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Limnol. Oceanogr., 52(1), 2007, 428–440
2007, by the American Society of Limnology and Oceanography, Inc.
E
In situ feeding and metabolism of glass sponges (Hexactinellida, Porifera) studied in
a deep temperate fjord with a remotