species of comparable size
(Table 3). This weak pumping rate implies a rather low specific carbon gain from the particulate pool (4.2 nmol C L21
(ml sponge)21 min21) despite the high removal efficiency.
However, when the removal of both particulate and dissolved
carbon (TOC) is considered, the specific organic carbon retention by T. swinhoei (30 nmol C L21 (ml sponge)21 min21)
is the highest ever reported (Table 3). Past studies, however,
had not measured DOC removal. In the present study, the
specific oxygen demand of T. swinhoei (23.4 nmol O2 L21
(ml sponge)21 min21) was slightly lower than its TOC gain,
as expected for an organism with phototrophic symbionts
(Magnino et al. 1999). Given the lack of information on the
T. swinhoei consortium growth rate and primary production,
it is not possible at this stage to estimate the relative con-
tributions of DOC and photosynthetically derived production
to body mass production of the sponge. The specific respiratory demand of three other tropical sponges (Reiswig
1974, 1981) was higher than that of T. swinhoei and higher
than the POC intake (Table 3), further strengthening Reiswig’s suggestion that a major carbon source in those sponges
was dissolved organic matter.
Considering the large amount of organic material T. swinhoei processes, a large efflux of remineralized nutrients was
expected (Gili and Coma 1998). Thus, the efficient removal
of ammonium by the sponge is surprising and may be attributed to the presence of photosymbionts. Nevertheless, at
this stage, our nutrient data set is too limited to allow further
discussion of the nutrient dynamics in this species.
This is the first report of in situ removal of bulk DOC in
invertebrates. The results of this study suggest the presence
of a previously undocumented pathway of carbon flux in
benthic habitats. If DOC is supplied by the sponge to symbiotic bacteria, the microbial loop in the reef would be accelerated. If, on the other hand, the DOC is directly utilized
by the animal, the loop would be‘‘short-circuited.’’ Because
most of the labile DOC in the reef is assumed to be released
locally, its removal by invertebrates would represent a major
pathway of carbon recycling, a key process in coral reef
communities. Our preliminary data (unpubl.) suggest that
other reef sponges also remove DOC. The abundance of
sponges in coral reefs and their filtration capacity might have
been grossly underestimated (Richter et al. 2001). These data
suggest that DOC may play a major role in the trophic dynamics of coral reefs.
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Received: 19 January 2002
Accepted: 16 July 2002
Amended: 5 September 2002
Chap. 6. Phytoplankton grazing in reef rocks
5.
Selective filtration of ultraplankton by three tropical suspension
feeders: a sponge, an ascidian, and a bivalve
By
Yahel G., D. Marie, S. Eckstein, and A. Genin
Submitted to Marine Ecology Progress Series
38
Chap. 6. Phytoplankton grazing in reef rocks
Abstract
Diet composition and prey preference patterns were studied in three benthic coral-reef suspension
feeders: a sponge (Theonella swinhoei), a mollusk (the boring bivalve Lithophaga simplex), and a
tunicate (the solitary ascidian Halocynthia gangelion). The water inhaled and exhaled by
undisturbed specimens was cleanly collected in situ. Flow cytometric comparison of these water
samples provided a direct measure of the grazer's filtration efficiencies over the course of four
years (1996-2000) covering a wide range of environmental conditions. Ultraplankton (<8 µm),
which dominated the planktonic biomass in the oligotrophic waters overlying the reef, was
removed efficiently by each of the three grazers exhibiting a unique species-specific selectivity
patterns. Synechococcus removal efficiencies were 70±14%, 76±16%, and 92±8% (mean±1SD),
for the bivalve, ascidian and sponge, respectively. Coccoid photosynthetic bacteria were preferred
by all three taxa over both the larger eukaryotic algae and the somewhat smaller nonphotosynthetic bacteria. The ascidian and the bivalve efficiently removed the minute
photosynthetic bacteria Prochlorococcus (~0.6 µm, 65±19% and 41±19%, respectively) but not
non photosynthetic bacteria (8±7% and 5±19%, respectively). The negative selection against non
photosynthetic bacteria was surprising considering their dominance in the planktonic community
and the considerable size overlap on non photosynthetic bacteria with the Prochlorococcus
population. The small fraction of non-photosynthetic bacteria retained by the bivalve and the
ascidian did not differ in size but had higher apparent nucleic acid content in comparison with the
ambient (inhaled) population. Considerable retention of non photosynthetic bacteria was exhibited
only by the sponge (84±8%) which was the most efficient and least selective suspension feeder
(T. swinhoei removal efficiencies ranged from 73±27% for eukaryotic algae to 95±7% for
Prochlorococcus). Selectivity for cell attributes within prey taxon was evident for the bivalve
which preferred Prochlorococcus and eukaryotic algae with higher chlorophyll content. The
sponge preferred the smaller photosynthetic cells but showed no preferential size selection for
non-photosynthetic bacteria. We suggest that for benthic tropical picoplankton grazers, selectivity
is not size dependent and probably relies on other cell attributes such as cell surface properties
and/or motility. The mechanisms underlying the observed selectivity patterns are still unresolved
and - as each phylum relies on a unique filtration mechanism – should vary between the three
phyla. As the selectivity did not appear to maximize carbon or energy gain, it is suggested that at
the reef, where carbon is not a rare commodity, suspension feeders have evolved to optimize the
gain of other nutrients or to avoid harmful prey taxa.
39
Chap. 6. Phytoplankton grazing in reef rocks
Acknowledgments
We thank R. Yahel, I. Ayalon, M. Ohevia, R. & C. Wyeth, B. Munkes, Efrat Gotlibe, R. Motro,
A. & G. Brandes, Y. Shif, D. Tchernov, and D. Weil, for help in the field and laboratory, the IUI
staff for logistic support, D. Lindell, A. Post, M. Kiflawi, E. Hadas, T. Katz, for useful
discussions, and the Rieger Foundation for supporting GY. This study was funded by grants from
the US-Israel Binational Science Foundations, The Israel Science Foundation and a grant from the
German Ministry of Science, Technology and Education (BMBF) through the Red-Sea Program.
The flow cytometry analysis was funded by the French program PROSOPE and the European
program PROMOLEC.
40
Chap. 6. Phytoplankton grazing in reef rocks
Introduction
Feeding by active pumping and filtration of the particles suspended in the water is a common
nutrition mode in many sessile invertebrates (Gili and Coma 1998). This mode of feeding has
received considerable attention since the onset of modern marine biology (review by Jørgensen
1966). However, the vast majority of these studies have focused on temperate zone suspension
feeders (mostly bivalves) characterized by adaptations for feeding on relatively large food
particles (>4 µm) in a highly dynamic, turbid and productive environment (Hawkins et al. 1998).
Much less attention has been devoted to tropical suspension feeders adapted for clear oligotrophic
waters and for feeding on the picoplankton and bacteria that typically dominate these habitats
(e.g., Sommer 2002). With the advent of modern flow cytometry, fluorescent staining, and
tropical research, there is a growing appreciation of the fundamental significance of bacteria and
ultraphytoplankton as a trophic link in benthic-pelagic coupling in tropical communities such as
coral reefs (e.g., Ayukai 1995;Yahel et al. 1998; Fabricius and Domisse 2000; van Duyl et al.
2002). However, little progress has been made in the study of the nutritional ecology of
suspension feeders in these systems (Pile 1997; Hawkins et al. 1998; Fabricius and Domisse
2000).
Selective feeding is an important mechanism by which animals can maximize their energy gain,
avoid harmful or toxic food items, and satisfy their nutritional needs (e.g., for nutrients or rare
elements). In animals that actively pump waters through their feeding organs or filtration
apparatus, selection can take place at several levels (Shumway et al. 1985): 1. Differential
retention of particles by the filtration apparatus. Particles that are rejected or not retained will be
carried out in the exhalant currents. 2. Pre-ingestion sorting of trapped particles. Bivalves, for
example, eject unwanted particles as pseudofeces while ascidians may use squirting for this
purpose (Armsworhty et al. 2001). 3. Differential digestion. Unwanted particles will be excreted
undigested in feces (e.g., Cucci et al. 1985).
While the mechanisms underlying selective feeding in bivalves are still controversial (e.g.
Beninger and St Jean 1997; Bayne 1998, Ward et al. 1997, 1998b; Benninger 2000, Riisgård and
Larsen 2000, Silverman et al. 2000; Ward et al. 2000) there is a general agreement that post
trapping sorting (No. 2 above) occurs under certain conditions (Jørgensen 1996; Bougrier et al.
1997; Ward et al. 1997, 1998a,b; Hawkins et al. 1998; Cranford and Hill 1999; Arifin and
Bendell-Young 2001). Differential retention in bivalves is mostly considered to be size dependent
with a decreasing filtration efficiency for particles < 4µm (Jørgensen 1996; Hawkins 1998;
Yukihira et al. 1999; Kreeger and Newell 2001). A few laboratory studies have reported sizeindependent differential retention (reviewed by Bougrier et al. 1997), e.g., for the oyster Ostrea
edulis (Shumway et al. 1985) and the mytilid Mytilus edulis (Newell et al. 1989), however, their
findings were criticized by Jørgensen (1996). Moreover, the validity of laboratory-based studies
and artificial diets for field situations has been recently questioned by several authors (review by
Cranford and Hill 1999; Riisgård 2001).
In comparison to a bivalve feeding that was addressed by numerous studies, much less attention
has been devoted to sponges and ascidian feeding and more to studies discussing sponge feeding
in tropical waters (reviewed by Yahel et al. 2003). Selective feeding in sponges was reported by
Reiswig (1971), Wilkinson et al. (1984), and Pile et al. (1996) (reviewed by Witte et al. 1997).
Ascidians removed particles from the water by continuously moving mucus sheets lining the inner
surface of the brachial basket. The filtration mechanisms of the ascidian Halocynthia pyriformis
41
Chap. 6. Phytoplankton grazing in reef rocks
was recently described in detail by Armsworthy et al. (2001). As a group, ascidians are
considered non-selective filter feeders (Ribes et al. 1998) and whereas the possibility of selective
feeding was suggested by several authors it has not been conclusively established to date
(Armsworthy et al. 2001).
The objective of this study was to examine for selectivity patterns in undisturbed animals feeding
over a wide range of environmental conditions in their natural habitat. Three common reef
suspension-feeders were selected: the sponge Theonella swinhoei, the bivalve Lithophaga
simplex, and the ascidian Halocynthia gangelion.
Methods
Study site
The study was carried out in the northern Gulf of Aqaba. Undisturbed animals were studied by
SCUBA diving in their natural habitat on the fore reef (3-18 m depth) of the Coral Reef Nature
Reserve of Eilat, Israel. For a full description of the study site see Yahel et al. (2002) and
references therein. Briefly, oceanographic conditions at the study site were calm with wave height
<0.3 m and bottom currents rarely exceeding 20 cm sec-1. Temperature ranged from 200C in
February to 26-280C in August-September. Due to the steep bathymetry and lack of external
sediment sources, the water in the Gulf is clear. The low chlorophyll values (1988-1998 annual
average 0.35±0.09 µg liter-1, measured daily at the reef, Genin, unpublished data), and low total
particulate organic carbon (0.09±0.02 mg litre-1, Yahel et al. 2003) are typical for oligotrophic
conditions. Total particulate carbonates amount to 0.7±0.9 mg liter-1 at night and are apparently
higher during the day (Yahel et al. 2002). The phytoplankton community in the Gulf of Aqaba is
dominated by ultraphytoplankton (<8µm, Yahel et al. 1998, Sommer et al. 2002). In the northern
tip of the Gulf the ultraphytoplankton community exhibits large seasonal fluctuations
concurrently with seasonal changes in water-column conditions (Lindell and Post 1995).
Eukaryotic algae (0.6 – 4.0 µm, Sommer et al. 2002) dominate in the nutrient-replete water during
the winter mixing. Synechococcus (0.9-1.2 µm, Sommer et al. 2002) is the major component of
the ultraphytoplankton during the spring blooms and retained high concentration (>104 ml-1)
throughout the year. Prochlorococcus (0.5-0.7 µm, Partensky et al. 1999) dominates
(numerically) in nutrient-depleted summer-stratified waters, and is not detected at the height of
the mixing in late winter. Due to the bathymetry, the Gulf’s fringing reefs are constantly flushed
with open-sea water (Genin et al. 2002) so that the ultraplankton composition over the reef
closely follows that of the open water. However, a thick (1-3 m) phytoplankton-depleted layer is
usually found over the reef (Yahel et al. 1998), apparently due to intense benthic grazing. This
grazing was recently quantified at the community scale by Genin et al. (2002). Apart from the
corals themselves, the three most conspicuous suspension feeder at the reef are sponges, ascidians
and boring bivalves (Yahel et al. 1998).
Studied taxa
Theonella swinhoei (phylum porifera, class demospongiae, order lithistida, family
theonellidae) is a common sponge in coral reefs throughout the Indo-West Pacific. The barrelshape body has a single osculum at its apex. The sponge typically occurs in clusters. In the Gulf
of Aqaba, clusters are generally small (<6 specimens per cluster) and specimen dimensions are
around 5-15 cm in length, 3-8 cm in diameter. The body material is dense and contains large
42
Chap. 6. Phytoplankton grazing in reef rocks
populations of symbiotic prokaryotic bacteria. Pumping rate average 230±69 ml specimen-1 min-1
(Yahel et al. 2003). This sponge removed bulk quantities of dissolved organic matter from the
water it pumps (Yahel et al. 2003).
Lithophaga simplex (phylum mollusca, order bivalvia, family mytilidae) is a boring bivalve
that inhabits only two species of live massive corals in the Red Sea: Astreopora myriophthalma
and, less frequently, Goniastrea pectinata (Mokady et al. 1992) but is specific to other corals in
other localities (Morton 1983). L. simplex reach's a few cm in length and is hidden within the
corals with only the distal end of the siphons (1-3 mm in diameter) visible. In the Gulf of Aqaba,
several hundred L. simplex specimens may colonize a single coral head with a mean density of
0.22±0.11 mussels per cm-2 coral surface (Mokady et al. 1998).
Halocynthia gangelion (phylum: chordata, order stolidobranchia; family pyuridaeis) is the
most conspicuous solitary ascidian in the coral reef of Eilat (Yahel et al. 1998). Pumping is
continuous, and averages 112±33 ml specimen-1 min-1 (Yahel, Marie and Genin, in prep).
Water sampling
An in situ, non-intrusive technique was used to directly measure the rate and efficiency of
prey removal from the water filtered by the studied animals. The technique, termed “InEx”, is
based on the simultaneous, pair-wise collection of the water Inhaled and Exhaled by the animal.
An important quality of the InEx technique is the lack of any manipulation of the studied
organisms, thus allowing realistic estimates of their feeding under natural conditions. The
difference in plankton cell characteristics (e.g. size) and concentration among a pair of samples
provides a measure of the retention efficiencies of different prey types by the animal.
A scuba diver sampled the exhaled water using an open-ended tube held within the exhalent
jet with the tube’s intake end positioned ~2 mm above the animal’s exhaling aperture (sponge’s
osculum, bivalve’s and ascidian’s siphons), using the ex-current jet to flush and then fill the tube.
Filling time was determined individually for each pair so that it would last 150% of the time it
took the exhalant jet to flush clear an identical tube pre-filled with fluorescein dye (measured a
few minutes prior to each sampling). A sample of the inhaled water was taken simultaneously by
slowly withdrawing water into an identical tube, attached at its proximal end to a syringe while
holding its open (intake) end 3-5 mm from the animal's inhaling apertures (sponge’s ostia,
bivalve’s and ascidian’s siphons). Sampling duration was at least two orders of magnitude longer
than the time (<2 seconds) it took the water to pass through the animal. Thus, each InEx pair
represented an integration of approximately 2-3 min of the animal's activity. Care was taken to
sample undisturbed and well-functioning individuals, i.e., fully open valves and extended siphons
(for bivalves and ascidians), indicating pumping in full capacity under optimal conditions
(Jørgensen 1996).
A total of 206 pairs of InEx samples were collected (87 for H. gangelion, 74 for L. simplex, and
45 for T. swinhoei) in 44 dives during 10 sampling sessions spanning October 1996 to September
2000 and covering all four seasons. An effort was made to sample 2-3 species per dive.
Flow cytometry
A flow cytometer was used to measure the concentration and cell characteristics of nonphotosynthetic bacteria (Bact) and the three dominant autotrophic groups in the studied waters
[Prochlorococcus (Pro), Synechococcus (Syn), and pico-eukaryotes (Euk)]. Taxonomic
43
Chap. 6. Phytoplankton grazing in reef rocks
discrimination was made based on cell side-scatter (SSC, a proxy of cell volume, Simon et al.
1994), forward-scatter (FSC, a proxy of cell size, Robertson et al. 1998), orange fluorescence
(Fl2) of phycoerythrin and red fluorescence (Fl3) of chlorophyll (Marie et al. 1999). In a typical
pico-phytoplankton analysis some 150 to 300 µl of sample waters (>2.5•104 cells) were analyzed
during 2-3 min on a FACSort (Becton Dickinson) with 15 mW laser power with the discriminator
set to the Fl3. A second run was used to analyze all DNA containing cells following a dark, 20
min incubation of 200 µl of sample water with the nucleic acid stain Syber Green I (1:104 of
commercial stock, Marie et al. 1999) at room temperature. About 50 µl of sample water (>4•104
cells) were analyzed during a two minute run with the discriminator set to green fluorescence
(Fl1). Prochlorococcus has very weak chlorophyll fluorescence near the surface, especially in
summer. Thus in some cases, when full separation from the noise was not possible, it was
necessary to apply a Gaussian fit to a density distribution plot of the SSC or Fl3; this
extrapolation allows better estimates of cell concentration. Only samples where the nonphotosynthetic bacteria and Prochlorococcus populations could be fully resolved both from the
noise and from each other were used for subsequent analysis of cell attributes populations.
Yellow-Green Beads (Polysciences™ 0.95 µm) were used as an internal standard in each
sample and (unless stated otherwise in the text) all cellular attributes were normalized to the beads
_______
____
_______
using the equation: Norm i , j = Pop i , j / Beads i , j where the normalized mean (Norm) of a population
attribute j (e.g., side scatter) of population i (e.g. Synechococcus) was calculated as the ratio of the
ith population mean of the jth attribute and the mean of the respective attribute for the beads
(Marie et al. 1999). This normalization allows proper comparison of results obtained using
different instrument settings.
List mode data were recorded using 4 decades log scale and 256 bins (channels) and
analyzed using Cytowin (Version 4.1 developed by D. Valuot, http://www.sbroscoff.fr/Phyto/cyto.html#cytowin or WinMDI (Version 2.8 developed by J. Trotter,
http://facs.scripps.edu/software.html). Log to linear transformation was done using the
4X
256 −1
equation X lin = 10 log
. No normalization to beads was applied to the density distributions
examples presented below; thus care was taken to average and compare only a few (>12) samples
from adjacent water samples, which were run consecutively using exactly the same instrument
settings.
In order to test the hypothesis that active bacteria are positively selected for by the studied
suspension feeders, the redox dye CTC, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining
technique (Sieracki, 1999) was used with aliquots of each of the InEx experiments undertaken in
September 2000. A third aliquot was taken from each water sample and incubated in the dark, at
room temperature with 2 mmol liter-1 CTC (Polysciences). Incubation was terminated after 15
min by the addition of 0.1% glutaraldehyde. The sample was then analyzed as a third run. In a
positive control established by enriching sample water with (1%) LB medium CTC positive cells
concentration was doubled within 20 min.
Statistical analysis
The sampling design (InEx) was specifically developed as a "pairwise comparison". Therefore, a
"within subject" design (i.e., paired t-test, repeated measure ANOVA, and their nonparametric
alternatives) was used throughout the analysis to test the null hypothesis of unselective filtration.
Unlike classical grazing experiments where the suspension feeder activity affects food
44
Chap. 6. Phytoplankton grazing in reef rocks
concentrations in the experimental vessel (Chesson 1983, Riisgård 2001), measuring grazing
using the InEx technique allows estimation of Chesson selectivity index (αi) as the maximum
−1
 m 
likelihood estimator α~i = Fi  ∑ Fi  (case 1 in Chesson 1983) where m is the number of prey
 i =1 
types and Fi is the filtration efficiency for the ith prey type, calculated as Fi=(Ini-Exi)/Ini (where
Ini and Exi are the concentrations of the ith prey type in the water inhaled and exhaled by the
studied animal, respectively). A separate αi was calculated for each InEx sample (pair). For better
visualization and in order to allow comparison of periods when Prochlorococcus was absent the
αi were rescaled to εi using the equation εi=(mαi-1)/[(m-2)αi+1] where m is the number of prey
type available (Chesson 1983). Values of εi range from -1 when none of the ith prey type is taken,
to 1 when the ith prey type is the only one selected for. However, since the statistical properties of
the εi are not fully resolved, statistical inference was done solely with the αis' (Chesson 1983).
Filtration efficiency and αis' were arcsine and square root transformed, respectively, to meet
ANOVA requirements of homogeneity of variance and normality (verified by Cochran and
Lilliefors tests, respectively). Pairwise, post-hoc comparisons were made using Tukey HSD test
for unequal n. Statistical analyses were done using STATISTICA for Windows (Ver 6.0, StatSoft,
Inc. 2002). Note that the terms "selectivity" and "preference" are used here in the narrow sense to
denote differential retention. Neither the underlying mechanisms nor assimilation or utilization of
the removed cells were addressed.
Results
The water temperature at the study site was relatively stable throughout the four years of data
collection, normally ranging between 200 –260 C with rare peaks up to 280C. Seasonal variations
in ultra-planktonic prey abundance and community composition was considerable, spanning
several order of magnitude, especially for Prochlorococcus (null to 2.5•105 cells ml-1) and the
eukaryotic algae (102 to 1.5•104 cells ml-1). Synechococcus and the non-photosynthetic bacteria
demonstrated much less variability and (except for a few days during the spring blooms) ambient
concentrations ranged from 1·104 to 3•104 Synechococcus cells ml-1, and 5•105 to 10•105 nonphotosynthetic bacteria cells ml-1. Phytoplankton seasonal succession generally followed the
pattern described by Lindell and Post (1995). The ambient (inhaled) concentration of CTC
positive cells in September 2000 was 6•104±3•104 ml-1 (n=32). Over the entire study period, the
mean forward scatter (FSC, a proxy for cell size, units are fraction of calibration beads mean in
the same run, see methods) of non-photosynthetic bacteria averaged 0.28±0.13 (N= 140) and
those of the Prochlorococcus and Synechococcus were 0.44±0.13 and 0.82±0.13, respectively.
Within each water sample the Prochlorococcus forward scatter distributions had typically »25%
overlap with the bacteria on the one hand and with the Synechococcus on the other.
Synechococcus scatter showed similar overlap with both the Prochlorococcus from below and the
eukaryotic algae from above. This size overlap between populations is reflected by the large CVs'
(>75%) of the population means (average 262%, 173%, and 206% for the non-photosynthetic
bacteria, Prochlorococcus, and Synechococcus, respectively).
Plankton removal was evident in each of the InEx samples undertaken with each of the
three suspension feeders studied. Synechococcus concentrations, for example, were reduced by >
30% in each of the 206 cases. Diet composition, food preferences, and the level of selectivity
varied considerably between the three suspension-feeders studied (Fig. 1). These interspecific
differences previaled despite large seasonal variations in ambient prey abundance and
composition. Comparison of the CTC positive cells in the InEx samples from each of the three
45
Chap. 6. Phytoplankton grazing in reef rocks
suspension feeders (n=13 for H. gangelion, 12 for T. swinhoei, and 8 for L. simplex, September
2000) showed no significant difference (paired t-test, P>0.05) or a trend in any of the grazers.
L. simplex
The bivalve L. simplex removed ultraplankton from the water it pumped in a highly
selective manner (repeated measure ANOVA, F3,117 =254.5; P<0.0001 Fig. 1B). Synechococcus
(FSC, 0.89±0.10 of beads FSC) was the preferred prey type with removal efficiencies of up to
90% (69±14%, Fig. 1A). Larger eukaryotic algae (FSC 7.4±3.28) were removed with somewhat
reduced efficiency (62±14%) but comparison of selectivity indices indicated they were not
significantly less preferred (Tukey HSD, P=0.066). The minute photosynthetic bacterium
Prochlorococcus (FSC 0.40±0.08) was also readily taken by the bivalve (41±19%) but its
removal was significantly lower (Tukey HSD, P<0.001) than that of the other two photosynthetic
groups. Surprisingly, only a small proportion of the non-photosynthetic bacteria (FSC 0.31±0.08)
was removed by the bivalve (5±19%) despite the large size overlap between Prochlorococcus and
the non-photosynthetic bacteria (Fig. 2, A and B). In fact, removal of more than 20% of the
Prochlorococcus population was evident in 50 (88%) of the 57 InEx samples when
Prochlorococcus was present (Fig. 3B) whereas removal of at least that magnitude of the nonphotosynthetic bacteria was recorded only in seven cases (10% of the 71 samples, Fig. 3D). In
34% of the sample 10-20% of the non-photosynthetic bacteria cells were removed and in 15% of
samples 5-10% removal efficiency was recorded. Null to "negative" (elevation of cell counts in
the exhaled waters) removal of non-photosynthetic bacteria was found in ~25% of the InEx
samples (whereas Prochlorococcus was always removed).
Despite the marked seasonal changes in prey abundance, the removal of photosynthetic prey
remained nearly constant, resulting in a quasi linear functional response (Fig. 3 A-C) and the food
preference ranking did not change throughout the different sampling seasons (Fig. 3E). Seasonal
variations in filtration efficiencies were evident only for the eukaryotic algae (Kruskal-Wallis
ANOVA, P<0.05) exhibiting no clear pattern. Filtration efficiencies of the non photosynthetic
bacteria were correlated with ambient (inhaled) concentrations (rp=0.58, P<0.001), indicating that
removal occurred mostly in higher than average bacterial concentrations (>5•105 ml-1, Fig. 3D).
The optical properties of the cells retained by the bivalve indicated a surprising selectivity
within prey populations. The examples in Fig. 4 clearly show a shift to the left in both the scatter
and fluorescence of the Synechococcus exhaled by the bivalve, indicating a preferential retention
of high scatter, high fluorescence cells. Cell distributions were always peaked (Kurtosis >0) with
a long right tail (Skewness>0) in both the inhaled and the exhaled waters, but the exhaled
populations were significantly less skewed and less peaked (except for the red chlorophyll
fluorescence). The same trend was also evident for the more diverse group of eukaryotic algae
(Fig. 5). Fig. 6A summarizes L. simplex preferences within each prey type for each of the relevant
cellular attributes as recorded by the flow cytometer. Most notable is the preferential retention of
Prochlorococcus and eukaryotic algae with higher chlorophyll content (Wilcoxon Matched Pairs
test, P<0.001). Larger (higher forward scatter) Synechococcus and eukaryotic algae but not
Prochlorococcus cells were also preferentially retained. No significant change was evident for the
chlorophyll and phycoerythrin content (orange fluorescence) of Synechococcus populations
exhaled by the bivalve. Further, the small fraction (5%) of non photosynthetic bacteria retained by
the bivalve had significantly higher green fluorescence (nucleic acids content).
46
Chap. 6. Phytoplankton grazing in reef rocks
T. swinhoei
The sponge T. swinhoei was the most efficient grazer studied, removing up to 100% of
certain prey types from the water it pumped (Fig. 1A, Fig. 7A,B) with averages ranging from
73±28% for eukaryotic algae to 95±7% for Prochlorococcus. T. swinhoei was also the only grazer
that removed a considerable portion of the non-photosynthetic bacteria populations (84±8%, Fig.
1A, Fig. 7D). Due to its high filtration efficiency, selectivity in the sponge diet was less prominent
(Fig. 1B) but still significant (repeated measures ANOVA, F3,84=19.9, P<0.001). The sponge's
preferred prey types were pico-cyanobacteria with some predilection for Prochlorococcus (Tukey
HSD test P<0.001, Fig. 1B). In September 1998 an usually large eukaryotic algae population
dominated (FSC 18.3±4.9) and were the less preferred food type for the sponge ((Tukey HSD test
P<0.001, Fig. 7C, E), which removed only 31±10% of these algae. In July and September 2000,
however, removal efficiencies resembled those of the other prey types (90±4% and 82±6%,
respectively) and preference ranking was Pro>Syn>Euk>Bact (repeated measures ANOVA,
F3,54=77.5, P<0.001). Relationships between prey removal and ambient prey concentration were
remarkably linear over the entire range of ambient concentrations encountered (R2>0.87, Fig. 7AD), indicating constant removal efficiencies.
The most notable feature of the sponge's selection for specific cell attributes within prey
type was the discrimination against larger (high scatter) photosynthetic cells (Fig. 6C). This
pattern was consistent throughout the three sampling seasons for both the larger eukaryotic algae
and the minute coccoid cyanobacteria (Prochlorococcus and Synechococcus). The selection
pattern for chlorophyll content (red fluorescence) was more complex with significant interaction
between the sampling period and the removal effcincies (repeated measure ANOVA F2, 36=13.2,
P<0.001). In September 1998, the removed eukaryotic algae had, on average, higher chlorophyll
content (despite their reduced scatter), whereas in 2000, the removed algae had reduced scatter
and reduced chlorophyll fluorescence.
H. gangelion
The filtration pattern of the ascidian H. gangelion resembled that of the bivalve (Fig. 1A, B)
with some subtle differences (Fig. 8). Removal efficiencies of photosynthetic cells were
intermediate, slightly higher than those of L. simplex and lower than those of T. swinhoei.
Average efficiencies ranged from 55±21% for eukaryotic algae to 76±16% for Synechococcus
(Fig. 1A, Fig. 8). Similar to the bivalve, the ascidian was highly selective (Friedman ANOVA,
χ2(61,3)=140.6, P<0.001) but its selectivity showed a significant interaction with sampling period
(repeated measure ANOVA, F15, 141=11.8, P<0.001). Synechococcus (FSC, 0.78±12) was the
most preferred prey throughout the research and this preference was elevated in the absence of
Prochlorococcus (Fig. 8E). In contrast to the high preference for the minute photosynthetic
bacterium Prochlorococcus (FSC, 0.50±0.13, αpro= 0.32±0.06), and its efficient removal
(65±19%, Fig. 1A, Fig. 8B), only a small fraction of the non-photosynthetic bacteria (FSC,
0.36±0.16, removal, 8±7%, Fig. 8D) was retained by the ascidian. The retention of
Prochlorococcus exceeded 20% in 63 of the 64 cases when Prochlorococcus was present,
whereas filtration of non-photosynthetic bacteria exceeded that value in only four of the 87
samples, with null to "negative" removals in about a third.
In H. gangelion, filtration efficiencies of Prochlorococcus and eukaryotic algae varied
considerably between different sampling periods (Kruskal-Wallis ANOVA, P<0.05, Fig. 8E). For
example, Prochlorococcus removal in fall (82±14%) and winter 1988 (77±6%), was significantly
higher (Tukey HSD test, P<0.01) than in the 1997 and 2000 summer sampling sessions (42%47
Chap. 6. Phytoplankton grazing in reef rocks
63%). Similarly, removal efficiency for eukaryotic algae in March 1997 (23±11%) was
significantly lower than those of several other sampling seasons (January and September 1998,
April and September 2000, Tukey HSD test, P<0.001) but not of July 1997, September 1997, and
February 2000. Moreover, while in most cases preference ranking was Syn>Pro>Euk>Bact, in
September 2000 eukaryotic algae were preferred over Prochlorococcus (Fig. 8E). The removal of
coccoid cyanobacteria remained remarkably constant despite the seasonal shifts in planktonic
community composition. The ascidian functional response with respect to Prochlorococcus and
Synechococcus was remarkably linear over the large range of ambient concentrations encountered
(R2>0.62, Fig. 8A, B, Fig. 8A, insert).
Similar to the bivalve, the small fraction (8%) of non photosynthetic bacteria retained by the
ascidian contained significantly higher nucleic acid than the ambient (inhaled) population (Fig.
6B). However, no selectivity for specific cell attributes within photosynthetic prey population was
found (Fig. 6B). The only exception was a preference for smaller Synechococcus (lower forward
scatter) that was evident in about two thirds of the cases (Wilcoxon Matched Pairs test, P<0.001).
Discussion
This study demonstrates selective, size-independent capture of planktonic heterotrophic and
autotrophic microorganisms by coral reef suspension feeders belonging to different phyla, each
with markedly different water transport and filtration systems. Selection for different types of
cells was evident both between (e.g., photosynthetic versus non-photosynthetic prokaryotes) and
within (e.g. cells with higher pigment content) taxa. The sponge T. swinhoei was the most
efficient and the least selective filterer. It was also the only one to remove large proportions of
non-photosynthetic bacteria. The bivalve L. simplex was the least efficient and most selective,
preferring the coccid cyanobacteria Synechococcus (~1 µm) over both the larger eukaryotic algae
and the smaller non-photosynthetic bacteria. The ascidian H. gangelion showed intermediate
filtration efficiency and selectivity. In contrast to the bivalve and sponge, H. gangelion exhibited
significant seasonal variation in prey preferences.
To minimize artifact, which confounded laboratory studies in temperate waters (review by
Jørgensen 1966; Riisgård 2001), this study was based on an in situ sampling technique. Direct
filtration efficiencies were measured in the field with undisturbed animals. Remote observations
and measurements using an online video system and acoustic current measurements of the excurrent jet velocities confirmed that the animal behavior was not affected by the sampling (Yahel
et al. 2003 and unpublished data). Our study tested only the first step in the feeding process,
namely, particle trapping and retention from the highly diluted medium in which the suspension
feeders reside. Post-trapping (pre- and post-ingestion) sorting was reported for bivalves (Ward et
al. 1997, 1998a,b; Cranford and Hill 1999), sponges (reviewed by Witte et al. 1997) and to some
extent for ascidians (Armsworthy et al. 2001). These mechanisms may provide the organisms
with further control of their diet but were beyond the scope of the present study.
The planktonic community in the water overlying the studied coral reefs in Eilat was typical for
warm oligotrophic surface waters (Shalapyonok et al. 2001) and both cell concentrations and
optical properties resembled those described in the nearby open waters by Lindell and Post (1995)
and Sommer et al. (2002). Since seasonal changes in filtration efficiencies and pumping rate were
relatively minor, changes in carbon or nitrogen gain were driven mostly by seasonal changes in
the plankton community. In-depth discussion of these aspects of the studied suspension feeders'
48
Chap. 6. Phytoplankton grazing in reef rocks
ecology is beyond the scope of the present article and will be presented elsewhere (Yahel, Marie
and Genin, in prep.)
The assertion that cell removal efficiency was not size-dependent was based upon the following
observations:
1.
2.
3.
4.
The ascidian and bivalve efficiently removed the minute photosynthetic bacterium
Prochlorococcus but not the non-photosynthetic bacteria despite a large overlap in the
scatter distribution of these two populations.
All three grazers significantly preferred the pico-planktonic coccoid autotrophs
(Synechococcus and Prochlorococcus) over both larger eukaryotic algae and the somewhat
smaller non-photosynthetic bacteria.
Cell size (scatter) distribution of the four prey taxa examined (non-photosynthetic bacteria,
Prochlorococcus, Synechococcus, and eukaryotic algae) typically overlapped by much more
than 25% (Fig. 2, compare also the scatter distribution of Synechococcus and the eukaryotic
algae in Fig. 4 and Fig. 5, respectively).
The patterns of size (scatter) and type selection did not match (Fig. 1, Fig. 6). For example,
among prey types, L. simplex preferentially selected Synechococcus over the eukaryotic
algae (that on average were larger); however, within the eukaryotic algae the bivalve
preferred the larger cells (Fig. 5, note the log scale on the x axis). Similarly, H. gangelion
rejected non-photosynthetic bacteria and preferred the smallest Synechococcus cells but
exhibited no size selection within either Prochlorococcus or eukaryotic algae (Fig. 6B).
Selective grazing in the micron and sub micron range has been documented in the pelagic realm
(Christaki et al. 1999, Sommer et al. 2002). The preference for Synechococcus by the benthic
suspension feeders reported here was also evident in nine other reef suspension feeders' taxa we
examined with the InEx technique, both in the Gulf of Aqaba and in the Indian Ocean (3 sponge
species, 3 bivalve species, 2 solitary ascidians, and a colonial tunicate, Yahel et al. in prep). The
only exception was Tridacana squamosa, which preferred larger eukaryotic algae. In Curacao,
van Duyl et al. (2002) reported that the concentration of Synechococcus pigments was
significantly reduced in comparison with other algal pigments in the water flowing over the reefs,
indicating its selective grazing. These findings are in sharp contrast to theoretical models
predicting that particles in the size range of 0.2-1 µm have the lowest probability to encounter
planktonic grazers feeding appendage (Shimeta 1993). Indeed, in a recent pelagic grazing sample,
Sommer et al. (2002) found Synechococcus to be the least preferred cell type in the open waters
of the Gulf of Aqaba. As the phytoplankton supply to the reef is mostly affected by pelagic
processes (van Duyl et al. 2002; Genin et al. 2002) it could be speculated that actively pumping
suspension feeders are more suitably adapted to filter ~1µm cells in comparison to pelagic
suspension feeders. This adaptation may have evolved in response to the relatively high
abundance and persistent populations of Synechococcus in oligotrophic waters.
Light scatter and cell size determination
Light-scatter of single cells as a proxy of size in natural planktonic assemblages analyzed by a
flow cytometer is widely used either as a relative measure (Cavender-Bares et al. 2001) or as an
absolute measure after proper calibration for cultured strains (Shalapyonok et al. 2001). The
scatter signal of large eukaryotic algae is sometimes obscure (e.g., Cavender-Bares et al. 2001),
largely due to the fact that these algal cells have complex shapes, and their component parts have
a wide range of refraction and absorption characteristics (Cunningham and Buonnacorsi 1992).
Simon et al. (1994) found that preservation severely affects the correlation of forward scatter, but
49
Chap. 6. Phytoplankton grazing in reef rocks
not side scatter, with cell size in pico-eukaryotic algae. These limitations, however, are much less
severe for the small, simply shaped pico-prokaryotes studied by us (Cunningham and
Buonnacorsi 1992, Robertson et al. 1998). In this study we made no attempt to calibrate the
scatter signal to cell volumes or size, rather we conservatively infer a positive, roughly linear
relationship of the forward scatter with picoplankton (<2µm) cell volume (e.g., Simon et al. 1994,
Cavender-Bares et al. 2001). This assumption may, however, underestimate smaller cell size (e.g.
coccid bacteria) as discussed by Shalapyonok et al. (2001). For the larger eukaryotic algae only a
monotonic, positive, relationship was assumed.
Grossart and Simon (2002) used DAPI staining and image analysis to record 20 size classes of
bacteria between <0.2 µm and 2 µm in the open water of the Gulf of Aqaba; however, only 15%
of all measured bacteria were larger than 0.4 µm. According to H.P. Grossart (pers. com.), larger
bacteria prevailed in the photic zone whereas in the a-photic zone bacteria were significantly
smaller. Thus the above size range may grossly underestimate the actual size range of surface
bacteria. In contrast Gundersen et al. (2002) using electron microscopy, found that >30% of the
bacteria in the Bermuda Atlantic Time-series Study (BATS) were larger than 0.65 µm and >12%
were larger than 0.85µm (calculated from Fig. 1 in Gundersen et al. 2002).
Our cell properties comparisons were especially robust due to the paired sampling design applied
in this study (the same populations are compared in the same water prior to and after the passage
via a grazer filtration apparatus). The normalization to the calibration beads provided additional
protection against instrumental drifts and shifts in sheath fluid properties. Discrimination between
the four prey types was not on the same taxonomic level. Moreover, the accuracy of
discrimination was also variable; it was close to 100% for Synechococcus and the eukaryotic
algae but much lower for Prochlorococcus and non-photosynthetic bacteria that on occasion
merged with the noise. To be conservative we have omitted from the cell property analysis any
samples where the non-photosynthetic bacteria, Prochlorococcus, and noise could not be fully
resolved (see methods). As a result, we have most likely overestimated the actual differences
between the non-photosynthetic bacteria and Prochlorococcus populations. Population size has an
important effect on the accuracy of statistical description of cell properties. Thus, larger
confidence intervals were usually associated with smaller populations such as the eukaryotic
algae (and in some seasons also Prochlorococcus), as well as with the decimated populations in
the exhaled water (especially for T. swinhoei).
L. simplex
L. simplex preferentially retained specific cell types, such as Synechococcus, over both larger
eukaryotic algae and smaller bacteria (Fig. 1, Fig. 3E). The minute Prochlorococcus was also
efficiently removed (up to 88%) despite a considerable size overlap with non-photosynthetic
bacteria that were retained in null to very low efficiencies (Fig. 1, Fig. 3A-D). Larger cells and
cells with higher chlorophyll content were preferred both within Synechococcus and eukaryotic
algae. These selectivity patterns prevailed regardless of large seasonal shifts in the planktonic
community composition and abundance. The most notable exception was the reduced removal
efficiency of Prochlorococcus in September 2000 (Fig. 3B, E).
A size-independent retention of phytoplankton was reported for some bivalves feeding on large
particles in temperate zones (e.g., Ostrea edulis, Shumway et al. 1985; Mytilus edulis, Newell et
al. 1989, Bougrier et al. 1997; juvenile scallops, Shumway et al. 1997; and Crassostrea gigas
50
Chap. 6. Phytoplankton grazing in reef rocks
Bougrier et al. 1997, reviewed by Bougrier et al. 1997). Based on gut content analysis, Loret et al.
(2000) report on selective, quality-based feeding by the tropical pearl oyster Pinctada
margaritifera with high preference for Cryptophytes (5-10 µm) and against similar sized
Chlorophytes and Prymnesiophytes. Synechococcus type cyanobacteria were only weakly
retained, presumably due to their small size (see also Yukihira et al. 1999). The methodology
used by Loret et al. did not allow discrimination between preferential retention and post trapping
(preingestive) selection. It should be noted that while only a insignificant portion of the nonphotosynthetic bacteria was retained by L. simplex, other bivalves such as Geukensia demissa
were capable of efficient filtration of such small cells (Kreeger and Newell, 2001). Most
interestingly, Pile and Young (1999), using a method similar to ours, found that median size
specimens of a cold seep symbiotic mussel (25-90 mm) showed a bimodal filtration efficiency,
preferentially retaining bacteria over the larger protozoans, but removing none of the intermediate
size Synechococcus cells.
A decrease in clearance rate in response to elevation in the supply of particulate organic matter
was commonly reported for suspension feeding bivalves (e.g., Yukihira et al. 2001 and references
therein). This reduction can be a consequence of reduced filtration efficiency, reduced pumping
rate, or both. Using direct filtration efficiency measurements, we found no reduction in filtration
efficiencies over the entire range of plankton concentration encountered. Thus, if L. simplex is
reducing its food intake under natural conditions, it should do so primarily by variation of
pumping rates. However, pumping rate measurements carried out concurrently with our InEx
measurements (averaged 10±3 ml specimen-1 min-1) indicated no correlation with particulate food
availability (Yahel, Marie, and Genin, in prep).
A full understanding of the mechanism underlying the above selectivity patterns is not possible at
this stage, as the fine details of particle capture are still being debated even for temperate bivalves
that feed on relatively large phytoplankton (Ward et al. 2000, and references therein). Earlier
reports on differential, size-independent selection by bivalves suggested that differences in algae
shape and flexibility may affect its capture. As the coccid Synechococcus was removed with the
highest efficiency, such an explanation seems less plausible in the case of L. simplex. Shimeta
(1993) suggested that the clearance of sub-micrometer particles should be greatly enhanced by
cells motility. Indeed swimming behavior was found in several cultured Synechococcus strains
(Brahamsha 1999). On the other hand, similar motility is not known in Prochlorococcus cells,
which appear to be lacking the necessary genes (A. F. Post, pers. com.). Thus some sort of cell
recognition mechanism may operate at the sites of cell interception on the bivalve gills.
T. swinhoei
The sponge T. swinhoei was the most efficient grazer studied, removing up to 100% of
Prochlorococcus and Synechococcus cells from the water it pumps. T. swinhoei was also the only
grazer that removed a considerable portion of the non-photosynthetic bacteria (Fig. 1A, Fig. 7D).
Both non-photosynthetic bacteria and eukaryotic algae were significantly less preferred. Similar
in situ feeding efficiency and preference patterns were found by Pile et al. (1996) in the boreal
sponge Mycale lingua. Interestingly, the reduced filtration efficiency of eukaryotic algae was
associated with a preferential retention of smaller cells (Fig. 6C).
Sponges utilize a unique filtration system, allowing a removal of a diverse spectrum of food
ranging from DOC (Yahel et al. 2003) to zooplankton (reviewed by Ribes et al. 1999). While we
are not aware of any study refuting selective feeding in sponges, the notion that sponges are
unable to filter selectively is common in the literature (e.g., Pile and Young 1999; Kowalke
51
Chap. 6. Phytoplankton grazing in reef rocks
2000). In fact, close examination of Fig. 2B in Pile et al. (1996) and Fig. 2 in Kowalke (2000)
indicate clear differential retention in both cases. Selective retention with preference for smaller
particles was reported by (e.g.) Reiswig (1971), Witte et al. (1997), and Ribes et al. 1999.
Wilkinson et al. (1984) used radioactive labeling to demonstrate discrimination between
symbiotic and 'food- type' bacteria in two tropical sponges. Some of the above ambiguity may
stem from the ability of sponges to ingest (into specialized phagocytes) many types of particles
(reviewed by Witte et al. 1997) and subsequently release unwanted ones into the exhalent currents
(reviewed by Wilkinson et al. 1984).
The comparison of within prey type selectivity in T. swinhoei should not be over applied to
Synechococcus and Prochlorococcus, as almost all cells were usually removed by the sponge.
Within the eukaryotic algae, however, a clear negative selection of larger cells was always
evident, in fact, in September 1998, when most of the eukaryotic algae were large (FSC
18.3±3.4); filtration efficiency of these cells was reduced by more than two-fold. (Note that
prescreening of larger cells due to ostia size would have resulted in apparent elevated filtration
efficiency for these cells.) Assuming that all the exhaled cells are captured by the sponge and
actively transported by archaeocytes to the exhalent canals, some changes in cell properties may
be a consequence of partial digestion during transport (Reiswig 1971; Wilkinson et al. 1984).
Nevertheless, some cell types have been reported to pass intact through some tropical sponges
(Reiswig 1971).
InEx samples with three other reef sponges (Mycale fistulifera, Subarites clavatus, Cliona sp.)
showed similar selectivity patterns, with prokaryotic phytoplankton being preferred over both
non-photosynthetic bacteria and eukaryotic algae, although M. fistulifera filtration efficiencies
were much lower than those of T. swinhoei whereas those of the boring sponge Cliona sp. were
significantly higher (Yahel, Marie and Genin, in prep.). As discussed above, carbon is not
necessarily a rare commodity in the reef and filtration mechanisms may have been adapted to
supply suspension feeders with other required nutrients. This is especially likely for T. swinhoei,
as living cells comprise only >10% of the sponge's diet whereas most of its respiratory demand is
supplied by the uptake of dissolved organic carbon (Yahel et al. 2003).
H. gangelion
The overall mean filtration efficiency of the H. gangelion ranged from 8% for non-photosynthetic
bacteria to 76% for Synechococcus, with the latter resembling published values for other solitary
ascidian (Fiala-Medioni 1978; Armsworthy et al. 2001). Filtration efficiencies in the range of 95100% such as those reported by Robins (1984) for some phleobranchiate ascidians were recorded
only 10% of our measurements and only for Synechococcus and/or Prochlorococcus. Similar to
the bivalve, the ascidian was highly selective, retaining only a few non-photosynthetic bacteria
but as much as 97% of the photosynthetic bacterium Prochlorococcus (Fig. 1, Fig. 2, Fig. 8).
Unlike the bivalve, which selected for larger eukaryotic algae, and the sponge, which preferred
smaller eukaryotic algae (Fig. 6 A, C, respectively), the ascidian showed no consistent overall
preference for a characteristic eukaryotic alga.
Using a recirculating bell jar, Ribes et al. (1998) found that the ascidian H. papillosa
indiscriminately removed all living particles ranging from non-photosynthetic bacteria up to 60
µm pennate diatoms. Indiscriminate feeding was also reported by Robbins (1984) for
phleobranchiate ascidians. Kowalke (1999) measured the size spectrum of particles retained by
four Antarctic ascidian species and reported decreasing removal efficiency below a certain size
threshold. In contrast, Armsworthy et al. (2001) found clear shifts in the retention efficiency by H.
52
Chap. 6. Phytoplankton grazing in reef rocks
pyriformis from preferential removal of large particles (>5 µm) in low sediment loads to small
particles (<3 µm) in higher sediment loads. Armsworthy et al. suggested that H. pyriformis was
capable of altering the structure and retentive capabilities of the mucus net through squirting.
Selective rejection of unwanted particles by squirting and evacuation of the atrial cavity was
noted in other ascidians (reviewed by Armsworthy et al. 2001). In the present study we tested
only the first step of filter feeding, namely, the capture of particles from the flowing waters.
Hence, we did not discriminate between ingested particles and those that may have been
subsequently rejected by squirting. On the fore-reef of Eilat, squirting by H. gangelion is rare (<3
squirts hour-1, Yahel, unpublished online video monitoring). In fact we noted correspondence
between squirting and disturbance and therefore in the few cases when squirting occurred, the
samples were discarded.
Active versus inactive bacteria
Gasol et al. (1995) reported that active bacteria (cells that take up and reduces the redox dye
CTC) were more likely to be larger and therefore should be most likely cropped if grazers select
for larger prey. A test of this hypothesis during a period (September 2000) when CTC positive
cells comprised 11±7% of the total bacterial population indicated no significant selectivity. This
observation is in accordance with the general lack of evidence for size-based selectivity in the
studied grazers. It should be noted, however, that the reliability of the CTC method in
discriminating active versus inactive cells was recently questioned (e.g., Servais et al. 2001).
There is also increasing evidence that high nucleic acid content is a better predicator for bacteria
activity and viability (Lebaron et al. 2001). Recently Bernard et al. (2000) suggested that the most
active bacteria are actually those with medium cell-size. Indeed, while the small fraction of nonphotosynthetic bacteria removed by both L. simplex and H. gangelion had a medium size (i.e.,
scatter) it had a significantly higher nucleic acid content (Fig. 6), suggesting that it corresponded
to the actively multiplying bacteria of Lebaron et al. (2001).
Conclusions
Significant, size-independent selectivity was exhibited by all three benthic tropical picoplankton
grazers examined. Filtration efficiency was generally invariable over a large range of ambient
concentrations but was affected by apparent shifts in the ultraplankton species composition. We
suggest that selectivity is not size-dependent and probably relies on other cell attributes such as
motility and/or cell surface properties. The mechanisms underlying the observed selectivity in the
different phyla are still unresolved. The overall preference pattern in the three phyla is
surprisingly similar, exhibiting preferences of coccoid photosynthetic prokaryotes over both
larger or similar sized eukaryotic algae and smaller or similar sized non-photosynthetic bacteria.
This preference pattern appears to control the planktonic community structure over coral reefs
(van Duyl et al. 2002). As the observed selectivity did not appear to maximize carbon or energy
gain, it is suggested that at the reef, where carbon is not a rare commodity, suspension feeders
have evolved to optimize other factors such as the gain of nutrients or avoidance from harmful
toxins.
53
Chap. 6. Phytoplankton grazing in reef rocks
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Chap. 6. Phytoplankton grazing in reef rocks
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58
Chap. 6. Phytoplankton grazing in reef rocks
Figure legends
Fig. 1 A. The average filtration efficiency of the bivalve L. simplex, the ascidian H. gangelion,
and the sponge T. swinhoei,. Error bars, 95% confidence intervals.
B. Average selectivity index (αi, Chesson, 1978) calculated for all InEx pairs where data for all 4
prey items were available. The expected value for non selective predation was 0.25 (dotted line).
Error bars, 95% confidence intervals.
Fig. 2 A representative comparison of the normalized frequency distribution of the cellular
properties of Prochlorococcus (open symbols) and non-photosynthetic bacteria (filled symbols) in
the ambient water next to the studied specimens based on 10 inhaled water samples collected on
four different days in September 1998. The samples were stained with the nucleic acid stain Syber
Green I. Prochlorococcus was distinguished from the non-photosynthetic bacteria on the side
scatter vs. red fluorescence cytogram based on their red chlorophyll fluorescence. Bin averages
were calculated for cell frequencies within each of 256 logarithmically spaced bins (channels) and
normalized as the percentage off the total number of cells in the respective population. A. Side
scatter is related primarily to cell volume and texture and to some extent also to cell fluorescence.
B. Forward scatter is related to cell size. Horizontal axes are plotted using arbitrary, log scale
units. Shaded area, 95% confidence interval of the mean
Fig. 3 Lithophaga simplex: A-D, Removal of each of the four ultra-planktonic preys plotted
against its ambient (inhaled) concentration. Pro – Prochlorococcus, Syn – Synechococcus, Euk Eukaryotic algae, Bact- non-photosynthetic bacteria. Dotted lines (X=Y) represent 100% removal.
Different symbols denote different sampling periods ( October 1996, ○ March 1997, ●
September 1997, □ January 1998, ∆ September 2000). R2 and slope are the linear regression
statistics for all data pooled.
E. Prey preference in the different sampling periods. Chesson αis were rescaled to εi so that it
would be independent of the number of prey types available. εi range from -1 to 1, where -1
indicate none of the ith prey type is taken and εi of 1 indicate cases when the ith prey type is the
only one selected for. Zero is the expected value for ε if there is no preference. Error bars were
omitted for clarity of presentation (Chesson 1983).
Fig. 4 An example of the normalized frequency distribution of the optical cell properties of
Synechococcus in the water inhaled (filled symbols) and exhaled (open symbols) by 12
Lithophaga simplex. As each sample was internally normalized, similar relative frequencies (Y
position) do not indicate similar concentrations (exhaled concentrations were significantly lower).
Samples were collected during two days in the 1997 eukaryotes spring bloom (18 and 25 March
1997). List mode data were transformed from log to linear and the Skewness (Sk) and Kurtosis
(Ku) were calculated separately for each sample before normalization. The difference in the
location of the distribution (population mean) was highly significant in all cases (paired t-test,
P<0.001). The shape of the distributions was also significantly different for all but the
phycoerythrin fluorescence distribution (Wilcoxon Matched Pairs test, P<0.05 for the comparison
of both the Skewness and Kurtosis within each pair). Orange fluorescence is related to
Synechococcus phycoerythrin content (a light harvesting and nitrogen storage pigment).
Horizontal axes are plotted using arbitrary, log scaled units. Symbol size roughly resembles the
standard error.
59
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 5 As Fig. 4 but for eukaryotic algae (<8µm) cells. Since these cells normally do not contain
phycoerythrin, the orange fluorescence signal was not plotted. The difference in the location of
the distribution (population mean) was highly significant in all cases (paired t-test, P<0.001). The
shape of the distributions was also significantly different for all but the side scatter distributions
(Wilcoxon Matched Pairs test, P<0.05).
Fig. 6 The average (±SE) normalized differences of cells characteristics between the inhaled and
exhaled waters within each prey type. (A) L. simplex, (B) H. gangelion, (C) T. swinhoei.
Normalized difference was calculated separately for each InEx pair as 100 (AIn-AEx) AIn-1 where
AIn and AEx denote the average attribute of the respective cell population in the water inhaled and
exhaled by the studied taxa, respectively. Positive values indicate selection for prey with higher
cell attributes (fluorescence or scatter) with values indicating the percent deviation from the
respective inhaled population. The significance of the difference between inhaled and exhaled
population attributes was tested using Wilcoxon Matched Pairs test: ns, no significant difference,
*,<0.05, **, P<0.01, ***, <0.001. Attributes that were not relevant for the respective prey
population are indicated as “er”. Error bars, SE. Note the scale differences between the panels.
Fig. 7 Theonella swinhoei: A-D, Removal of ultra-plankton plotted against its ambient (inhaled)
concentrations. (■ September-October 1998, ▼ July 2000, ∆ September 2000). E. Comparison of
prey preference patterns for each of the sampling periods. See Fig. 3 legend for details).
Fig. 8 Halocynthia gangelion: A-D, Removal of ultra-plankto plotted against their ambient
(inhaled) concentrations (○ March 1997, ● July-September 1997, □ January-February 1998, ■
September-October 1998, ¤ November 1999, ▲ February 2000, ∇ April 2000, ∆ September
2000). E. Comparison of prey preference patterns for each of the sampling periods. See Fig. 3
legend for details).
60
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 1
Pro
Syn
Euk
Bact
Filtration efficiency (%)
A
100
80
60
40
20
0
Chesson selectivity index (αi)
L. simplex
H. gangelion
0.6
T. swinhoei
Pro
Syn
Euk
HBac
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
L. simplex
H. gangelion
61
T. swinohei
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 2
A
Pro
Bact
3
Relative frequency (%)
2
1
0
10
100
Side scatter
2
B
1
0
1
10
Forward scatter
62
100
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 3 Lithophaga simplex
A. Syn
150
2
R =0.81
Slope=0.67
20
100
10
50
0
0
B. Pro
2
R =0.62
Slope=0.36
0
10
20
30
0
900
C. Euk
100
150
2
R =0.79
Slope=0.47
10
50
D. Bact
2
R =0.32
Slope=0.54
600
300
5
0
0
-300
0
5
10
0
3
300
600
900
-1
Cells inhaled (x 10 cells ml )
Chesson electivity index (εi)
3
-1
Cells removed (x 10 cells ml )
30
E
Pro
Syn
Euk
Bact
0.5
0.0
-0.5
Oc
M
S J
t-9 ar-9 ep-9 an-9
6
7 8
7
63
Se
p-0
0
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 4
Inhaled
Exhaled
A
3
2
1
0
10
Relative frequency (%)
2
B
100
Side scatter
1
0
10
100
Forward scatter
C
3
2
1
0
3
D
100
Red (chlorophyll) fluorescence
2
1
0
100
1000
Orange (phycoerythrin) fluorescence
64
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 5
A
Inhaled
Exhaled
3
2
Relative frequency (%)
1
0
2
100
Side scatter
B
1
0
100
1000
Forward scatter
C
3
2
1
0
100
Red (chlorophyll) fluorescence
65
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 6
ns
ns
***
*
er
***
ns
ns
er
-10
*
*
***
*
10
er
ns
ns
ns
er
ns
ns
ns
B
***
er
ns
ns
0
-10
*
*
***
***
30 C
er
**
**
*
-20
er
*
***
***
Green (Fl1) / Orange (Fl2)
Red (Fl3)
Forward scatter
Side Scatter
Euk
Pro
Syn
0
ns
er
ns
**
Celular attribute change (%)
0
***
**
ns
10
A
**
er
ns
ns
20
-30
-60
-90
-120
Bact
Prey type
66
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 7 Theonella swinhoei
50
A. Syn
B. Pro
2
R =0.98
Slope=0.93
2
R =0.99
Slope=1.00
200
100
0
0
0
25
50
0
2
R =0.87
Slope=0.93
R =0.97
Slope=0.98
5
500
0
0
0
200
1000 D. Bact
2
C. Euk
10
100
5
10
0
3
300
600
-1
0.4
0.2
E
Pro
Syn
Euk
Bact
0.0
-0.2
98
-0.4
Se
p-
Chesson electivity index (εi)
Cells inhaled (x10 Cells ml )
67
Ju
Se l-00
p00
3
Cells removed (x 10 cells ml-1)
25
900
Chap. 6. Phytoplankton grazing in reef rocks
Fig. 8 Halocynthia gangelion
40
300
A. Syn
200
200
B. Pro
2
R =0.62
Slope=0.41
3
-1
Cells removed (x 10 cells ml )
100
20
0
0
100 200 300
100
2
R =0.80
Slope=0.92
0
0
15
20
0
40
0
1000
C. Euk
2
2
R =0.81
Slope=0.16
500
5
0
0
0
5
10
15
0
3
300
-1
Cells inhaled (x 10 cells ml )
0.5
E
0.0
Pro
Syn
Euk
Bact
Fe
A b0 0
pr
00
Se
p0
0
Se
p9
8
-0.5
M
ar
97
Ju
S e l97
p9
7
Ja
n9
8
Chesson electivity index (εi)
200
D. Bact
R =0.40
Slope=0.26
10
100
68
600
900
Chap. 6. Phytoplankton grazing in reef rocks
6.
Phytoplankton grazing by epi- and in-fauna inhabiting
exposed rocks in coral reefs
By
Gitai Yahel*, Tania Zalogin, Ruthy Yahel#, and Amatzia Genin
Coral Reefs – in press
69
Chap. 6. Phytoplankton grazing in reef rocks
Abstract
Exposed rocks with no visible macro-fauna are abundant in all coral reefs. Depletion of
phytoplankton cells and pigments by the minute crypto fauna inhabiting the outer few centimeters
of such rocks was experimentally studied over an annual cycle in the Gulf of Aqaba, Red Sea.
Different substrata were introduced into small (3.6 L), well mixed, tanks that were fed by running
seawater pumped directly from the reef at a rate of 11±1 L hr-1. A steady-state reduction in
phytoplankton abundance and chlorophyll a concentration of 38±26% (mean ± 1 SD) was found
for untreated rocks but not for sand, gravel, or killed controls. Average areal clearance rate by
untreated rocks was 17.3±8.0 ml cm-2 hr-1. Conservative extrapolation of this rate to the whole
reef community, suggests that the fauna inhabiting exposed rocks clears 2.1± 0.9 m3 m-2 d-1 at
Eilat. Phytoplankton removal by untreated rocks varied from 1.5 ng chlorophyll a cm-2 hr-1 during
the oligotrophic summer conditions to 6 ng chlorophyll a cm-2 hr-1 during the spring bloom. These
values correspond to a potential nitrogen gain of 1.3 and 5.2 mmol N m-2 day-1, respectively.
Cryptic reef-rock fauna can have a key role in the biogeochemical functioning of coral reef
communities.
70
Chap. 6. Phytoplankton grazing in reef rocks
Introduction
Benthic grazing on phytoplankton is a principal trophic pathway in shallow, temperate coastal
habitats (Riisgård et al. 2004 and references therein). The dominant grazers (phytoplanktivores) in
those communities include bivalves, ascidians and polychaetes (Riisgård 1998 and references
therein). Studies of benthic-pelagic coupling in coral reefs, where the phytoplanktonic community
is dominated by minute prokaryotic cells (Ferrier-Pages and Gattuso 1998), have focused on
zooplankton, rather than phytoplankton, as the principal source of prey (reviewed by Yahel et al.
1998). However, following the advent of technologies that allow identification and counting of
micron-size picoplankton in aquatic ecosystems, many recent studies demonstrated the significant
role of phytoplanktivory and bacteriovory in the trophic dynamics of coral reefs (Ayukai 1995;
Ferrier-Pages and Gattuso 1998; Gast et al. 1998; Yahel et al. 1998; Richter and Wunsch 1999;
Fabricius and Dommisse 2000; Richter et al. 2001; van Duyl and Gast 2001; van Duyl et al. 2002;
Genin et al. 2002; Houlbrèque et al. submitted). Nevertheless, unlike temperate and boreal coastal
habitats, the identification of the key benthic phytoplanktivores and the assessment of their
feeding rates have, by and large, remained elusive. To our knowledge, phytoplanktivory has been
investigated only in 5 coral reef dwellers: an Octocoral (Fabricius et al. 1998), two sponges (Pile
1997; Yahel et al. 2003) and commercially reared bivalves (Yukihira et al. 1999; Loret et al.
2000).
Potential sinks for phytoplankton in coral reefs could be physical mechanisms such as
entrainment and burial by high pressure surge in the reef framework (Haberstroh and Sansone
1999) and wave driven filtration via sand ripples (Huettel and Rusch 2000). However, such
mechanisms pertain primarily to the surge zone and cannot account for removal processes
occurring in moderate and low flow environments below the wave action zone (Genin et al. 2002;
Reidenbach et al. submitted). Several biotic guilds have been suggested by various authors to be
the dominant phytoplankton grazers in different reefs. For instance, following the discovery of
phytoplanktivory in the octocoral Dendronephthya hemprichi (Fabricius et al. 1995),
phytoplankton depletion over soft coral dominated reefs was attributed to the corals (Yahel et al.
1998; Fabricius and Dommisse, 2000). Yahel et al. (1998) have speculated that epibenthic active
suspension-feeders may be responsible for the phytoplankton depletion over fringing reefs at the
Gulf of Aqaba where soft corals are scarce. In follow-up studies, Richter and Wunsch (1999) and
Richter et al. (2001) suggested that the entire removal of phytoplankton can be accounted for by
cavity-dwelling fauna, primarily sponges. Yet, reef cavities were extremely rare at the
experimental sites of Genin et al. (2002) in the Gulf of Aqaba where intense phytoplankton
grazing was documented. Moreover, up scaling calculations of individual grazing rate
measurements made at the same sites (Yahel et al. in prep.) suggest that visible active suspension
feeders (including, sponges and boring sponges, Lithophaga and other bivalves, and tunicates)
account for only ~15% of the observed phytoplankton removal.
In a recent compilation of >1000 surveys of different coral reefs, Hodgson and Liebeler (2002)
reported that "exposed" rocks, that is, hard substratum (excluding recently killed corals) not
occupied by live corals, sponges, fleshy algae, or other conspicuous macrofauna, comprise on
average 26% of the Indo-Pacific and 18% of the Atlantic reefs surface area, compared with mean
live coral cover of 35% and 23%, respectively. Sand patches and coral rubble (0.5-15 cm) are the
two other predominant exposed substrata in coral reefs (Hodgson and Liebeler 2002). Aside from
the role infauna and epifauna have in reef bioerosion, their ecological functioning in the reef
community is poorly understood (Hutchings 1978, 1983; Gischler 1997). Several reports
describing the community inhabiting exposed rocks in coral reefs indicated that populations of
minute suspension feeders (such as polychaetes, foraminifers, tunicates, and boring sponges) are
71
Chap. 6. Phytoplankton grazing in reef rocks
highly abundant in this microhabitat (e.g., Bonem 1977; Peyrot-Clausade 1977; Vasseur, 1977;
Hutchings 1978, 1983; Gischler 1997, Holmse et al. 2000 and references therein). In reefs under
stress, where the abundance of dead coral, rocks and rubble is high (e.g., Edinger et al. 2000) and
where sometime anthropogenic eutrophication co-occurs, bioerosion becomes intense and the
abundance of boring sponge increases (Holmes et al. 2000 and references therein).
The objective of this study was to experimentally test the hypothesis that the cryptic fauna that
inhabits exposed rocks in coral reefs removes considerable amounts of phytoplankton from the
overlying waters, rendering significant its role in pelagic-benthic coupling in coral reefs.
72
Chap. 6. Phytoplankton grazing in reef rocks
Methods
Phytoplankton removal by the fauna inhabits different reef substrata (exposed rocks, gravel, and
sand) was studied at the fringing coral reef in front of the Steinitz Marine Laboratory of Eilat,
Gulf of Aqaba, Red Sea (29°30’N, 34°56’E). Reef substrata were brought to the laboratory and
placed in small tanks fed by running waters pumped from the reef. Phytoplankton concentration
was measured by using either chlorophyll a or cell counts with a flow cytometer. The sampling
period spanned the seasonal variation in the northern tip of the Gulf of Aqaba (Lindel and Post
1995) starting during summer stratification (August 2001) and extending through autumn
(September 2001) and the winter mixing (October-December 2001), to the spring bloom (March
2002). See Yahel et al. (1998, 2002) and references therein, for a description of the study site.
Experimental setup
The setup included 10 polypropylene tanks (3.6 L, Figure 1) into which different reef substrata
(untreated rock, gravel, sand, and controls, see below) were randomly allocated in each
experiment (Table 1). Each tank was supplied with running seawater via a 1 m long, 6 mm
diameter pipe from a common header tank (20 L) that supplied a constant flow of 11±1 L hr-1
(mean ± SD). To ensure thorough mixing of the water in the tanks (Riisgård, 2001), an adjustable
aquarium pump (100-300 L hr-1, Chosen Aquarium Equipment Corp. model AT005) was
mounted in each tank, and the tank water outlet was installed near the tank bottom (Figure 1). To
simulate natural flows, the pumps were adjusted so that the flow around the substratum surfaces
would be in the natural range encountered in the reef (5-15 cm sec-1, Yahel et al. 2002). It took a
few seconds for drops of fluorescein dye injected with a syringe to the tanks to be thoroughly
mixed. The water was pumped to the header tank from the near bed boundary layer, 0.5 m above
the reef surface, in front of the Marine Biology Laboratory of Eilat (Yahel et al. 2002). To
minimize the effect of fouling organisms within the pipes, a dual water supply system was
installed. Every few days, the systems were switched and the recently used system was flushed
for 30-60 min with fresh water to kill the fouling fauna. The tanks were immersed in a shaded
water table that was filled by the overflow from the header tank. The temperature and
illumination in the tanks were similar to those at the adjacent reef at 5 m depth.
Rocks, sand, and gravel
For each experiment, a fresh set of substrata was collected from 6-14 m depth on the fore reef and
transferred submerged in seawater to the tanks. Untreated rocks without visible macro-fauna
(mean displacement volume of individual rocks 390±209 ml, range 80-750 ml) covered most of
the tank bottom with a vertical relief of 5 – 15 cm. Whenever possible, the original vertical
orientation of the rock was maintained. To facilitate haphazard selection, volunteer divers were
asked to collect the rocks from the reef surface. In order to avoid unnecessary damage, no chisels
were used so that only loosely attached rocks were collected. Therefore, most rocks were
degraded coral skeletons, some still retaining the original coral form, while several were large
reef rubble (>10 cm e.g., Gischler 1997). Few rocks were a conglomerate composed of dead coral
skeleton together with non-carbonated cobbles originating from the nearby mountains. The
volume of the rocks in each tank was measured by the displacement volume method. In
September 2001 and March 2002, the entire surface area of each rock was also measured by
carefully fitting an aluminum foil to the rock surface and subsequently weighing the dried foil.
Gravel (408±181 ml per tank) and sand (414±239 ml per tank) were collected by scooping the
upper 5 cm of the substratum into a plastic bag at the same site of the rock collection. The gravel
(~0.5-5 cm) and sand (0.3-0.05 cm) at our site both contained more than 50% of non-carbonated
73
Chap. 6. Phytoplankton grazing in reef rocks
material. As these substrata are routinely perturbed by burrowing fish (Yahel et al. 2002), no
effort was made to retain their original orientation in the tanks.
For the control, untreated rock fauna was killed by drying the rocks outdoor for several days or
baking them at 3500C for three hours. In September and October 2001, control rocks were treated
with 0.5% NaOCl for 24 hours to minimize nutrient release from decomposing organic matter
(see below).
Water sampling and analysis
Each experiment lasted 2-4 days, with one sampling session during the day (see below),
sometimes followed with a second sampling session during the night starting 1-3 hrs after sunset
(Table 1). The only exception was the September experiment where sampling session were
disperse over a three weeks time.
After setting up the experimental setup, the substrata were allowed to acclimate within the tanks
for at least 24 hours. An hour before the beginning of a sampling session, the tanks inflows were
cleaned by mean of injecting tank water at high pressure through the tubing. Sampling
commenced by collecting duplicate water samples from the common header tank. Then duplicate
tank water samples were collected from the outflows of all tanks simultaneously into 300 ml
opaque BOD glass bottles. A second duplicate water sample was collected from the header-tank
by the end of the sampling session (~10 min). The mean of the four header-tank water samples
was considered representative of the inflow concentration for all tanks. The volume of the BOD
bottles was individually measured to the nearest 10 µl. Water samples were immediately
transferred to the laboratory, pre-filtered using a 100 µm net to remove large zooplankton and
fragments of benthic algae, filtered on GF/F filters and analyzed for chlorophyll a by cold acetone
extraction and fluorometric determination on a TD-700 (Turner design) fluorometer as in Yahel et
al. (1998). The precision of our method (measured as the average deviation of duplicate
chlorophyll measurements from their mean) was 5 ng chlorophyll L-1 (<2%, N=214).
In March 2002 a flow cytometer (FACScan, Becton Dickinson) was used to measure the
concentration of the two dominant autotrophic groups in early spring at our study site
(Synechococcus, and pico-eukaryotes) and the non-photosynthetic bacteria as described by Marie
et al. (1999). Taxonomic discrimination was made on the basis of cell side scatter, orange
fluorescence of phycoerythrin, and red fluorescence of chlorophyll (Marie et al. 1999).
In order to detect possible remineralization processes, ammonium concentrations were measured
during a daytime sampling session in September and October 2001 using the fluorometric method
(Holmes et al., 1999). Ammonium concentration was determined on a DyNA Quant 200
fluorometer (Hoffer) after 3 hours dark incubation with the reaction mixture in room temperature.
To improve the method accuracy, an internal standard curve was obtained for each sample by
adding 0, 100, 200, and 300 µl of a standard ammonium solution (2.5 µmol L-1) into 4 ml aliquots
of the original water sample (David, 2003).
Using a thoroughly mixed system, plankton concentrations in the tanks outflow were equal to the
concentration within the tank. Assuming steady state condition for the duration of the sampling
(Riisgård, 2001), we could calculate the substratum clearance rate in each tank (a virtual volume
of water filtered clear of plankton per a time unit) as CR =Q*(Ci-Co)/Co (see equation 6 in
Riisgård, 2001) where Q [L hr-1] is the flow rate through the system and Ci and Co are the
plankton concentrations in the inflow and the outflow respectively (measured either as ng
74
Chap. 6. Phytoplankton grazing in reef rocks
chlorophyll L-1 or cell counts with a flow cytometer). Following a standard engineering approach
for calculating mass transfer rates (plankton or ammonium) between the rough rock surfaces and
the overlying water (Wildish and Kristmanson 1997; Thomas and Atkinson 1997), we scaled the
observed clearance rate to the planar area occupied by the rocks in the tank (~240 cm2). Thus,
unless stated otherwise, CR was normalized to the planar tank area and is reported as ml seawater
cleared per cm2 area per hour.
Algal growth experiments
The hypothesis that phytoplankton grazing and subsequent nutrient mineralization by the
untreated rock fauna would facilitate the growth of benthic algae was tested in September and
October 2001 by incubating four small transparent plastic cylinders within each experimental
tank. Cylinders were prepared by chopping 1.5 ml Eppendorf microtubes that were suspended
with small plastic coated wires 5 cm below water surface. After 14 or 6 days incubations (in
September and October, respectively) the cylinders were removed and the algae chlorophyll was
extracted by soaking the cylinder in 90% buffered acetone for 24 hours at 40C in the dark.
Chlorophyll was measured fluorometrically as above. Chlorophyll a accretion was used as a
proxy for the benthic algae standing crop developed on the cylinder during the incubation.
Rock fauna
Attaining a full quantitative taxonomic description of infauna and the epifauna on the substrata
was beyond the scope of this study. In order to quasi-quantitatively describe the fauna, each rock
was visually examined and photographed and then a sub-sample (0.5-16 ml) was carefully
chiseled from the outer 3 cm of the rock surface and examined under a dissecting microscope.
Sub samples were carefully taken apart within a seawater bath, using a small chisel, and the
dismantled material was filtered (200 µm) and preserved in 4% buffered formalin for later
microscopical analysis. This procedure was repeated for each tank in the first three experiments
(August-October 2001). To examine the natural faunistic variability, three rock samples were
taken and screened in three cases. No attempt was made to quantify the numerous algae
(endolithic, coralline, encrusting) found on the rocks. For sand and gravel, a 10 ml sample was
preserved as above and the infauna inhabiting one ml aliquot was microscopically counted.
The dismantled rocks were sorted under a dissecting microscope and identifiable organisms were
photographed and counted (Hutchings et al. 1992). The separation and isolation of the organisms
was difficult as many animals were fractured or deformed during rock dismantling (Hutchings
1978, 1983). Since colonial organisms could not be quantified (Kiene and Hutchings 1994), only
presence/absence was noted for these taxa. The organisms were identified to the species level
whenever possible; however, more often we were able to sort organisms only to order or family.
Polychaetes with mouth parts indicating a suspension feeding mode of feeding were counted
separately.
Statistical analysis
Throughout the text, means are reported ±1 standard deviation (SD) unless otherwise stated. Each
experiment was analyzed separately using a Repeated Measures ANOVA with the sampling
sessions as repeated measures crossed with the substratum type. As ANOVA assumptions of
homogeneity of variance and normality were usually met, no transformation was used. We used
planned comparisons to compare the clearance rates of sand and gravel with the control, and of
untreated rocks with all other substratum types pooled. Nighttime clearance rates were compared
75
Chap. 6. Phytoplankton grazing in reef rocks
with the preceding daytime results using Wilcoxon match paired test when the comparison was
carried only once (experiment 1 and 2) or a two levels repeated measured ANOVA when the
comparison was repeated twice (experiments 4 and 5) with Day/Night as first repeated measure
level and the sampling sessions as a second repeated measure. The countable organisms'
abundance in each tank was estimated as the product of normalized abundances of each taxon in
the corresponding sub-samples and the rocks volume in each tank. To examine possible
relationships between the fauna of each tank and the mean tank clearance rate we used a multiple
backward stepwise linear regression analysis. A complete analysis of residuals was performed to
validate the robustness of the resulting model. Statistical analysis was undertaken using Statistica
6.0 (2002, Statsoft Inc.). The faunistic composition of the different substratum was compared
using the ANOSIM (Analysis of Similarity) routine and the Primer statistical package (Ver. 5.22,
Primer-e Ltd.).
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Chap. 6. Phytoplankton grazing in reef rocks
Results
Ambient condition
Typical oligotrophic conditions prevailed at our study site during summer stratification period
(August -October 2001), with surface water temperature (SST) ranging from 250 - 260C and
chlorophyll concentration ranging from 150-220 ng L-1. Prokaryotes dominated the
phytoplanktonic community. With the deepening of the winter mixing at the Gulf, eukaryotic
algae became more abundant and in December 2001 (SST, 210C) chlorophyll concentration rose
above the annual mean (400 ng L-1). The spring bloom (>600 ng chlorophyll L-1) followed the
initiation of water warming in March 2002 (SST, 21.90C). Typical bloom conditions were evident
in March 2002 when the Prochlorococcus abundance was extremely low (Lindel and Post 1995,
Yahel unpublished data), Synechococcus (1.65x105 cells ml-1) were 4-16 fold the non-bloom
concentration (1–4 x104 Synechococcus cells ml-1) and eukaryotic algae (1.1x104 cells ml-1) were
3-10 times above the non bloom concentration (1–4 x103 cells ml-1, Yahel, unpublished data).
Phytoplankton removal
Throughout the experiment, substantial phytoplankton removal was evident only in the tanks
containing untreated rocks (38±26% , clearance rate of 17.3±8.0 ml cm-2 hr-1, N=23; Figure 2).
Due to the lack of phytoplankton removal by any of the baked, bleached, or sun dried rocks, these
controls were pooled in subsequent analyses. Planned comparisons indicated that the clearance
rate in the tanks containing untreated rocks was significantly higher than in the tanks containing
other substrata in each of the experiments (F>12.5, P<0.05). The tanks with gravel showed some
removal in the summer but barely any later on. In all five experiments, both sand (areal clearance
rate: 0.7±7.9 ml cm-2 hr-1, N=8) and gravel clearance rates (4.5±7.6 ml cm-2 hr-1, N=8, Figure 2)
were not significantly different from the controls (Planed comparison, P>0.1). Averaging the
clearance rates of each tank over all sampling sessions in each experiment (N=3-7) yielded a
measure of the mean uptake of the corresponding substratum. A Kruskal-Wallis test over these
means indicated a significant difference in the clearance rates of the different substratum types (H
3, N= 47 =33.4, P <0.001). As in the parametric test, a post-hock, non-parametric (pairwise) multiple
comparison of the medians indicated that the clearance in the tanks containing untreated rock was
significantly higher than all other treatments (P<0.001) while the gravel and the sand were not
significantly different from each other or from the control (P=1.0).
Differences in chlorophyll concentration greater than three times the precision of our method (i.e.,
>15 ng chlorophyll L-1) occurred in 97 of the total of 103 individual measurements carried with
tanks containing untreated rocks. Mean clearance rate in the tanks containing untreated rocks
ranged from 5.0±2.5 ml cm-2 hr-1 to 37.8±13.3 ml cm-2 hr-1 and the average difference between
inflow and outflow (tank water) concentrations was 86±75 ng chlorophyll L-1 (38±26%). Gravel
showed detectable phytoplankton removals in 67% of the cases, but the average difference
between inflow and outflow was only 9±46 ng chlorophyll L-1.
In March 2002 the removal of Synechococcus sp. and eukaryotic algae was highly correlated
(r=0.90, P<0.001, N=16). Phytoplankton clearance measured as cell counts was significantly
correlated with chlorophyll clearance rate (r> 0.57, P<0.01, N=16) and the three measures were
not significantly different (Two level Repeated Measures ANOVA, F2,47 = 0.4, P=0.69). Mean
clearance rates were 15±8, 13±7, and 14±7 ml cm-2 hr-1 for Synechococcus, eukaryotic algae, and
chlorophyll, respectively. The grazing rates of non-photosynthetic bacteria was also considerable
(8±6 ml cm-2 hr-1), although significantly lower (Two Level Repeated Measures ANOVA, F1,7=8,
77
Chap. 6. Phytoplankton grazing in reef rocks
P=0.02) in comparison to phytoplankton grazing. Clearance rates of non-photosynthetic bacteria
were well correlated with chlorophyll clearance (r=0.60, P=0.01, N=16) but less so with
phytoplankton cell counts (r<0.3, P>0.2, N=16).
Clearance rates in the untreated rock tanks varied significantly among sampling sessions in all but
the spring experiment. The comparison of the nighttime and daytime clearance-rates did not
reveal any difference except in December 2002 when nocturnal grazing was significantly higher
(F1,2=39, P=0.02, data not shown). It should be noted that the statistical power associated with
this null observation is low due to the small number of day/night comparison (1-2 per
experiment). Clearance rates were positively correlated with rock surface area (Figure 3a,
Spearman Rank Order Correlation rs=0.65, N=12, P=0.02), but not with its displacement volume
(Figure 3b, rs=0.25, N=23, P=0.2) or ambient (inflow) chlorophyll (rs=-0.22, N=23, P=0.3). The
same trend was also evident when each experiment was analyzed separately. Since clearance rates
were not correlated with ambient chlorophyll, the phytoplanktonic biomass gained by the rock
fauna (ng chlorophyll a cm-2 hr-1, calculated as the product of clearance rate and the average
chlorophyll concentration in the tank) was mainly determined by ambient concentration (Figure 4,
rs=0.78, N=23, P<0.001). Thus, during the spring bloom, the rock fauna removed four times more
phytoplankton than during the oligotrophic summer (Figure 4).
Rock fauna
The rocks were inhabited with a highly diverse and rich fauna (Table 2, Figure 5). Most of the
animals were found in the outer ~3 cm. The average density of individual animals (excluding
colonial taxa) in samples taken from the outer (<3 cm) layer was 15.6 per ml, reaching up to 60
individuals per ml. Foraminifera and polychaetes were the most abundant non-colonial taxa
(>80% occurrence), followed by nematodes, gastropods, and other worms. The abundances of
nematodes and polychaetes were highly correlated (Spearman r=0.80, P<0.05). Encrusting
sponges had an overall low coverage with high abundance (> 30% cover) found only in 3 of the
18 rocks examined. Boring sponges were present in 88% of the sub-samples microscopically
examined (Table 2) but were absent in five of the tanks showing clear phytoplankton removal.
Boring bivalves, a highly abundant occupant on living corals (Yahel et al. 1998, Holmes et al
2000), were almost absent in the rocks. Different rocks varied in their faunistic composition
(Figure 5), with no correlation between the abundances of the different taxa (except nematodes
and polychaetes). However, similarity analysis (Bray Curtis similarity of untransformed counts,
Clark 1993) indicated that the rock surface fauna (average similarity 40.5%) had unique
characteristics that differed significantly from the communities living in both the sand and gravel
(Analysis of Similarity, R=0.3, P=0.001, Clark 1993). The sand and gravel communities were
more diverse (within groups similarity <23%) and thus, the two groups were not significantly
different from each other at the crude taxonomic resolution used (Analysis of Similarity, pairwise
comparison P>0.5). The gravel, dominated by pebbles of magmatic origin, was the poorest
substratum in our study site. The fauna of the few non-carbonate rocks examined was also rather
sparse with very few infaunal organisms.
Despite the low taxonomic resolution employed, multiple regression analysis indicated the
existence of a relationship between the faunistic composition of the tanks (estimated as the
product of organism densities in sub-samples and the substratum volume) and the clearance rate
(Adjusted R²=0.39, F2,23=9.0, p=0.001). When the entire data set was analyzed, regression
analysis suggested that the abundance of polychaetes (β=1.39), and crustaceans (β=-1.1)
accounted for 39% of the observed clearance rate variability between tanks. When untreated rock
78
Chap. 6. Phytoplankton grazing in reef rocks
tanks were considered separately, polychaete (β=1.7) and crustacean (β=-1.6) abundances
accounted for 50% of the variability (Adjusted R²=0.50, F2,9=6.7, p=0.016).
Ammonium
Ammonium concentrations in the inflow were in the typical range of the reef bottom water during
the stratified season (Rasheed et al. 2002). In September, ammonium concentrations were
somewhat higher (median 205 nmol NH4 L-1) than in October (181 nmol NH4 L-1). Chlorophyll a
concentration showed the same trend (197 and 156 ng L-1, respectively). Three of the four
untreated rock tanks examined in each experiment showed small ammonium regeneration
(median 0.5 and 7.4 nmol cm-2 hr-1 for September and October, respectively) whereas a fourth
tank showed net uptake (<3.8 nmol NH4 cm-2 hr-1). These values corresponded to a regeneration
of 0.3 and 2.0 nmol NH4 per ng chlorophyll removed in the September and October experiments,
respectively. Gravel and sand (n=4 for each treatment) showed net NH4 uptake in one experiment
and net increase in the other.
Algae growth experiments
Benthic algal growth rate on plastic cylinders in the untreated rock tanks (56±40 ng chlorophyll
cylinder-1 day-1) were the lowest, compared with 107±70, 148±135, and 211±184 ng chlorophyll
cylinder-1 day-1 on cylinders in tanks containing sand, bleached control, and gravel, respectively
(Two Way ANOVA over tank's means, F3,54=11.5, P<0.001). The latter three substrata were not
significantly different from each other (Tukey Unequal N HSD post hock pairwise comparison,
P>0.8).
79
Chap. 6. Phytoplankton grazing in reef rocks
Discussion
The crypto fauna that inhabits exposed reef rocks removed up to 60 % of the phytoplankton from
the water flowing above it in our laboratory tanks (Figure 2, Figure 3). The 95% confidence
interval for the annual mean clearance rate of untreated rock was 13.9 – 20.7 ml cm-2 hr-1. The
lack of removal by the control (killed) rocks (Figure 1. An illustration (side view) of an
experimental tank with 3 rocks (r). Tank dimensions are 15.5 x 15.5 x 15 cm. The water inlet is
via a 6 mm hose from a common head tank, water outlet is from near the tank bottom via a 20
mm hose. An electric pump (p) ensured through mixing of the tank water. Each experiment
consisted of 10 identical tanks attached to the common head tank.
Figure 2) clearly ruled out hydraulic filtration or other abiotic removal mechanisms as a
significant phytoplankton sink under the experimental conditions. Such mechanisms may,
however, have a significant role in situ, primarily within the wave action zone and in high flow
environments (Haberstroh and Sansone, 1999; Huettel and Rusch, 2000). We therefore conclude
that the observed plankton removal in the tanks was due to a biotic process, most likely grazing
(phytoplanktivory) by minute, unidentified, phytoplanktivorous suspension-feeders that inhabit
the outer few cm of the rocks. Due to the small sample size, the statistical power of our
experimental design was insufficient to reject H1 of significant plankton removal by the gravel or
sand. However, the low to null removals recorded for these substrata indicate that they are far less
important sinks for phytoplankton in comparison with exposed rock surfaces on the Eilat fringing
reef.
The reef rocks we studied were selected by many volunteer divers asked to choose rock samples
with no visible macro-fauna cover (e.g., corals, sponges, tunicates, and fleshy algae). Thus it is
most probable that these rocks would have been classified as 'rock' in most reef surveys (e.g.,
Reef Check, Hodgson and Liebeler, 2002). As in previous reports (e.g., Bonem 1977; PeyrotClausade 1977; Vasseur, 1977; Hutchings 1978, 1983; Gischler 1997, Holmse et al. 2000 and
references therein), close examination of the rocks' surface under the dissecting microscope
revealed a highly diverse and rich fauna (Table 2, Figure 5) with a mean total density of 15.6
individuals per ml of rock surface (outer 3 cm, excluding colonial forms). However, the cryptic
nature of many rock dwellers precluded accurate visual counts of all the specimens present in a
sample (Hutchings, 1983). For example, serpulid polychaetes create a maze of microscopic tubes
within minute rock crevices. It was sometimes impossible to separate individual tubes or to verify
whether the tube contained a living worm. Boring sponges (e.g., Cliona spp.) posed another
complication as there was no simple measure for their abundance (Kiene and Hutchings 1994).
Moreover, due to methodological limitations (Wickham et al. 2000), ciliates and flagellates were
not enumerated. Thus, data reported here should be considered conservative. As indicated by the
multivariate analysis, rock surface fauna had unique characteristics and was both richer and more
diverse in comparison with the gravel and sand communities. The regression analysis suggests a
dependence of removal rate on the substratum community composition.
Robust inference from the experimental results to the field should rely on a proper measure of the
amount of rock surface or phytoplanktivorous grazers in the experimental tanks, establishing
proper methodology for measuring these parameters in the field, and good replication of field
hydrodynamics in the lab. Unfortunately, none of the above can be easily established. Rock
volume was clearly irrelevant as the vast majority of the fauna was found within five cm from the
rock surface (see also Hutchings et al. 1992 and references therein). Indeed, rock volume and
80
Chap. 6. Phytoplankton grazing in reef rocks
clearance rate were not correlated (Figure 3b). Rock surface area is probably a better predictor of
the grazing rates as indicated by the significant, albeit not very high (rs=0.65), correlation with
clearance rates (Figure 3a). This low correlation may have partly been a result of the inclusion of
rocks bases and measurement inaccuracies; however, the most likely explanation is the high
variability of the grazer's abundance and community composition (Figure 5).
Due to the aforementioned limitations, our laboratory data can not be directly applied to infer
grazing rates in the field. Nevertheless, scaling the observed clearance rate to the planar area
occupied by the rocks in the tank can provide reasonable first order estimate for the potential of
reef rock fauna to remove phytoplankton at the field. Note that since the relief of rocks within the
tanks was limited to 0.15 m, our calculated areal clearance rates for the reef should be considered
as a lower bound estimate. Moreover, the rather high difference in phytoplankton concentration
between the inflow and outflow waters (38±26%) may indicate that the rate of consumption by
the fauna in the tanks was limited by phytoplankton availability. If the feeding by the rock surface
fauna was indeed limited by insufficient supply of planktonic food (as suggested by Figure 4),
then potential grazing rates were further underestimated.
Scaled up, the annual mean clearance rate by untreated rock fauna was 4.2±1.9 m3 m-2 day-1 (95%
confidence interval: 3.3 – 5.0 m3 m-2 day-1). Considering a 50% rock cover at our study site
(Yahel et al. 1998) and boundary layer flow similar to the field (Yahel et al 2002, Reidenbach et
al., submitted) the exposed rock fauna may be estimated to have cleared 1.6-2.5 m3 m-2 day-1. This
clearance rate is in the lower range of reported values for beds of benthic suspension feeders in
temperate eutrophic waters (e.g., Riisgård 1998) and accounts for a small percentage of the
observed phytoplankton removal reported by Genin et al. (2002) for the same study site. Clearly,
other suspension feeders such as macro-sponges and tunicates and the fauna inhabit live corals
(e.g., Lithophaga spp.) contribute substantially to the total grazing by the reef community. In
March 2001 when direct cell counts were available, nitrogen and carbon gain via plankton
removal (including bacteria) could be estimated using published conversion factors (Caron et al.
1995). The nitrogen removal rate was 5.27 mmol m-2 reef day-1, exceeding values reported by
Fabricius and Dommisse (2000) for the total particulate nitrogen removal by a soft coral
dominated reef, whereas the phytoplanktonic carbon removal rate was 0.14 g C m-2 reef day-1,
about half the Fabricius and Dommisse (2000) value. It should be noted that the March
experiment was done during a spring bloom with elevated plankton concentrations. A more
realistic estimate of the annual carbon gain was calculated using the annual chlorophyll average
for 1988-1998 (353±91 ng L-1, daily measured at the reef, Genin et al. 1995) and chlorophyll:
carbon ratio of 60 (Yahel et al. 1998). Using these values the estimated annual import of
phytoplanktonic carbon to the reef via rock surfaces fauna was at least 16 g C m-2 year-1. While
this value is an order of magnitude lower than those recorded over soft corals dominated reefs
(Yahel et al. 1998; Fabricius and Dommisse, 2000), it is comparable to values reported for
oligotrophic barrier reefs in Australia (4-20 g C m-2 year-1, Ayukai 1995).
In contrast to reports of nutrient release in coral reefs rock cavities (Gast et al. 1998; Richter and
Wunsch 1999), ammonium regeneration within the untreated rock tanks was marginal, and no
enhancement of benthic algae growth could be detected. Dense populations of epilithic and
endolithic algae were present in all the rocks examined. Such algal population are usually absent
or diminished in reef cavities (Richter et al. 2001). It is therefore possible that the majority of the
remineralized nitrogen (and phosphorus) was consumed by autotrophic and microbial
communities on the untreated rocks and could not be available to the experimental plastic tubes
located few cm from the rocks.
81
Chap. 6. Phytoplankton grazing in reef rocks
In areal terms, exposed rock is the second most important niche in coral reefs world wide
(Hodgson and Liebeler, 2002). Although the presence of a diverse infauna in these rocks had been
known for several decades (reviewed by Hutchings 1983, Holmes et al. 2000), only little attention
had been devoted to its ecological function. This study demonstrates the potential importance of
rock surface fauna in importing allochtoneous nutrients (as phytoplankton) into the reef. Further
studies are required to establish the role of different taxa and the overall contribution of reef rocks
fauna to biogeochemical fluxes in coral reefs.
Acknowledgements
We thank M. Ohavia, R. Motro, S. Inbidner, D. Weil, S. Bitton, I. Handel, D. Fuchs, I.
Linchevski, G. Niv, Y. Gesser, Y. Weiss, I. Ginzburg, E. Mahlab, M. Shitrit, I. Shub, R. Schindler
for help in the field work; E. David, D. Golan, and A. post for help with the ammonia analysis; E.
Avilev for English proofing; and the IUI staff for logistic support. We are grateful to Dr. N. BenEliahu for the identification of polychaetes and to E. Halicz for the identification of foraminifera.
This study was founded by the US-Israel Binational Science Foundation (BSF).
82
Chap. 6. Phytoplankton grazing in reef rocks
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85
Chap. 6. Phytoplankton grazing in reef rocks
Figures and tables
Figure captions
Figure 1. An illustration (side view) of an experimental tank with 3 rocks (r). Tank dimensions
are 15.5 x 15.5 x 15 cm. The water inlet is via a 6 mm hose from a common head tank,
water outlet is from near the tank bottom via a 20 mm hose. An electric pump (p)
ensured through mixing of the tank water. Each experiment consisted of 10 identical
tanks attached to the common head tank.
Figure 2. Average areal phytoplankton clearance rate (ml of seawater filtered clear of chlorophyll
a per cm2 of the tank bottom per hr) in tanks containing three different coral reef
substrata (reef-rocks, gravel, and sand) and a control (baked, sun-dried or bleached
rocks) during 5 independent experiments. Averages were calculated over tank means
clearance rate. Error bars, SE, N=3-8 tanks containing rocks and 2-3 tanks containing
sand and gravel per experiments (see Table 1 for more details).
Figure 3. Areal phytoplankton clearance rate by untreated rocks as a function of: a - surface area
of the untreated rocks and b - volume of untreated rock in the experimental tanks. Each
point indicates the average of 3-7 repeated measures of the same tank. Error bars, SE.
See Table 1 for number of replicates. Rock displacement volume was measured in all
experiments, while surface area was measured only in September and March. ♦August
2001 ▲September 2001 ■October 2001 ▼December 2001 ●March 2002. Phytoplankton
clearance rate was positively correlated with the rock surface area during both
September 2001 and March 2002 (Spearman r >0.66) but no correlation with rock
volume was found in any of the experiments.
Figure 4. Average rate of phytoplankton grazing by the rocks fauna (ng chlorophyll a cm-2 hr-1),
calculated as the product of clearance rate and the average chlorophyll concentration in
the tank, as a function of ambient phytoplanktonic biomass. Vertical error bars are SE
calculated over tank means. Horizontal error bars are SE calculated over the mean
ambient chlorophyll of all sampling sessions in an experiment (period). See Table 1 for
number of replicates.
Figure 5. An example of the faunistic composition in sub samples (1-3) taken from the untreated
rock surface of tanks 7(A), 8(B), and 10 (C) used in the August 2000 experiment. Note
the high intra rock variability.
86
Chap. 6. Phytoplankton grazing in reef rocks
Table 1.
The dates and design of the tank experiments carried out during the study.
Date
August 2001
September
2001b
October 2001b
December 2001
March 2002
Total
a.
b.
c.
d.
No. of Sampling sessions
Day
Night
Total
4
1
5
6
1
7
Rocks
4
4
No. of experimental tanks
Gravel Sand
Control
3
3
a
2
2
2c
3
2
2
17
4
3
8
23
2
1
0
8
0
2
2
6
3
4
4
23
2
1
0
8
2c
2c,d
2d
8
In August 2001 sun dried and empty controls were run prior to substratum experiments.
Benthic algae growth experiment
Bleached controls
Combusted controls
87
Total
10
10
10
7
10
47
Chap. 6. Phytoplankton grazing in reef rocks
Table 2
Average (SE) and maximum densities (# ml-1 of rock) of the fauna identified in sub-samples taken
from the outer 3 cm of reef rocks surfaces used in the August, September, and October 2001
experiments. Percent values indicate the proportion of the sub-samples in which the taxon was
present (sub-sample volume ranged 0.5-16 ml). Colonial organisms, such as sponges, ascidians,
and bryozoans were not quantified, thus only presence/absence data are reported for these taxa.
Endolithic, encrusting, and coralline algae were present in most rocks but were not quantified.
Visual observations of the rocks indicated the occurrence of additional taxa that were absent in
the sub-samples, including: boring bivalves (Lithophaga spp.), holothurians, unidentified
ophiuroids, nemerteans, small sea anemones and a few coral polyps (new recruits).
Untreated rock
(N=18)
Gravel (N=7)
Sand (N=7)
Taxon
Density
Max
%
Density
Max
%
Density
Max
%
Foraminifers
5.4 (1.7)
21
82
0.4 (0.2)
1
43
4.7 (2.2)
15
71
Polychaetes
3.8 (1.0)
19
94
0
0
0
2.6 (1.1)
6
57
Filter-feeding
Polychaetes
0.8 (0.2)
3
59
0.3 (0.2)
1
29
0.3 (0.3)
2
14
Nematodes
3.4 (1.4)
19
76
0.3 (0.2)
1
29
0.6 (0.3)
2
43
Other worms
0.7 (0.2)
3
59
0.1 (0.1)
1
14
0.3 (0.2)
1
29
Mollusks
0.8 (0.2)
4
71
0.1 (0.1)
1
14
1.7 (0.8)
5
43
Crustaceans
1.1 (0.5)
8
76
0.1 (0.1)
1
14
0.6 (0.2)
1
57
Sponges
88
29
0
Tunicates
47
29
0
Bryozoans
23
0
0
88
Chap. 6. Phytoplankton grazing in reef rocks
Figure 1
Head
tank
inlet
outlet
r
r
p
89
r
s
Chap. 6. Phytoplankton grazing in reef rocks
Figure 2
Areal clearance rate (ml hr-1 cm-2)
30
20
Control
Gravel
Reef Rock
Sand
10
0
-10
Aug 01
Sep 01
Oct 01
90
Dec 01
Mar 02
Chap. 6. Phytoplankton grazing in reef rocks
Figure 3
50
a
40
-1
-2
Areal phytoplankton clearance rate (ml hr cm )
30
20
10
0
0
500 1000 1500 2000 2500 3000 3500
2
Rock surface area (cm )
50
b
40
30
20
10
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Rock volume (l)
91
Chap. 6. Phytoplankton grazing in reef rocks
Figure 4
6
-2
-1
Chlorophyll gain (ng cm hr )
Mar 02
Dec 01
4
Nov 01
Sep 01
2
Aug 01
0
100
200
300
400
500
600
Ambient chlorophyll (ng liter-1)
92
700
Chap. 6. Phytoplankton grazing in reef rocks
Figure 5
Animals density (no. ml rock-1)
25
Foraminifers
Polychaetes
Nematodes
Other worms
Mollusks
Crustaceans
20
15
10
5
0
A1
A2
A3
B1
B2
93
B3
C1
C2
C3
Chap. 7. Conclusions
7.
Discussion and conclusion
The significance of this interdisciplinary study spans several scientific disciplines and
ecological scales. The major findings and their significance are presented and outlined below.
Phytoplankton grazing in coral reefs
During this work, we have comprehensively investigated phytoplankton-grazing processes
at the coral reef on several levels, from individual phytoplankter-grazer interactions, to whole
community fluxes. At the level of the individual grazer we have extensively documented in situ
phytoplankton grazing rates, selectivity pattern and annual dynamics for a diverse group of
passive (2 soft- and one stony-corals), and active (4 sponges, 3 tunicates, 5 bivalves) suspension
feeders. The yet un-described cryptic fauna inhabits the upper surfaces of apparently "bare" reef
rock was also found to be an important sink for phytoplankton. Phytoplankton fluxes into large
reef section, dominated by either asymbiotic octo-corals or stony corals, were quantified, and the
spatial distribution of phytoplankton over and around coral reefs was thoroughly described.
Phytoplankton was demonstrated to be a major allochthonous source of carbon and nutrients to
the coral-reef community, constituting an important link in the benthic-pelagic coupling of this
ecosystem. The idea that phytoplankton grazing (and not only zooplankton), is an important
source for allochthonous carbon in coral reefs would require a new outlook at the way we budget
benthic-pelagic coupling in this community.
Fabricius and Dommisse (2000) had recently used a Lagrangian method to measured
fluxes of phytoplankton and small particulate matter into coastal reef communities dominated by
zooxanthellate soft corals in Australia. Their estimated phytoplankton fluxes were in the range of
fluxes measured by us for different reef communities. Gast et al. (1998), have reported intense
mineralization and enhanced bacterial growth rates but reduction in bacteria abundance within
reef cervices. They suggested that filter-feeders activity might underlie these observations.
Indeed, Richter et al. (2001) confirmed this suggestion in their recent study of phytoplankton
grazing by coelobite (caves) communities in the Gulf Aqaba. Their finding suggests that,
wherever such cavities are abundant, their cryptic dwellers are an important sink for
phytoplankton (and presumably bacteria) at the reef.
94
Chap. 7. Conclusions
Table 1. Reported phytoplanktonic fluxes into coral reefs (reproduced from Yahel et al. 1998). Number in
bold were originated from subsequent studies (after 1998) and were add to the original table.
* denotes contributions of this work.
Size and properties
Reference
719
Soft-corals “Perforated” reef
*Yahel et al., 1998
414
Dendronephthya thicket
*Fabricius et al., 1997
124
By reef dominated by
Fabricius and Dommisse,
zooxanthellate soft corals (GBR)
2000
Carbon
Carbon Flux
Source
g C m-2 yr-1
Phytoplankton
109
By a fringing reef slop community
(Control volume exp.)
30
*Genin et al. 2002
By active suspension feeders (Using InEx)
at an Indian Ocean atoll lagoon
*Yahel unpublished
4-20
GBR reef
Ayuaki, 1995
4
Diatoms > 68 µm
Glynn, 1973
Bulk DOC removal by marine metazoans
Our study of bulk dissolved organic matter removal by reef suspension feeders indicated
that DOC is a major carbon source for a reef sponge and possibly some tunicates and bivalves. To
the best of my knowledge, a robust quantification of DOC removal in situ by invertebrates has
not been reported. This discovery of a previously undocumented nutritious pathway would require
a reformation in our understanding of trophic dynamics in coral reefs communities and beyond.
Benthic-boundary hydrodynamics characterization
An additional, important novelty of this research lies within the realm of the physicaloceanographic investigations. Complementing the biological work, our detailed hydrodynamic
measurements (Reidenbach, Monismith, Koseff, Yahel, and Genin, Submitted to J. Phys.
Oceanogr.) elucidated the role of flow patterns in controlling phytoplankton supply and removal
on a wide range of scales, from hydrodynamics in the benthic-boundary layer up to the regional
coastal circulation. To the best of my knowledge, this hydrodynamic study was the most
advanced study to date of the detailed structure of near-bed turbulence and flow over coral reefs.
Our finding indicated that the coral reef is several times hydrodynamically rougher than a flat
sandy bottom. The topographic roughness greatly enhances turbulence and mixing, replenishing
the depleted benthic boundary layer with plankton-rich waters from above. This study also
supplied the first ever documentation of the physics of meso-scale flow variability that drives
overall exchanges between the reef and the adjacent sea. The mechanism is a thermal flow, driven
by differential heating and cooling of the shallow water column over a slope, much like the
pattern observed in lakes (Monismith et al 1990). The cross-shore flows, although much weaker
than the predominant long-shore currents, play a special role in maintaining the overall flux of
phytoplankton biomass to the reef from offshore.
95
Chap. 7. Conclusions
Methodological innovation
Although this was not our major objective, we believe this work conveys a significant
contribution to the methodological arsenal of benthic marine ecology in two fields.
At the individual level, we consider the development and evaluation of an in situ
technique (the InEx) to measure feeding rates in active suspension feeders an important
contribution. Leading authors currently hotly debate this field (e.g., Riisgård and Larsen 2000;
Beninger 2000; Silverman et al. 2000; Ward et al. 2000; Riisgård 2001a; Riisgård 2001b;
Cranford 2001). A key point repetitively rose in these discussions is the urgent need for a reliable
in situ technique - one we believe our InEx provides. The InEx technique is also suitable for
sampling small and/or cryptic suspension feeders that are specifically common in coral reefs. It is
often impossible to sample some of them (e.g., boring sponges and bivalve) using traditional
laboratory techniques.
At the community level, we have adapted a standard, well-established engineering control
volume approach to measure mass fluxes over a complex benthic community (e.g., Hatcher
1997). The application of a control volume approach over other reef localities (as well as other
benthic habitats) will allow the first ever, reliable assessment of community metabolism for
habitats other than reef flats (e.g., Barnes 1983) and mad flats (e.g., Asmus and Asmus 1991).
Selectivity among picoplanktonic prey
Our individual based studies are the first modern and detailed account of the feeding of
active tropical benthic suspension feeders feeding on picoplankton. The combination of flow
cytometry with our InEx technique had furnished us with unprecedented resolution, and yielded
some unexpected results. Most striking was the selective, size-independent removal of certain
bacterial types (photosynthetic and those with higher DNA content). Regardless of the underlying
evolutionary and ecological driving forces (e.g., adaptation for the selection of active bacteria),
this size independent selectivity must rely on a yet unknown cell recognition mechanism.
Analysis in progress
Due to the large scope of this study, few papers are still in various stages of preparation.
Due to time limitation, these will not be included in the submitted thesis. These include:
1. Yahel G., Marie D., and Genin A. The role of bivalves, tunicates, and sponges as a
sink for ultra-plankton in coral reefs: Abundance, grazing rates, diet composition, and
functional response.
2. Monismith S.G., Reidenbach M.A., Koseff J.R., Yahel G and Genin A. Boundary
Layer Mixing and Circulation Over Rough Topography: Flow over Coral Reefs
3. Reidenbach M.A., Monismith S.G., Koseff J.R., Yahel G., Ohevia M. and Genin A.,
Flow and plankton dynamics in the rough boundary layer over coral reefs: I: Effects of
topographic roughness on turbulent transport and mixing processes
4. Genin A, Reidenbach M.A., Yahel G., Ohevia M., Monismith S.G., and Koseff J.R.
Flow and plankton dynamics in the rough boundary layer over coral reefs: II:
Phytoplankton fluxes
96
Chap. 7. Conclusions
Unresolved questions and future work
The work on DOC was limited to testing whether or not ‘bulk’ DOC is removed (or
produced) by the animals, without an attempt to examine the chemical/molecular composition and
the structural characteristics of the removed fraction (which is a task for another PhD thesis). It is
my sincere hope that studies of the latter subject will follow this pioneering work. Likewise, I did
no attempt to address the biological aspects of DOC uptake by invertebrates. For example, very
interesting questions such as whether symbiotic bacteria facilitate DOC uptake by invertebrates
(e.g., Ilan and Abelson, 1985), an examination of the morphological structures and/or biochemical
mechanisms that allow such an uptake (Reiswig 1985), and whether (or what proportion of) the
removed DOC and ultraplankton end up being assimilated by the animals, will be left for followup studies. Most importantly, we still do not know whether, and to what extent, DOC feeding is
unique to the reef. I see the accomplishment of InEx DOC experiments with sponges and other
suspension feeders from other benthic habitats as my first priority for the near future. Similarly,
neither the mechanism, nor the exact nature of bivalve's, ascidians, and sponge's preference
patterns has been resolved.
The finding of intense phytoplankton grazing in "bare" reef rocks seems to be only the 'tip
of the iceberg'. The role of live-rock fauna in mediating phytoplankton fluxes in the reef, the
identity of the grazers, and the role of this fauna other geochemical fluxes in coral reefs awaits
future study.
97
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Yamamuro, M. and Koike, I. (1994) Diel changes of nitrogen species in surface and overlying
water of an estuarine lake in summer: Evidence for benthic-pelagic coupling. Limnol.
Oceanogr. 39, 1726-1733.
Zlotnik, I. and Dubinsky, Z. (1989) The effect of light and temperature on dissolved organic
carbon excretion by phytoplankton. Limnol. Oceanogr. 34, 831-839.
105
Feeding on ultraplankton and dissolved organic carbon in coral
reefs: from the individual grazer to the community
By
Gitai Yahel
Appendices
A thesis submitted in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
Submitted to the senate of the Hebrew
University of Jerusalem
2003
106
9.
Appendix I
In situ depletion of phytoplankton by an azooxanthellate soft
coral
By
Fabricius K.E, G. Yahel, and A. Genin
1998
Limnology and Oceanography 43, 354-356
107
10. Appendix II
Intense benthic grazing on phytoplankton in coral reefs revealed
using the control volume approach
By
Genin A., G. Yahel, M.R. Reidenbach, S.K. Monismith, and J.R.
Koseff.
2002
Oceanography 15, 90-96
111
BREAKING WAVES
Intense Benthic Grazing on Phytoplankton in Coral
Reefs Revealed Using the Control Volume Approach
Amatzia Genin, Gitai Yahel
The Hebrew University • Eilat, Israel
Matthew A. Reidenbach, Stephen G. Monismith and Jeffrey R. Koseff
Stanford University • Stanford, California USA
Abstract
This article has been published in Oceanography,
Volume 15, Number 2, a quarterly journal of
The Oceanography Society. Copyright 2001 by
The Oceanography Society. All rights reserved.
Reproduction of any portion of this article by
photocopy machine, reposting, or other means
without prior authorization of The
Oceanography Society is strictly prohibited. Send
all correspondence to: [email protected], or 5912
LeMay Road, Rockville, MD 20851-2326, USA.
A major objective of biogeochemical studies of coral reefs is to quantify fluxes of particulate and dissolved matter
between the reef and overlying waters. However, direct measurements of these fluxes are hard to obtain due to the
typically small concentration changes as the water flows over the bottom and due to shear, turbulent mixing and concentration gradients characterizing benthic boundary layers. Using state-of-the-art underwater technology, we were
able to apply the “Control Volume” approach to measure in situ phytoplankton grazing on a scale of a whole coralreef community. The results indicate that the import of carbon and nutrients via this grazing is a major, previously
underestimated, trophic pathway in coral reefs. The amount of phytoplankton grazed by 1 m2 of reef is similar to the
total phytoplankton produced in the entire water column of the surrounding sea under 1 m2 of sea-surface. The import
of allochthonous nutrients into the reef via this grazing balances the downstream leak of dissolved nutrients.
Physically, the flow over the rough topography of the reef produces enhanced turbulence, enabling high grazing rates
to be sustained, while on larger scales, the exchange between the offshore ocean and the reef is supported by buoyancy-driven flows. With the advent of underwater technology, the control volume technique is no longer limited to
unique situations (e.g. closed lagoons, shallow flats), but should be generally applicable for measurements of benthicpelagic fluxes in oceans and lakes.
Introduction
The answers to many key ecological questions in
benthic ecology depend on accurate measurements of
fluxes across the benthic boundary. The coral reef is one
of many benthic ecosystems for which such measurements are scarce (reviewed by Hatcher, 1997). Available
estimates are questionable since they either represent
unique situations (e.g. mixed waters over a shallow
flat), or are based on indirect measurements (e.g.
extrapolation from chambers, laboratory flumes). We
are still unsure, for example, whether coral reefs are a
net sink or source for atmospheric CO2 (e.g. Kayanne et
al., 1995 vs. Gattuso et al., 1996), nor is it clear to what
extent nutrients in coral reefs are recycled (Odum,
1971), absorbed directly from the flowing water
(Atkinson, 1992), or imported via intense benthic grazing on plankton (Glynn, 1973; Fabricius and
Dommisse, 2000). Even the measure of carbon input via
community grazing on plankton is imprecisely estimated for coral reefs, except for a few unique situations
such as closed lagoons or perforated reefs (Hatcher,
1997; Yahel et al., 1998). Nevertheless, it is generally
agreed that the gross productivity of coral reefs, rivaling that of the most productive ecosystems in the
ocean, is an order of magnitude higher than that of the
typically poor, oligotrophic sea around the reef.
Obviously, nutrient scarcity, that limits the phytoplankton productivity in the ambient waters, is somehow
relaxed in coral reefs. Earlier investigators (e.g. Odum,
1971) considered tight recycling within the reef community to be the main mechanism allowing high gross
productivity to be maintained. This old paradigm has
recently shifted,due in great part to the studies of
Atkinson and colleagues (e.g. Atkinson, 1992; Hearn et
al., 2001). Their work showed that direct removal of
dissolved nutrients by the reef community, a process
that depends on flow and bottom drag, is sufficiently
high to account for the ecosystem’s productivity, specifically at sites where the bottom is rough and the currents are strong. However, neither the “recycling” nor
the “nutrient uptake” explanations account for the leak
of nutrients from the ecosystem to the open sea, which
can be substantial (e.g. Delesalle et al., 1998; Hata et al.,
1998). “Exotic” sources of nutrients, such as groundwater and upwelling, were found at some sites (e.g. D’Elia
et al., 1981) but their general prevalence is questionable
(Tribble et al., 1994).
Clearly, accurate measurements of mass fluxes
across the bottom-water interface are required in order
to understand the trophic functioning of coral reefs (as
well as many other benthic ecosystems). However, such
Oceanography • Vol. 15 • No. 2/2002
90
in situ measurements are almost always difficult to
obtain since gradual changes of concentrations in the
water flowing over the bottom are usually hard to
detect, while at the same time, those concentrations
may change considerably with distance from the bottom (e.g. O’Riordan et al., 1993; Yahel et al., 1998).
Generally, mass fluxes between stationary objects and
moving fluids can be straightforwardly measured
under conditions of fairly homogeneous or confined
flow. Common examples include studies conducted in
flumes and wind tunnels, to more complex measurements made in streams and rivers (Street et al., 1996).
Various techniques (reviewed by Wildish and
Kristmanson, 1997) have been used to overcome the difficulties in measuring mass fluxes in such habitats. One
such technique is the “chamber” experiment, where a
single organism or a small section of the benthic community is enclosed in situ in a water-tight container (e.g.
Hopkinson et al., 1991). However, this highly intrusive
method is size-limited, blocks natural flows, prevents
food replenishment and potentially introduces serious
artifacts. Other techniques can be applied in unique situations where the geometry is advantageous, such as
lagoons and channels. In yet another technique,
Eulerian measurements at one spot (e.g. Atkinson,
1992) or Lagrangian measurements aboard a raft (e.g.
Barnes and Lazar, 1993) are made at a place where the
water column is assumed to be fully mixed - e.g. the
reef flat. However, in many cased such measurements
are made in situations where the flow is vertically
sheared and the water is not well mixed, so that calculations of mass fluxes that are based on measuring the
gradual changes in the concentration of elements at a
fixed depth as the water flows down-stream (e.g.
Fabricius and Dommisse, 2000) could be erroneous.
The Control Volume Experiment
The artifacts, restrictions and assumptions underlying the above techniques render them non-usable in
most benthic habitats, particularly the wide-open, subsurface parts of ecosystems, such as the coral reef slope
(Hatcher, 1997). Here we introduce a novel application
of a standard, well-established engineering approach,
the “Control Volume” (Street et al., 1996), to measure in
situ biogeochemical fluxes over large sections of coral
reefs. Underlying this approach is the designation of an
imaginary box (Figure 1) in which the base is a section
of the benthic community and the four “virtual walls”
extend through the water column up to either a clear
boundary (e.g. sea surface) or an imaginary “ceiling”.
By calculating net fluxes through the walls and ceiling
one can determine (through integration) the net sink
(or source) of a substance at the volume’s “floor”, i.e.
across the benthic-water interface. Fluxes are calculated based on simultaneous measurements of currents
and concentrations across the volume.
Our objective was to evaluate the extent to which
allochthonous carbon and nutrients are imported to
the reef via benthic grazing on water-born plankton.
Figure 1. A schematic drawing of the Control Volume
(approximately 20 m x 10 m) at the coral reef. Black
squares indicate the pumps, 10 on each of four mooring
lines, with the bottom-most pump at 20 cm above bottom
and the uppermost at 1 m below the surface (see Photo 3).
Sub-surface floats (red, top) were used to keep the moorings
taut. Forty PVC pipes, each 100 m long and 12 mm in
diameter, were used to run the water from each pump to the
shore (see Photo 4). The blue instruments at the center of
the “box” represent the 3 ADVs (on a tripod) and an uplooking ADCP (on the bottom) (see Photo 5). A video camera (blue, front) was used to assure the absence of divers
during the runs and that the instruments’ position and orientation remained unchanged. Wide arrows on the left
indicate flow direction.
Rich populations of benthic phytoplankton feeders,
including sponges, tunicates, bivalves, bryozoans and
polychaetes, are ubiquitous in all coral reefs, both on
the bottom (Photo 1; Yahel et al., 1998) and in numerous
cryptic crevices within the substrate (Richter et al.,
2001). Our working hypothesis was that the feeding
rate by this guild of benthic grazers was sufficiently
high so that the amount of nutrients imported to the
benthic community, in the grazed phytoplankton alone,
could balance the leak of dissolved nutrients from the
studied reef (measured by Korpal, 1991). Furthermore,
we hypothesized that the flux of phytoplankton biomass to the benthic community should be supported by
turbulent mixing which serves to renew depleted
waters near the bottom and to enhance mass transfer
over the rough topography of this ecosystem (Thomas
and Atkinson, 1997). Accordingly, our control volume
experiment was designed to simultaneously measure
the rate of phytoplankton grazing by the coral reef
community and the governing hydrodynamic characteristics of the reef.
The work was carried out in summer 1999 on the
open, fore-reef slope in front of the H. Steinitz Marine
Biology Laboratory in Eilat, Israel (Figure 2; Photo 2). A
Oceanography • Vol. 15 • No. 2/2002
91
Photo 1. A picture of a rich coral reef in the Red Sea,
showing the high diversity of corals and invertebrates.
diverse reef community covered about a third of the
rocky bottom, where upright corals and knolls created
a typical bottom relief of the order of 0.5 m (Photo 3).
The reef is exposed to moderate currents and the surface waves are almost always small. The open sea is
oligotrophic, primarily during summer, with integrated water-column productivity of 0.05-0.2 g C m-2 d-1
(Reiss and Hottinger, 1984), while the productivity at
the reef is 10–20 times higher (Barnes & Lazar, 1993).
Since the distribution of plankton is notoriously
patchy, we instrumented the coral reef using an array
of densely-spaced samplers. Replicated samples were
Photo 2. An aerial photograph of the fringing reef along
the Nature Reserve of Eilat. The H. Steinitz Marine
Laboratory is seen behind the white tower at the far background. The bathymetry of this coastal area is described
in Figure 2.
Figure 2. The bathymetry of
the coastal waters near the
coral reefs of Eilat, Israel. The
study sites (marked as black
rectangles) were located on
the slope at 7–17 m depth off
the H. Steinitz Laboratory,
where the bottom slope was
20–30°.
Oceanography • Vol. 15 • No. 2/2002
92
Photo 4. Sampling the pumped water on the shore. Four
persons sampled the water simultaneously from all 40
pumps.
Photo 3. One of the four pump arrays deployed at the corners of the Control Volume over the coral reef in Eilat. The
pumps delivered the water to the shore via the pipes seen
along the mooring line.
simultaneously obtained from 40 different points in the
control volume using submerged pumps (Photos 3, 4),
while currents and hydrodynamic parameters were
measured with 5 different current meters (Photo 5),
covering the entire water column. A control experiment was performed at one of the sites by covering the
reef with a contour-fitting nylon sheet so that the benthic community was physically separated from the
overlying waters (Photo 6). Figure 3 is an example of
the measurements obtained in a single run, showing
the typical current and chlorophyll profiles in the coral
reef and the up-down stream difference in chlorophyll
concentration.
The overall average grazing rate at the reef (calculated from 60 separate experiments) was 0.3 g C m-2d-1,
similar to the entire phytoplankton productivity in the
adjacent open sea. No significant grazing was
Photo 5. The tripod with the three down-looking Acoustic
Doppler Velocimeters (background) and the bottommounted, up-looking Acoustic Doppler Current Profiler
(foreground).
Oceanography • Vol. 15 • No. 2/2002
93
observed when the reef was covered with the sheet of
nylon (the “null” experiment, see Photo 6). Based on
the local C:N ratio, the observed grazing rate accounted for over 90% of the leakage of nitrogen-nutrients
from this reef (Korpal, 1991). Rates of phytoplankton
grazing are frequently expressed using the surrogate
term “clearance rate”: the volume of ambient sea water
(m3) that contains a quantity (gr) of phytoplankton
equal to the total amount removed by the benthic community. Our measurements indicated that 1 m2 of coral
reef “cleared” about 25 m3 of water per day, more than
the dense beds of mussels in the St. Lawrence Estuary
(Fréchette et al., 1989) and 3–4 times higher than typical clearance rates estimated for bivalve beds in San
Francisco Bay (Cloern, 1982; Cole et al., 1992). Given
that velocities over many benthic habitats such as the
San Francisco Bay are much larger than what we
observe over the Eilat reef, how can the reef community sustain such high fluxes to the bed? The most likely
explanation is the unusual topographic roughness of
coral reefs that greatly enhances turbulence and mixing within the benthic boundary layer (O’Riordan et
al., 1993; Thomas and Atkinson, 1997). Is roughness the
sole explanation?
From the standpoint of mass transfer, the bottom
roughness can be characterized by the drag coefficient
CD, i.e., the proportionality between bottom stress and
the square of the current speed. Using our near-bottom
current measurements to calculate CD, we found that
CD ~~ 0.01. This value is about four times higher than the
canonical value typically found over flat bottoms
(Gross and Nowell, 1983), although not at large as values suggested by others which can be as high as 0.1
(e.g. Hearn, 1999). However, while reef roughness
enhances vertical exchange, turbulent mixing by itself
cannot maintain high fluxes to the bed. Particularly in
a system such as a long fringing reef where the current
is predominantly along the reef (Figure 4a), a progressive depletion of food should have been found far
downstream. Our observations indicated that on a
large scale (km) no such gradient occurred, either in
phytoplankton or their benthic grazers. Thus, an additional mechanism must replenish waters over the reef
by transporting phytoplankton-rich waters from the
open sea (Yahel et al., 1998) onto the reef. Such a mechanism would allow the reef community to maintain
high grazing rates over large temporal and spatial
scales.
In the gradually sloping Red Sea reefs, the mechanism is a thermal flow, driven by differential heating
and cooling of the shallow water column over a slope,
much like the pattern observed in lakes (Monismith et
al., 1990; Farrow and Patterson, 1993). During the day,
inshore waters, because they are shallower, become
warmer than those offshore, driving a surface outflow.
At night, surface cooling causes the inshore regions to
become colder and flow down the slope out into the
stratified waters offshore. Our ADCP records clearly
show this pattern (Figure 4b). The cross-shore flows,
Figure 3. Profiles of currents (top) and concentration of
chlorophyll a (mid, bottom) during a single run of the
control volume experiment (4 September, 1999, 08:57
local time). In the top panel, positive values of the longshore (blue) and cross-shore (red) currents indicate current directions to the north and east, respectively. Thus,
during the time of this control volume measurement the
current direction was mostly along the reef to the south
with a weaker cross-reef component to the east. Note the
general correspondence between the flow direction and a
decline in chlorophyll concentration downstream.
Patchiness of phytoplankton and spatial non-uniformity
in the flow sometime disrupt such correspondence, so that
the measurements must be repeated many times.
Oceanography • Vol. 15 • No. 2/2002
94
although much weaker than the predominant longshore currents (Figure 4a), play a special role in maintaining the overall flux of phytoplankton biomass to the
reef from offshore. The operation of such a thermal
flow over coral reefs was first suggested by Boden
(1952), who observed that the lagoon defined by
Bermuda and its system of reefs was 0.2°C warmer than
the nearby oceanic waters.
Conclusions
Our study provides the first direct measurements
of plankton grazing by a benthic community over a
deep open slope with a vertically sheared benthic
boundary layer. The application of a physical-biological
Control Volume with the use of a diverse array of
advanced instrumentation, showed that phytoplankton
grazing, a previously underestimated factor in coral
reefs, constitutes a major source of carbon and nutrients
to the reef community at our Red Sea site. Furthermore,
the high topographic roughness, an ubiquitous characteristic of all coral reefs, plays a key role in enhancing
Photo 6. The control experiment: The coral reef was covered with a wide plastic sheet to separate benthic animals
from the overlying water.
(a)
(b)
(c)
(d)
Figure 4. Velocities (cm s-1; a-c) and Reynolds Stresses (cm2 s-2; d) during the Control Volume Experiment at the shallower site
(2–7 September, 1999). The three velocity panels describe the vectors in the directions a) parallel to the reef, b) perpendicular to
the reef and c) vertical, with positive values indicating northward, eastward and upward flow, respectively. For better visualization, different color scales were used for each component. Note the strong semi-diurnal pattern in the long-shore, and the diurnal pattern in the cross-shore and vertical components. Vertical lines in d) indicate the times of control volume measurements.
Oceanography • Vol. 15 • No. 2/2002
95
turbulence, hence replenishing depleted waters near
the bottom. In addition, the thermal cycling, a phenomenon generally expected in a shallow system with
sloping bottom, plays a major role in replenishing the
water over the whole reef, allowing high grazing rates
to be maintained. Finally, because of its fundamental,
basic nature, the classical Control Volume approach
should be generally applicable for most benthic habitats, from the subtidal zone to the abyss.
Acknowledgements
This study was supported by the US-Israel
Binational Science Foundation and the Stanford
University Bio-X Interdisciplinary Research Initiative.
We thank G. Weil for drawing Figure 1, D. Fong for
assistance with the bathymetric chart, and M. Ohevia,
I. Ayalon, R. Holzman, R. Yahel, R. Motro, S. Ekstein, S.
Nielsen, E. Dunkelberger, Y. Shif, and A. Brandes for
their help with the field work.
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11. Appendix III
InEx” – an in situ method to measure rates of element intake
and excretion by active suspension feeders
By
Yahel G., D.M. Marie, and A. Genin
Limol. Ocemogr. Methods, 3, 46-58
119
LIMNOLOGY
and
OCEANOGRAPHY: METHODS
Limnol. Oceanogr.: Methods 3, 2005, 46–58
© 2005, by the American Society of Limnology and Oceanography, Inc.
InEx—a direct in situ method to measure filtration rates, nutrition,
and metabolism of active suspension feeders
Gitai Yahel1*, Dominique Marie2, and Amatzia Genin1
1
The Interuniversity Institute for Marine Sciences of Eilat and Department of Evolution, Systematics & Ecology, The Hebrew
University of Jerusalem, Israel
2
Station Biologique, CNRS, INSU et Université Pierre et Marie Curie BP 74, Roscoff Cx 29682, France
Abstract
Sponges, bivalves, and tunicates play an important role in the trophic dynamics of many benthic communities.
However, direct in situ measurements of their diet composition, filtration, and excretion rates are lacking for most
species, and knowledge of these rates is based mostly on indirect, in vitro measurements. This paper presents and
evaluates an in situ, nonintrusive technique of direct measurement of the rate and efficiency by which an active
suspension feeder removes (or discharges) substances from (to) the water it filters. The technique, termed “InEx,” is
based on the simultaneous, pair-wise collection of the water inhaled and exhaled by the animal. It was specifically
adapted to allow reliable sampling of common, small suspension feeders with excurrent aperture as small as 2 mm.
The difference in concentrations of a certain substance between a pair of samples provides a measure of the retention (or production) of the substance by the animal. Calculations of feeding (or production) rates are obtained
through multiplying the concentration difference by the pumping rate. The latter is concurrently measured by
recording the movement of a dye front in a transparent tube positioned within the excurrent jet. An important quality of the InEx technique is that it does not manipulate the studied organisms and thus allows realistic estimates of
the organism’s performance under natural conditions. Preliminary results showing the diet composition, feeding
rates, and removal efficiencies of some coral reef sponges, bivalves, and tunicates are presented and discussed.
The paramount role of active suspension feeders in benthic
food webs (Gili and Coma 1998), as well as their commercial
value (Bayne 1998), has inspired a host of studies concerning
their diet composition, food acquisition mechanisms, feeding
rates, and water transport (reviewed by Jørgensen 1966; Bayne
et al. 1976; Bayne and Newell 1983; Levinton et al. 1996;
*
Corresponding author’s current address: E-mail ([email protected]). University
of Victoria, Department of Biology, PO Box 3020 Stn CSC, Victoria, BC,
Canada V8W 3N5. Telephone: +1-250-721 8858, fax: +1-250-721-7120.
Acknowledgments
We thank R. Yahel, E. Hadas, I. Ayalon, M. Ohevia, R. and C. Wyeth,
B. Munkes, E. Gotlibe, R. Motro, S. Eckstein, A. Inditzky, N. Cohen, E. Hazan,
T. Cohen, L. Gutman, I. Meallem, A. and G. Brandes, D. Tchernov, Y. Shif,
and D. Weil, for help in the field and laboratory, G. Weil for the InEx
drawing, G. Fletcher, M. Stoeckle, and Jonathan Rose for help with
the multimedia files, the IUI staff for logistic support, H.M. Reiswig,
P. Beninger, B. Lazar, M. Ilan, D. Lindell, A. Post, M. Kiflawi, T. Katz,
J. R. Koseff, and S.G. Monismith for helpful comments on earlier versions, and the Rieger Foundation and Marom, Rapport, Reshef, Weil,
and Perry families for supporting G.Y. This study was funded by grants
from the US-Israel Binational Science Foundations, The Israel Science
Foundation, and a grant from the German Ministry of Science,
Technology, and Education (BMBF) through the Red-Sea Program. The
flow cytometry analysis was funded by the French program PROSOPE
and the European program PROMOLEC.
46
Bayne 1998; Gili and Coma 1998; Iglesias et al. 1998; Riisgård
and Larsen 2000; Riisgård 2001a). Our current understanding
of the nutritional ecology and physiology of active suspension
feeders is based, by and large, on studies conducted in closed
vessels (Ribes et al. 2000; Riisgård 2001a; Petersen et al. 2004),
using robust taxa adapted to the highly dynamic, turbid, and
productive environments of temperate and boreal coastal
waters (Hawkins et al. 1998). As such, these taxa are “preadapted” to laboratory conditions.
The vast majority of these closed vessel studies (reviewed by
Jørgensen 1966; Wildish and Kristmanson 1997; Riisgård 2001a),
have relied on indirect laboratory measurements (see Jørgensen
1966; Petersen et al. 2004), where removal/production rates are
commonly deduced from concentration shifts of markers such
as algal cells, colored beads, radioactive label, or excretion
products in the vessels containing the experimental organisms. Since indirect methods cannot differentiate pumping
rates and retention efficiency, both parameters are indistinguishably combined and reported as “clearance rate,” that is,
the volume of water cleared (100% removal) of suspended particles per unit of time (Jørgensen 1966; Riisgård 2001a). Similar indirect incubation methods have also been applied in the
field (e.g., Frost and Elias 1985) for the measurements of sus-
Yahel et al.
pension feeders’ feeding rates. Hopkinson et al. (1991) used in
situ bell jars to study the metabolism and nutrient fluxes of
benthic organisms. More recently, Ribes et al. (1998, 1999,
2000) introduced a recirculating bell jar system. This indirect
technique uses natural seawater under natural temperature
and illumination and considerably minimizes disturbance to
the studied animals. Bio-deposition methods (reviewed by
Iglesias et al. 1998; Petersen et al. 2004) estimate intake rates
by comparing the inorganic (and thus indigestible) content of
seston with the inorganic content of bivalve bio-deposits.
A direct laboratory method for the study of the intake and
production of active suspension feeders that involves minimal
interference was suggested by Wright and Stephens (1978).
Under this method, the exhaled water is sampled directly after
a single passage across the animal’s filtration apparatus using a
capillary tube. Unfortunately, this method has not gained much
attention since its initial use (but see Yukihira et al. 1999).
While indirect techniques lump together the pumping
rates and filtration efficiency, a considerable effort has also
been devoted to the study of water transport rates of various
active suspension feeding taxa (as reviewed by Jørgensen 1966;
Famme and Riisgård 1986; Jones et al. 1992; Wildish and Kristmanson 1997; Riisgård 2001a). The methodology used in
these laboratory studies ranges from the straightforward quantification of dyed water movement within a tube inserted into
a sponge osculum (Parker 1914; Gerrodette and Flechsig 1979),
an algal clearance rate method (assuming 100% removal efficiency, e.g., Møhlenberg and Riisgård 1979; Petersen and Riisgård 1992; Riisgård et al. 1993; Riisgård 2001a), a constantlevel tank method (in which the exhaled water is channeled
into a measuring flask, Jørgensen 1966 and references
therein), to complex mechano-optic devices (e.g., Jones et al.
1992; Famme and Riisgård 1986; Riisgård 2001a). Tank head
pressure (e.g., Jørgensen et al. 1990; Riisgård et al. 1993), temperature differences between the animal interior and its environment (Defossez et al. 1997), microscopic observations (e.g.,
Turon et al. 1997), and video endoscopy (Ward and MacDonald 1996) have also been used in laboratory studies.
There have been far fewer attempts to measure active suspension feeder pumping rates in situ. These attempts have
relied mostly on the use of hot wire or hot film thermistors
(Reiswig 1974; Vogel 1974, 1977; Fiala-Medioni 1978a;
Savarese et al. 1997). These instruments allow a continuous
record of excurrent velocity but estimates of water transport
rates require additional information on aperture area and
ambient flow (Fiala-Medioni 1978b). When such information
is available, pumping rates can be recorded for prolonged periods. Reiswig (1974) estimated instantaneous pumping rates
with a hand-held flow meter over the oscula of the giant
Caribbean sponges he investigated, whereas Savarese et al.
(1997) injected dye into inhalant canals and subsequently
measured the dye jet trajectories within the ambient flow
field. Most recently, an Acoustic Doppler Velocimeter was
used to measure the excurrent velocity of a coral reef sponge
Pumping and fluxes in suspension feeders
(Yahel et al. 2003), and time-lapse videography of exhalant
siphon area was suggested as a proxy for bivalve feeding activity in the field (MacDonald and Nodwell 2003).
Despite intense research efforts, many basic aspects of the
nutritional ecology and physiology of active suspension feeder
are not yet fully resolved, and the optimal methodology for
measuring these parameters is still hotly debated (e.g., Jørgensen 1996; Bayne 1998; Ward et al. 2000; Cranford 2001;
Riisgård 2001a, 2001b; Petersen et al. 2004). As pointed out by
Riisgård (2001a) and Petersen et al. (2004), closed vessel assay
results are sensitive to the experimental conditions, including
vessel geometry, flow characteristics, and the type of suspended particle diet offered. Comparisons between coral reef
suspension feeders transferred to the laboratory and undisturbed in situ animals indicated that pumping behavior was
considerably altered in the laboratory even when animals
were positioned in very large tanks with ample supply of fresh
reef water (Yahel unpubl. data unref.). Some coral reef sponges
and ascidians alter their feeding rate, modify their food preference, and most often stop feeding when placed in closed
vessels (Yahel and Hadas unpubl. data unref.). In several experiments, clearance rates were independent of food availability
in the vessels and, instead, varied as a function of the supply
of fresh seawater into the vessel. For example, the clearance
rate of the Synechococcus cyanobacteria by the reef sponge
Negombata magnifica was reduced by 4-fold when water residence time in the experimental tank was increased from 5 to
20 min (Hadas unpubl. data unref.). Moreover, numerous
active suspension feeders in coral reefs are either boring or
encrusting (Yahel et al. 1998) and cannot be separated from
their substrate and brought to the laboratory without severely
damaging their body.
In summary, drawing conclusions from results of closedvessel experiments regarding the actual performance of the
animals in the field is controversial, even under optimal
experimental conditions (e.g., Cranford and Hill 1999; Cranford 2001, and references therein).
The in situ indirect method developed by Ribes et al.
(1998, 2000) bypasses some of the problems inherent in the
laboratory system. Nevertheless, it suffers from the same limitation of the laboratory-based clearance method discussed
above (see Riisgård 2001a), and it is quite demanding in terms
of the amount of equipment and work required per specimen
sampled (Ribes et al. 2000).
Reiswig, in his comprehensive fieldwork on the ecology of
Caribbean sponges (Reiswig 1971a, 1971b, 1974), pioneered
the use of direct in situ techniques for the study of active suspension feeder diet composition, metabolic performance, and
water transport rates. His direct methods were based on comparing the content of the water inhaled and exhaled by
sponges and, therefore, were free of most of the methodological pitfalls associated with both indirect and in vitro experiments. Combined with pumping rate estimates obtained by
hot wire thermistors or hand-held flow-meters, Reiswig’s data
47
Yahel et al.
Pumping and fluxes in suspension feeders
Fig. 1. (A) Schematic representation of the InEx technique as applied to a solitary ascidian. Exhaled water is collected passively using the positive pressure at the exhaled water jet to flush and replace the water within an open-ended tube carefully positioned within the excurrent jet. Inhaled water is
sampled into an identical sampler attached to a syringe. In this example, the samplers are Pt10 (Table 1) designed for the collection of relatively large
water quantities (7 mL) from organisms with minute excurrent aperture (~3 to 5 mm). Arrows indicate the direction of water flows. (B) Photograph of
a Pipette-shaped (Pt) InEx sampler. (C) Photograph of a tube-shaped (Tu) InEx samplers (for sampler make-up, see Materials and procedures).
allowed calculation of the sponge’s actual grazing rates. Unfortunately, the methodology used by Reiswig for his giant
Caribbean sponges (e.g., trapping the exhaled water in large
plastic bags) cannot be easily applied to smaller sponges
(Wilkinson 1978) and even less so for more sensitive organisms such as bivalves and ascidians (Yahel unpubl. data
unref.). Use of samples obtained by active suction of the
exhaled water (e.g., using a syringe, Reiswig 1974, 1985;
Wilkinson 1978; Pile et al. 1996; Pile 1997) requires proper
control over the risk of “contaminating” the exhaled water
sample by sucking in ambient water, thus the suction rate
should be negligible in comparison to the excurrent flow rate
(e.g., Wright and Stephens 1978). Alternatively, suction samples can be obtained from the excurrent atrial cavity, provided
that the sample volume is negligible with respect to the cavity
volume. Unfortunately, many common benthic suspension
feeders do not possess large excurrent cavity or high pumping
rate. Therefore, hand-held syringe sampling is inappropriate
for most active suspension feeder species. If suction sampling
is to be used with small suspension feeders, water samples
should be withdrawn using a device that allows slow and wellcontrolled suction (e.g., Wright and Stephens 1978).
In the present article, we present a combination of earlier in
vitro methods used by Parker (1914), Wright and Stephens
(1978), and Møhlenberg and Riisgård (1979), and the pioneering in situ approach of Reiswig (1971a, 1971b). Our aim is to
develop a reliable, rapid, and nonintrusive sampling strategy
48
that enables both individual and community-scale studies of
the diet composition, pumping rates, grazing rates, and metabolism of a wide range of active suspension feeders in situ.
Materials and procedures
Water sampling—The InEx method is based on a simultaneous collection of paired water samples from the water
inhaled and exhaled by active suspension feeders. Two scuba
divers carry the sampling underwater. Samples of inhaled
water are drawn slowly by one diver with the use of a small,
open-ended tube attached to a syringe as the diver holds its
inlet next to the animal’s inhaling aperture (Fig. 1, see Video clip 1
<http://www.aslo.org/lomethods/free/2005/XXXXa1.html>). The
exhaled water is sampled by a second diver with an identical
tube held within the exhalent jet and aligned with it, as close
Video clip 1. Highlights from an InEx sampling at Race Rocks, British
Columbia, Canada. The sampled organism is the sponge Isodictya rigida.
Video courtesy of G. Fletcher, Pearson College, www.racerocks.com. Video
can be seen at http://www.aslo.org/lomethods/free/2005/0046a1.pdf.
Yahel et al.
Fig. 2. Examples of typical parameters measured with the InEx method.
(A) Concentrations of Synechococcus (Syn) in the water inhaled () and
exhaled () by five specimens of the solitary ascidian H. gangelion (two
consecutive replications collected from each animal). Lengths of connecting lines indicate number of cells removed. (B) Three consecutive
excurrent jet velocity measurements (dye-front speed) of the same specimens as in (A) obtained immediately after water sampling. (C) Instantaneous grazing rate estimates for the same specimens calculated as the
product of the data presented in A and B and the Tu10 sampler cross-section (Table 1). Error bars (SD) represent the total variation contributed by
individual animals (variation of pumping rate and retention efficiency),
plankton patchiness in the ambient water, and sampling error: SD composite
= (SD2cell removal × pumping rate2 + SD2pumping rate × cell removal2)0.5.
as possible (<2 mm) to the animal’s exhaling aperture, but with
no physical contact (Fig. 1, Video clip 1). When sampling is
accomplished, the inhalant sampling tube is plugged first,
starting at its proximal end, then the exhalant sampling tube
is carefully plugged, starting at its distal end, the proximal end
is then plugged while still positioned within the excurrent jet.
Filling time is determined individually for each pair as 1.5 × the
time it took the exhalent jet to flush clear the sampling tube
prefilled with dyed seawater (~5 mg L–1 fluorescein). If the dye
is suspected to interfere with water analysis, flushing time
should be measured with an identical tube a few minutes
Pumping and fluxes in suspension feeders
prior to sampling. Fluorometric measurements of dye concentrations in these tubes indicated that this filling time
ensured a replacement of greater than 99% of the water contained in the tube prior to the start of sampling (see Sampler
flushing time). The difference between the inhaled and the
exhaled water samples (InEx pair) indicates the net retention (or
production) of the substance of interest.
Multi-oscular organisms—To ensure genuine pairing of InEx
samples for multi-oscular organisms (e.g., sponges), entry and
exit points of the water path through the animal are visualized
with vital dye (fluorescein, 10 mg L–1); their locations are
marked for subsequent sample collection. Sampling is delayed
to minimize dye effect on water properties or animal behavior.
The duration of water passage through the filtration apparatus
may also be determined by the same method. However, with
the exception of glass sponges, all organisms surveyed so far
exhibited a passage time of less than 2 s, which is an order of
magnitude smaller than our typical sampling duration.
Excurrent velocity measurement—Excurrent jet speed and
water transport rates can be measured concurrently with each
InEx sample. A small amount of dyed seawater (fluorescein,
100 mg L–1) is placed just inside one end of a transparent,
cylindrical tube while the sampler’s finger seals the other side.
The tube is positioned within the excurrent jet and aligned
with it, as close as possible to the exhaling aperture (but avoiding physical contact) and the finger is released to allow the
exhalent jet to flow through the tube. The movement of the
dye within the tube is videotaped, and the dye-front speed is
determined with frame-by-frame analysis using marks on the
tube as a scale. This rapid measurement (a few seconds) is typically replicated 3 to 5 times.
Rate calculations and statistical analysis—The method yields
two independent measurements: the difference between InEx
(Inhaled-Exhaled) samples (Fig. 2A) and instantaneous water
flux per excurrent aperture. The latter is calculated as the product of the mean dye-front speed (Fig. 2B) and the tube or excurrent aperture cross-section area (see Pumping rate measurements). These parameters can be combined to produce
instantaneous filtration rate estimates (Fig. 2C). Alternatively,
traditional “clearance rate” estimates can be calculated as the
product of filtration efficiency [(In – Ex)/In)] and the water
transport rate. Due to the inherent patchy distribution of
plankton and other water constituents, care should be taken to
collect the InEx samples in a way that will ensure genuine pairing of the samples and facilitate true pairwise analysis. In cases
in which several aliquots are being analyzed from each water
sample, the appropriate statistical test is a repeated measures
analysis of variance (ANOVA) or its aparametric equivalents.
Materials for InEx samplers—A variety of sizes and shapes of
sampling tubes were examined in preliminary trials. The final
design samplers were easily fabricated from standard disposable polystyrene pipettes (e.g., Bibby Sterilin Ltd, Staffordshire, U.K.), and they consist of a shortened pipette with the
upper half of a chopped Micro-Tube (e.g., Eppendorf) fitted
49
Yahel et al.
Pumping and fluxes in suspension feeders
Table 1. Types of InEx samplers used
Sampler name
LPt 1
LPt 2
Pt 5
Pt 10
Tu 5*
Tu 10*
DFS 1
DFS 2
G 32
Length (mm)
Volume (mL)
Inlet
Inner diameter (mm)
Outer diameter (mm)
Body
Inner diameter (mm)
Outer diameter (mm)
Outlet
Inner diameter (mm)
Outer diameter (mm)
Cross-section area (cm2)
Number of stoppers
282
1.6
282
2.7
65
2.5
160
6.9
145
3.9
111
6
128
0.7
130
5.5
151
32.0
2.0
3.0
2.0
3.0
2.0
3.0
3.0
4.0
6.3
14.5
9.4
13.7
2.8
4.5
3.6
8.3
10.0
12.3
2.8
4.6
3.6
5.0
5.8
7.1
8.0
10.0
5.8
7.1
8.0
10.0
2.8
4.6
3.6
5.0
24.6
22.5
4.9
8.3
4.6
8.3
6.3
14.5
9.4
13.7
1
2
1
9.4
13.7
0.50
2
2.8
4.5
0.06
0
3.6
5.0
0.10
0
12.0
16.3
1
6.3
14.5
0.26
2
2
Samplers were fabricated from polystyrene Sterilin pipettes fitted with chopped micro-tube as stoppers, except for the G 32, which was custom-made
from glass. LPt samplers were intact pipettes fitted with a stopper on the distal end. Pt samplers had the same design but the pipette was shortened
(see Fig. 1B). Tube-shaped samplers were simple cylinders with stoppers fitted on both sides (see Fig. 1C). DFS samplers were simple transparent tubes
with no stoppers, used solely for pumping rate measurements.
*Samplers that were used for both water collection and dye-front speed measurements.
directly or with an external Tygon tube as a stopper to one or
both ends. Pipette-shaped samplers with only one stopper (LPt
and Pt, see Fig. 1B) were kept sealed by inserting their narrow
tip (2 to 3 mm) into a compatible 15 mm deep hole drilled in
a 30 mm silicon pad. Custom-made precombusted glass samplers were used for special applications (e.g., dissolved organic
carbon [DOC] sampling, Yahel et al. 2003). Some relevant
examples of InEx samplers are described in Table 1.
Statistical analysis—Statistical analysis was carried out using
STATISTICA, version 6 (data analysis software system, StatSoft
Inc. 2002). Parametric tests were used whenever possible, and
data were transformed when necessary to meet the requirements of normality and homogeneity of variance. The equivalent nonparametric tests were used whenever data did not
meet the ANOVA assumptions. For repeated measure ANOVA,
we also tested the compound symmetry and sphericity
assumptions (i.e., cases in which differences between levels
were correlated across subjects) and compared the results of
the univariate test with Wilks’ λ (a multivariate criterion).
Assessment
Comparison of inhaled and exhaled water—The InEx technique was used for the measurement of a variety of metabolic
parameters, including respiratory gases, plant nutrients, plant
pigments, total organic carbon (TOC), DOC, and alkalinity
(Yahel et al. 2003; Yahel et al. unpubl. data unref.). Ultraplankton (<10 µm, Murphy and Haugen 1985) was used to
examine the technique’s efficiency. Ultra-plankton cell counts
are ideal for this purpose as they are reliably made with a flow
cytometer and require a small sample volume (1 mL). Moreover, ultra-plankton consists of several cell populations that
are selectively removed by different active suspension feeder
50
taxa (Pile et al. 1996; Ribes et al. 1998, 1999) thus providing
“internal control” for the method.
Three locations were used to evaluate the method. Our primary location was the coral reef in front of the Steinitz Marine
Laboratory of Eilat, Gulf of Aqaba, Red Sea (29°30′N, 34°56′E).
Further evaluation was carried out at the reefs of Alphonse
Island, Seychelles (06°59′S, 52°43′E), and at Race Rocks
(123°32′W 48°18′N), British Columbia, Canada. The latter site
is characterized by high currents (>2 m s–1) and cold water (6°C
to 10°C). All field samples were collected by two divers using
standard SCUBA techniques. A flow cytometer (FACSort, Becton Dickinson) was used to estimate the concentrations of 4
groups of ultra-plankton: nonphotosynthetic bacteria (Bact,
median size ~0.4 µm) and the three major autotrophic groups,
Synechococcus (Synechococcus, ~1 µm), Prochlorococcus (Pro, ~0.6 µm),
and pico-eukaryotes (Eukaryotic algae, ~2 to 3 µm), as
described in Yahel et al. (1998).
InEx measurements of removal efficiencies exhibited high
repeatability. For example, the average deviation between
duplicate InEx samples obtained from 5 specimens of the solitary ascidian Halocynthia gangelion was 4% (Fig. 2A). In this
case, the mean removal efficiency of the Synechococcus was 70%
and the standard deviation was 6% (coefficient of variance
[CV] = 9%). In a similar experiment, 11 oscula from 3 specimens of the boring sponge Cliona mussae were sampled. The
average deviation between replications was 1%, and the mean
removal efficiency was 94% ± 4% (CV = 4%). The fact that very
few cells were found in the samples of the exhaled water indicated that the exhaled water was sampled cleanly, with negligible “contamination” by ambient, nonfiltered water.
The presence of different ultra-planktonic groups in the
ambient water provided internal control for the method. To
Yahel et al.
Pumping and fluxes in suspension feeders
Fig. 4. A comparison of two methods of sampling the water inhaled and
Fig. 3. Sampler type effect: Comparison of ultra-plankton retention efficiency of various active suspension-feeder taxa using two types of InEx
samplers. Two organisms possessing small (<3 mm) excurrent apertures,
the bivalve Lithophaga simplex (, n = 4), and the sponge Cliona mussae
(, n = 6) were sampled by a pipette-shaped LPt 1 sampler (open symbols). A cylindrical Tu 10 sampler (filled symbols) was used for the sponge
S. clavatus (, n = 8) and the ascidian H. gangelion ( , n = 3), both possessing much larger excurrent apertures (>9 mm). All samples were collected at the same site during a single sampling dive (23 September
1997). Pro, Prochlorococcus; Syn, Synechococcus; Euk, eukaryotic algae;
Bact, nonphotosynthetic bacteria. Error bars = SE.
test the significance of these differences, a within-between
repeated measure ANOVA design was used, with the removal
efficiencies [(In – Ex)/In] for each prey taxon (Pro, Syn, Euk,
Bact) as the within-subject, repeated measure factor, and the
grazer taxa as the between-subject, fixed factor. The analysis
showed that removal efficiencies of the different ultra-planktonic prey types varied significantly both within and between
active suspension feeder species (Fig. 3, F3,54 = 131.2, P < 0.0001;
and F3,18 = 25.5, P < 0.0001, respectively). For example, the
overall retention efficiency measured for the sponge Suberites
clavatus (80%) was almost twice that of the ascidian H. gangelion (44%), both sampled by an identical sampler. A consistent
removal pattern was exhibited by each of the active suspension feeder species regardless of the sampler type used (Fig. 3).
Hence, no significant removal (P > 0.2) of nonphotosynthetic
bacteria was measured for the ascidian (Tu 10 sampler) and
bivalve (Pt 10 sampler) samplers. Using the same samplers at
the same time on nearby sponges revealed high filtration efficiencies (>77%) of nonphotosynthetic bacteria by these organisms (S. clavatus, Tu 10 sampler; and C. mussae, Pt 10 sampler).
The repeatability of the inhaled water collection method
was tested by comparing three sets of triplicate samples collected in ~3-minute intervals next to three different incurrent
exhaled by two large reef sponges. Removal efficiency of nonphotosynthetic bacteria and eukaryotic algae was measured concurrently over the
same sponge oscula. Exhaled water was captured using both the InEx
technique (empty symbols) and slow active suction by a syringe (filled
symbols). For the purpose of this comparison, sponges possessing relatively large volume oscula (>5 mL) with strong excurrent gets (>120 mL
min–1, Yahel unpubl. data unref.) were selected. For each osculum, a
syringe sample was slowly drawn immediately after an InEx sample. The
apparent high removal efficiency of bacteria by M. fistulifera may be a
consequence of accidental suction from within the loose tissues of this
sponge (see text).
siphons (an ascidian and two bivalves). In all cases, cell counts
of each of the pico-planktonic groups were almost identical
(CVs ranged from 0% to 6%). Comparison of more spatially
dispersed samples taken next to different individuals located
5-30 m from each other over a similar time scale indicated
much higher CVs (ranging from 13% to 22%). This finding
highlights the strengths of the paired-sample design, in which
there is an inhaled sample for every exhaled sample.
To compare the passive InEx sampling technique to active
syringe suction of exhaled water (e.g., Reiswig 1985; Pile
1997), immediately after the collection of an InEx pair, water
was carefully and slowly drawn with either 10 or 30 mL
syringes next to the ostia (inhalant aperture) of some large
sponges as well as from within their oscula (exhalant aperture). The samples were analyzed with a flow cytometer and
an epi-fluorescence microscope (a method described by Lindell and Post 1995). Flow cytometry analysis yielded indistinguishable estimates of pico-plankton removal efficiency for
samples collected from the relatively large sponges Mycale fistulifera (n = 3), S. clavatus (n = 3), and Theonella swinhoei (n = 2)
(repeated measure ANOVA of pooled, arcsine transformed
data, F1,5 = 4.46, P = 0.883). These results (Fig. 4) indicate that
sampling using controlled suction could be an efficient
approach for sufficiently large taxa with strong excurrent jets
and/or large excurrent atrial cavities, provided that the sampling rate is negligible in comparison to the excurrent flow
51
Yahel et al.
Pumping and fluxes in suspension feeders
Table 2. Average sampler flushing time (SFT) required for the excurrent jet to displace and flush the ambient water contained in the
sampler and the average ratio of sampler volume (SV) to the volume of water pumped via the sampler during that time (WV)*
Sampler type and species
Tu 10
Didemnum candidum
Halocynthia gangelion
Mycale fistulifera
Suberites clavatus
Pt 10
Cliona mussae
Lithophaga simplex
LPt 2
Cliona mussae
LPt 1
Lithophaga simplex
Taxonomic grouping
n†
Colonial tunicate
Solitary tunicate
Sponge
Sponge
13
28
49
57
Sponge
Bivalve
SFT (s)
73
53
42
27
SV/WV ratio
(15)
(16)
(15)
(10)
14.1
16.7
14.7
16.4
20
20
95 (20)
152 (32)
3.4
3.4
Sponge
14
132 (32)
28.3
Bivalve
28
113 (44)
12.0
*Pumping rate was determined by the dye-front speed method (see text).
†n is the number of excurrent jets sampled.
rate. However, efforts to apply the syringe method for taxa
possessing smaller excurrent apertures (e.g., Lithophaga
bivalves or boring sponges) were unsuccessful. In such small
taxa, there was no simple way to control the sampling rate,
and the results yielded no interpretable signal.
Microscopic analysis of syringe samples collected from
within the sponge’s oscula revealed a dense population of large
eukaryotic algae (>10 µm) in samples taken from M. fistulifera.
These cells, too large to be detected by the flow cytometer, are
presumably similar to the eukaryotic algae reported by Pile
(1997, and references therein) to be “produced” and expelled
by several sponge species. Such algae were absent from the
corresponding exhaled (and inhaled) water samples obtained
by the InEx method, as well as from the ambient water samples obtained by syringes. It is suggested that the apparent
production of eukaryotic algae reported by Pile (1997) was an
artifact of using the syringe method due to forceful suction of
either periphyton or endo-symbionts (Wilkinson 1978) from
the oscula walls. Suction from within the oscula wall tissues
may also explain the apparent high removal efficiency of bacterial cells measured with the syringe technique for M. fistulifera (Fig. 4).
Sampler flushing time—Field experiments. The time required
for the excurrent jet to displace and flush the ambient water
contained in the sampling tube was designated “sampler
flushing time.” Flushing efficiency was measured in the field
by holding the tube (prefilled with dyed seawater) within the
excurrent jet, 1 to 2 mm from the excurrent aperture, until no
dye was visible within the tube. The tube was then sealed and
the residual dye concentration was determined fluorometrically (Turner AU-10, Turner Designs). An identical tube was
simultaneously held in a similar position over an inert surface
as control. By the time the samplers were determined “clear”
by underwater visual inspection, they contained, on average,
0.4% ± 0.7% of the initial fluorescein concentration (10 to
52
100 mg L–1). Despite the somewhat subjective manner of this
method, its repeatability was fairly high with 8% ± 7% deviation from the mean (n = 110 duplicate sampler flushing time
measurements). This convenient method was thus adopted as
the standard mean for sampler flushing time determination in
subsequent experiments. The ratio of sampler volume to the
volume of water required to flush it clear was nearly identical
for the same sampler type and independent of the sampled
taxa (Table 2). Sampler volume (Table 2), sampler geometry, and
ambient hydrodynamics affected sampler-flushing time.
When samplers were left open, a gradual exchange of the sampler and ambient water occurred also in the absence of an
excurrent jet as a consequence of wave action, ambient currents, and sampler movements. However, even under extreme
conditions (e.g., the excurrent jet oriented into a strong ambient current), whenever a sampler was located over an excurrent jet, the bulk of the dyed water was flushed out by the
excurrent within seconds (see Video clip 1), while identical
samplers simultaneously held over inert surfaces retained
>20% of the initial dye concentrations for several minutes.
Flow tank experiments. To test the method under controlled conditions, an artificial excurrent jet was fitted into a
flume flow chamber with transparent walls (Fig. 5; Kiflawi and
Genin 1997). The excurrent model was built with a 20-mm
internal diameter (ID) hose capped by a flat (1 mm) replaceable lid into which an orifice was drilled. This arrangement
assured an excurrent profile as close as possible to “plug flow”
(Charriaud 1982; Savarese et al. 1997). Ambient current velocity, jet flow rate, jet orientation, and aperture diameter were
adjustable. A flow meter (Gravity ball, Gilmon STD nr 13)
installed between the head tank and the excurrent model was
used to measure excurrent jet flows. Flow visualization was
carried out by dyeing either the tank (ambient) water or jet
water, or by injecting small amounts of dyed water into or
around the samplers. A video camera (Sony Handycam, High-8
Yahel et al.
Pumping and fluxes in suspension feeders
Fig. 5. Schematic representation of the setup used for testing and calibrating the InEx technique in the flume. a. Head tank; b. Flow regulator; c. Flow
meter; d. Excurrent model with interchangeable orifice; e. Dye injector; f. Video camera; g. Video monitor and recorder; h. Controlled propeller unit; i.
Return circuit; j. Water outlet; k. Additional measuring flask; l. Thermostat (drawing is not to scale, see Kiflawi and Genin [1997] for tank specifications).
CCD-V700E) mounted in front of the excurrent model was
used to record and analyze the fluid motion.
Sampler flushing time measurements were carried out in
the flume by filling a set of identical samplers with tank-water
containing a known dye concentration; samplers were then
Fig. 6. The time course of sampler flushing (SFT) of two samplers (in percentage of initial dye concentration) as a function of the time the sampler
was held open over the artificial excurrent jet. Double-sided arrows mark
the gap between the observed flushing time-courses and the prediction
of an exponential decay model. The Tu 10 sampler was held over a 10-mm
excurrent model (flow rate = 6.7 mL s–1). Half of the sampler content was
replaced within the first 5 s (L1) because of piston effect of the excurrent
jet, followed by an exponential dilution. The Pt 10 sampler was held over
a 2-mm excurrent model (flow rate = 0.88 mL s–1). The exponential dilution started after a typical lag required for the flow field to develop within
the sampler (L2). See Table 1 for sampler descriptions.
positioned above the excurrent jet for different flushing durations (3 to 100 s). At the end of each run, the sampler was
sealed, and the concentration of the dye remaining in the tube
was determined fluorometrically. Ratio of initial-to-final concentration was used as a measure for flushing efficiency. As
depicted in Fig. 6, sampler flushing-rate approximated exponential decay after an initial phase characterized by either a
short delay (pipette-shaped samplers) or an abrupt concentration drop (tube-shaped samplers). This exponential mode of
sampler flushing, a consequence of boundary layer formation
along the sampler walls (J.R. Kossef pers. comm. unref.),
enforced prolonged sampling whenever clean exhaled samples
were sought. For ~6 mL samplers, sampler flushing time
ranged from 0.5 min for high-pressure sponges (jet flow rate
~250 mL min–1) to 2.5 min for small bivalves (~10 mL min–1).
Contamination by ambient water—Intrusion of ambient
water into the exhaled water sampler during sample collection
(hereafter “contamination”) is of major concern. We used
visualization of ambient water invasion by dying either
“exhaled” or “ambient” tank water and testing various combinations of ambient conditions, excurrent models, and sampler
types. In cases in which the sampler’s inlet internal diameter
(ID) was larger than the excurrent diameter, dyed tank water
was entrained by the Venturi effect (Vogel 1978) into the sampler. This effect led to significant underestimation of the
retention efficiency, as demonstrated in Fig. 7A. Nevertheless,
dye visualization and fluorometric measurements indicated
that as long as the sampler ID was similar to or smaller than
the diameter of the animal’s excurrent aperture, only exhaled
water entered the Ex sampler. Upward dye movement and
flushing of the samplers may be evident, even in cases in
53
Yahel et al.
Pumping and fluxes in suspension feeders
Fig. 7. An example of proper and improper fitting of sampler ID to excurrent aperture and their effect on ultra-plankton retention efficiency estimates
(error bar, SD). (A) Two samplers of the same type but with different ID were
used to measure the filtration efficiency of the colonial tunicate Didemnum
candidum. Tu 10 ID > excurrent orifice; Tu 5 ID = excurrent orifice. MannWhitney U test indicated that efficiency estimates obtained with the Tu
10 sampler (ID > excurrent orifice) were significantly lower (*P < 0.05;
***P < 0.001; n = 13 and 12 for Tu 10 and Tu 5, respectively). (B) Two different sampler types, both with inlet ID ≤ excurrent orifice were used to estimate filtration efficiency of the boring bivalve Lithophaga simplex. No significant difference was found between the samplers (Mann-Whitney U test,
P > 0.15 for all comparisons; n = 23 and 36 for LPt 1 and Pt 10, respectively). Note that the Pt 10 sampler has a conical, pipette-shaped inlet end
and its volume is ~4 times larger than the cylindrical LPt 1.
which active pumping by the studied organism is absent. This
artifact is generated by higher ambient water velocities away
from the boundary at the outlet of the sampler (Bernoulli
effect, Vogel 1974). Various capping devices and configurations aimed to offset this effect were tested in the flow tank
with no success. However, this problem was significant only
with long samplers (>25 cm, e.g., LPt type, see Table 1) oriented perpendicular to the ambient flow or in the presence of
strong currents. This shortcoming can be resolved by selecting
sampling positions where the sampler is oriented with a slight
54
angle (<5°) into the ambient flow. In general, pipette-shaped
samplers and samplers with a smaller diameter-to-length ratio
were less susceptible to contamination by ambient water
intrusion.
To assess the potential of ambient water contamination, a
comparison of direct filtration efficiencies measurements was
carried out in the lab using the Wright and Stephens (1978)
method, with InEx measurements for the same individuals.
Five specimens of N. magnifica previously attached to
polyvinyl chloride plates and grown suspended over the reef
were transferred to a 4-L aquarium with a fresh reef water supply (residence time < 3 min). Using a micromanipulator, a
micro-capillary tube was mounted inside one of the sponge’s
oscula without touching the sponge. A similar tube was
located within 2 mm of the sponge’s ostia. The slow suction
through the capillary tubes ensured clean collection of
exhaled and inhaled water (Wright and Stephens 1978). Water
droplets from the distal end of the tube were collected and
analyzed by a flow cytometer. The sponges were then returned
to the field and sampled by the InEx method 3 d later, using
the minute InEx sampler (Pt 5, volume 2.5 mL, length-to-outlet ID ratio = 10, Table 1), which is predicted to be most susceptible to ambient water contamination. Filtration efficiencies measured at the laboratory were on average 6% ± 4%
higher than those measured in the field (Hadas unpubl. data
unref.). Therefore, InEx-based estimates are expected to underestimate the “true” filtration efficiencies by < 10%.
Pumping rate measurements—Calibration. Calibration of
dye-front speed measurements was carried out in the flume
(Fig. 5) under low ambient flow conditions (0 to 3.5 cm s–1),
with the excurrent model jet in vertical position. The water
flux from the model excurrent jet ranged from 6 to 160 mL
min–1 (jet speed 3 to 83 cm s–1) for the 2 mm orifice, and from
10 to 2100 mL min–1 (jet speed 0.2 to 45 cm s–1) for the 10 mm
orifice. Dye-front speed measurements were replicated 10
times for each flow rate.
The dye-front was first tracked without a tube (as suggested
by Savarese et al. 1997). However, for realistic flow rates (e.g.,
2.5 cm s–1 for a 6-mm orifice, Savarese et al. 1997), significant
deflection of the excurrent jet was evident even in very low
ambient current velocities (<2 cm s–1). Due to the extreme sensitivity of this measurement to ambient currents and the
necessity of an external scale to be held within the plane of
the dyed jet, this technique was not further employed.
Dye-front speed measurements through conducting tubes
were short (<5 s) and exhibited good repeatability (average CV
18% ± 10%, n = 59). Calibration of dye-front speed to water
velocity via the excurrent model exhibited good linear correlation (R2 > 0.96, Fig. 8) up to flow rates of 100 mL min–1, for
a Tu 10 sampler (ID 9.4 mm over a 10-mm orifice) and 65 mL
min–1, for a DFS 1 sampler (ID 2.8 mm over a 2-mm orifice).
Sampler size effect. Since a perfect match between the tube
diameter and the animal’s excurrent aperture is not practically
feasible, the same protocol was used to test the performance of
Yahel et al.
Fig. 8. An example of a pumping rate (dye-front speed) calibration
experiment. Dye front speed was recorded by video analysis within a
transparent tube located just above an artificial excurrent orifice. DFS 1
sampler (ID 2.4 mm) was used to sample a 2-mm orifice. The experiments
were performed in the flume. Error bars = SD.
sampling tubes that differed from the excurrent model orifice.
This comparison was undertaken using all possible combinations of samplers with 2.8-, 6.3-, and 9.4-mm ID (DFS 1, Tu 5,
and Tu 10, respectively) over 2-, 6-, 10-, and 12-mm excurrent
orifices. Results indicated that for IDs smaller than the model’s
orifice, the dye-front speed within the tube overestimated the
predicted (average) excurrent jet speed by no more than 15%.
For sampler IDs larger than the excurrent orifice, dye-front
speed was lower than the actual excurrent speed. Nevertheless,
for IDs up to 40% larger than excurrent orifice, dye-front
speeds were in good agreement with the predicted speeds
within the tube, calculated as actual excurrent flow rate/tube
cross-section area. Even when the samplers’ cross-section area
was twice that of the excurrent orifice (e.g., Tu 10 sampler over
a 6-mm orifice), dye-front speed deviated from the predicted
speed by less than 20% (average 12%, SD 17%, n = 12). Thus,
pumping rates should be calculated using measurements of
dye-front speed with tube IDs similar to or slightly larger than
the excurrent orifice. Water flux could then be calculated as
the product of sampler cross-section area and dye-front speed
within the tube. Alternatively, tubes with ID smaller than the
excurrent orifice can be used. In this case, the excurrent flux
should be calculated as the product of the dye-front speed and
the aperture cross-section area (which should be measured
separately). If a plug flow is assumed (e.g., Savarese et al. 1997),
this method may overestimate the actual flow rate. The use of
a dye-front speed sampler with a much smaller ID is not rec-
Pumping and fluxes in suspension feeders
ommended as, in such case, knowledge of the velocity distribution across the excurrent jet is also required (Charriaud
1982; Savarese et al. 1997; Riisgård 2001a).
Ambient flow effect. The effect of sampler orientation
with respect to the ambient flow was examined using the
same protocol as above for three different orientations: 90°
(sampler perpendicular to the flow), 45° (sampler outlet facing into the flow), and 135° (sampler outlet facing downstream). In each orientation, three different samplers were
tested: DFS 1, Tu5, and Tu 10 over 2-, 6-, and 10-mm orifices,
respectively. Current velocity was fixed to 4 cm s–1. Under
such moderate flows, the effect of the sampler’s orientation
relative to the flow was negligible. For instance, a Tu 10 sampler directed 45° into or along with the flow had a mean
deviation of –4% and +5% from the predicted dye-front
speed, respectively. This deviation was not significantly different from zero (t test, P > 0.22).
The effect of flow velocity over dye-front speed flux estimates was examined for DFS 1 and Tu 10 sampler in an
upright 90° orientation. Four ambient velocities were examined: 0, 4, 6, and 11 cm s–1, with the sampler oriented perpendicular to the flow. No influence of flow on dye-front speed
was found (one-way ANOVA, F3.35 < 2.2, P > 0.1). Nevertheless,
the power of the performed tests was small (>0.3) and thus
these negative findings should be interpreted cautiously. In
the setup used here, stable excurrent velocity could not be
maintained with higher ambient flows and therefore, no
quantitative data are available for the effect of higher ambient
flows. Nevertheless, qualitative observation under higher
ambient flow velocities with various sampler orientations
indicated that under such conditions excurrent flow rates
could be grossly underestimated.
Comments and recommendations
The direct in situ nature of the InEx technique circumvents
many pitfalls inherent to traditional methods used thus far in
the study of the physiology and ecology of active suspension
feeders (Riisgård 2001a). The organisms are studied in their
natural habitat with no manipulation or disturbance. When
properly applied, the InEx technique provides independent
estimates of both water transport rates and removal/production
efficiencies. Sampling is rapid and requires simple equipment.
The InEx technique can be used with a wide range of common
suspension feeders, including fairly small animals with excurrent jet aperture as small as 2 mm in diameter. It can be applied
to a wide range of measurements including feeding and respiration (e.g., Yahel et al. 2003), exchange of heavy metals and
toxic compounds, and nutrient uptake. InEx-based retention
efficiencies, pumping rates, and clearance rates (a product of
water flux and retention efficiency) are comparable to published values (see Table 3 in Yahel et al. 2003) and are less susceptible to contamination and/or sampling bias. Measurements of dye-front speed were in excellent agreement with
continuous in situ excurrent measurements made with
55
Yahel et al.
Pumping and fluxes in suspension feeders
Acoustical Doppler Velocitometer (MicroADV, 16 MHz) over
several individual sponges in Eilat (Yahel et al. 2003).
Since the InEx technique requires divers to hold the sampler in a stable position, it is less applicable in rough environments, such as the surge zone. Sampling in extremely
turbid water may be problematic since adequate visibility is
also required. Nevertheless, underneath the surge zone,
sampling is limited mostly by diving safety considerations,
and the method was effectively applied in low temperatures
(6°C, using dry suits) and high tidal currents (<1 m s–1).
Note, however, that while reliable water sampling collection
can be carried under such conditions, pumping rate estimates may be affected to an unknown degree by high ambient current. In addition, the InEx technique is not suitable
for active suspension feeders that do not have a defined
excurrent aperture.
The “pureness” of exhaled water samples depends to a large
extent on sampling time and sampler type (see below). However, as some contamination of exhaled water with ambient
water is unavoidable, the resulting estimates of removal/production efficiencies can be considered to be lower bound. A
minimum of four sampler volumes should be pumped
through a sampler to flush it clear (Table 2). Samples from
organisms with low pumping capacity are thus restricted to
few milliliters. In order to use a larger sample volume, which
is required for some analytical procedures (e.g., particulate
organic carbon [POC], attempts to adapt the plastic bag
method of Reiswig (1971a) to minute active suspension feeders were made, but with no success. In such cases, the optimal
solution seems to be to use a controlled suction device (Yahel
unpubl. data unref.).
The selection of sampler size and type for water collection
has significant bearing on sampling duration and quality. A
smaller sampler reduces sampling time and improves the sample’s quality. However, it is recommended that the ratio
between the sampler’s length and its outlet ID (upper-end) will
be kept between 10 and 25. Whenever possible, samplers with
narrow tips (e.g., Pt 10, Table 1) are preferable.
Several studies used the product of jet speed and aperture
area to estimate water-transport rate (e.g., Fiala-Medioni 1978a,
1978b, Jones et al. 1992; Savarese et al. 1997). Jet speed–based
estimates assumed a known distribution of water velocities
across the excurrent aperture (Charriaud 1982). This latter
assumption may not be valid for many sponge species (e.g.,
Mycale spp., Suberites spp., and Theonella spp.) in which large
exhalent canals are merged from different directions just
below the osculum opening. Our observations with dyed
water revealed that excurrent jets in many suspension feeders
may be highly turbulent and are frequently composed of several independent jets. In such cases, reliable estimates of the
actual water flux may best be achieved through measuring the
pumping rate with a short tube that captures the excurrent jet
(either with the DFS method as described above or with a flow
meter that measures the flow velocity within the tube). This
56
method may be better than measuring the unconfined flow
and multiplying it by the excurrent aperture area.
A perfect match between the dye-front speed sampler’s
inlet and the animal excurrent aperture is hard to achieve
under field conditions. Nevertheless, fairly good estimates can
be obtained using a set of commercially available glass tubes
with ID increments of 1 or 2 mm. If only excurrent jet velocities are of interest, we recommend the use of a dye-front speed
sampler with ID smaller than excurrent orifice. However, if
pumping rates are under study, the sampler ID should be as
close as possible but slightly larger than the diameter of the
excurrent orifice. The water flux in this case will be calculated
as the product of sampler ID and dye-front speed. This procedure circumvents the need for an additional measurement of
excurrent orifice cross-section area (Charriaud 1982; Savarese
et al. 1997) and the complications associated with the variable
velocity distribution across the excurrent jet.
Rates of uptake (or release) of commodities by active suspension feeders are traditionally reported per animal mass or volume units (Wildish and Kristmanson 1997). The ability to
attribute such values to field populations is hampered in many
cases when quantification of biomass or living volume of active
suspension feeders in the field is impractical, inaccurate, or
excessively destructive. Furthermore, colonial taxa often accumulate and incorporate large quantities of exogenous material
over or into their tissues (e.g., Reiswig 1985), while the quantification of the biomass or living volume of boring taxa is practically impossible. An important advantage of the InEx technique is that all fluxes are estimated per excurrent jet (Fig. 2), in
situ, with no manipulation of the studied organism. This technique thus allows realistic estimates of the organism’s performance under natural conditions. Hence, InEx results are suitable
for extrapolations to community fluxes, simply by surveying
the density of relevant excurrent apertures in the system.
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Submitted 22 April 2004
Revised 14 November 2004
Accepted 15 November 2004
‫תקציר‬
‫צריכת פיטופלנקטון על ידי יצורים שוכני קרקעית הינה מסלול תזונתי עיקרי בבתי גידול ימיים רדודים באזור‬
‫הממוזג‪ .‬מחקרים שנערכו בשוניות אלמוגים‪ ,‬שם נשלטות בד"כ האוכלוסיות הפלנקטוניות על ידי תאים פרוקריוטים‬
‫זעירים‪ ,‬התמקדו באופן מסורתי בזואופלנקטון כמקור מזון חיצוני עיקרי לאוכלוסיית השונית‪ .‬מטרת עבודתי הייתה‬
‫לחקור את תפקידם של פיטופלנקטון‪ ,‬חיידקים‪ ,‬וחומר אורגאני מומס במערכת האקולוגית של שונית האלמוגים‪ ,‬מרמת‬
‫הצרכן הבודד ועד רמת החברה כולה‪.‬‬
‫מיפוי המפזר המרחבי של פיטופלנקטון סביב שוניות אלמוגים )‪ (Limnol. Oceanogr., 43, 551‬גילה כי שכבת‬
‫גבול מדולדלת מפיטופלנקטון )‪ 1-3‬מטר( נמצאת בד"כ על פני השונית‪ ,‬אך לא על פני אתרים סמוכים בעלי קרקעית‬
‫חולית‪ .‬עובי השכבה המדולדלת והבדלי הריכוזים היו תלויים במשטר זרימת המים על פני השונית ובקרבתה‪ .‬פיתוח‬
‫והתאמה של שיטת ה"נפח המבוקר"‪ ,‬תוך כדי שימוש במערך מכשירי מדידה אוקינוגרפיים מתקדמים‪ ,‬אפשר לראשונה‬
‫מדידת קצבי צריכת פיטופלנקטון במדרון השונית ובקנה מידה של חברת השונית כולה )‪.(Oceanography, 15, 90‬‬
‫התוצאות מעידות כי יבוא פחמן וחומרי דשן )נוטריינטים( על ידי צריכת פיטופלנקטון‪ ,‬הינו מסלול מרכזי במארג המזון‬
‫של שוניות אלמוגים‪ ,‬שחשיבותו לא הוערכה עד כה כיאות‪.‬‬
‫פיתוח ושיכלול שיטות למדידת קצבי אכילה בשדה )‪ (Limnol. Oceanogr. Methods., submitted‬אפשר‬
‫מדידת הרכב הדיאטה‪ ,‬דגמי ברירת טרף‪ ,‬וקצבי אכילה של ‪ 15‬יצורים מסננים ישיבים בשונית‪ .‬אלמוגים רכים‬
‫)‪ ,(Limnol. Oceanogr., 43, 354‬ספוגים )‪ ,(Limnol. Oceanogr., 48, 141‬צדפות קודחות ואצטלניים ‪(Mar.‬‬
‫)‪ ,Ecol. Progr. Ser., submitted‬נמצאו כצרכנים יעילים וחשובים של פיטופלנקטון‪ .‬הספוגים לבדם נמצאו גם‬
‫כצרכנים יעילים של חידקים לא פוטוסינטתים‪ .‬אוכלוסיות היצורים זעירים השוכנות בשכבות העליונות של סלעי שונית‬
‫"חשופים" לכאורה‪ ,‬עדיין לא נחקרו בפרוטרוט‪ ,‬עם זאת מתברר‪ ,‬כי גם לאוכלוסיות אלה תפקיד חשוב כצרכני‬
‫פיטופלנקטון בשונית האלמוגים )‪ .(Coral Reefs, in press‬דגמי העדפת הטרף של צרכני האולטראפלנקטון )< ‪10‬‬
‫מיקרון( בשונית היו דומים‪ ,‬והצביעו על העדפה לכחוליות עגולות זעירות על פני תאים אאוקריוטים שווי גודל או‬
‫גדולים יותר‪ ,‬כמו גם על פני חידקים שווי גודל או קטנים יותר שאינם פוטוסינטתים‪ .‬מכיוון שדגם העדפה שכזה אינו‬
‫מביא את צריכת הפחמן או האנרגיה אל המרב האפשרי‪ ,‬מוצע כי בשונית האלמוגים‪ ,‬בה פחמן מחוזר אינו נמצא בחסר‪,‬‬
‫יצורים מסננים התפתחו במקביל לצריכה מיטבית של חומרי מזון אחרים כגון חנקן או זרחן‪.‬‬
‫התאמת השיטות שפיתחנו למדידה של חומר אורגני מומס בים‪ ,‬אפשרה לנו לכמת באופן ישיר את קצבי ההסרה של‬
‫חומר אורגאני מומס על ידי הספוג ‪ .(Limnol. Oceanogr., 48, 141) Theonella swinhoei‬הספוג הסיר עד ‪26%‬‬
‫)‪ (12±8%‬מסה"כ הפחמן האורגני )מומס וחלקיקי( במים אותם שאב במהלך מעבר יחיד של המים דרך מערכות הסינון‬
‫של הספוג‪ .‬כמות הפחמן האורגאני המומס המוסרת גדולה בסדר גודל מזו של הפחמן בתאים החיים המוסרים על ידי‬
‫הספוג‪ .‬עדויות להסרה דומה של חומר אורגאני מומס התקבלו גם לגבי צדפה קודחת‪ ,‬איצטלן יחידאי‪ ,‬ושני מיני ספוגים‬
‫אחרים‪ .‬ממצאינו מדגימים כי לחומר אורגאני מומס עשוי להיות תפקיד חשוב בתזונתם של בעלי חיים רב‪-‬תאיים‪ ,‬וכי‬
‫תפקידם של רב‪-‬תאיים במחזור חומר אורגאני מומס עשוי להיות חשוב יותר מכפי שסברו עד כה‪.‬‬
‫‪133‬‬
‫‪.12‬‬
‫תוכן עניינים עברי‬
‫‪ .1‬תמצית‪7 ...................................................................................................................... .‬‬
‫‪ .2‬רקע מדעי‪10 ...................................................................................................................‬‬
‫שטפי פחמן‪ ,‬חומרי דשן )נוטריינטים( ופיטופלנקטון בשונית האלמוגים‪10 ............................. .‬‬
‫השפעת ההידרודינמיקה על הסעת מסות‪11 ..................................................................... .‬‬
‫קצבי רעיה פרטניים‪12 ............................................................................................... .‬‬
‫תפקיד הפחמן האורגני המומס‪14 .................................................................................. .‬‬
‫מטרות המחקר‪15 ...................................................................................................... .‬‬
‫‪ .3‬מפזר מרחבי של הפיטופלנקטון ורעייתו בקרבת שונית האלמוגים‪17 ......................................... .‬‬
‫‪ .4‬תזונה והסרת יסודות בספוג שוכן השוניות ‪ Theonella swinhoei‬באתרו‪:‬‬
‫המקור העיקרי לפחמן הוא הפחמן האורגני המומס )‪30 ............................. .(DOC‬‬
‫‪ .5‬סינון בררני של אולטראפלנקטון על‪-‬ידי שלושה יצורים מסננים טרופיים‪:‬‬
‫ספוג‪ ,‬איצטלן וצדפה‪40 ................................................................................ .‬‬
‫‪ .6‬רעיית פיטופלנקטון בסלעים "חשופים" בשונית האלמוגים‪71 ................................................... .‬‬
‫‪ .7‬דיון ומסקנות‪96 ...............................................................................................................‬‬
‫‪ .8‬מראי מקום – רשימה כללית ‪100 ..........................................................................................‬‬
‫נספחים‪:‬‬
‫‪ .9‬דלדול פיטופלנקטון על ידי אלמוג רך‪ ,‬נטול אצות שיתופניות באתרו ‪109 .......................................‬‬
‫‪ .10‬יישום שיטת ה"נפח המבוקר" מגלה רעיה נמרצת של פיטופלנקטון בקרקעית שונית האלמוגים ‪113 ..‬‬
‫‪ - "InEx” .11‬שיטה למדידה של קצבי קליטה והפרשה של יסודות על‪-‬ידי‬
‫יצורים מסננים פעילים באתרם ‪121 .....................................................................................‬‬
‫‪134‬‬
‫עבודה זו נעשתה בהדרכתו של ד"ר אמציה גנין‬
‫המחלקה לאקולוגיה‪ ,‬אבולוציה וסיסטמאטיקה‬
‫האוניברסיטה העברית בירושלים‬
‫והמכון הבין‪-‬אוניברסיטאי למדעי הים באילת‬
‫צריכת אולטרה‪-‬פלנקטון וחומר אורגאני מומס בשונית האלמוגים‪:‬‬
‫מרמת הפרט ועד לחברה‬
‫חיבור לשם קבלת תואר דוקטור בפילוסופיה‬
‫מאת‬
‫גיתי יהל‬
‫הוגש לסנט האוניברסיטה העברית בשנת תשס"ג‬
‫צריכת אולטרה‪-‬פלנקטון וחומר אורגאני מומס בשונית האלמוגים‪:‬‬
‫מרמת הפרט ועד לחברה‬
‫חיבור לשם קבלת תואר דוקטור בפילוסופיה‬
‫מאת‬
‫גיתי יהל‬
‫הוגש לסנט האוניברסיטה העברית בשנת תשס"ג‬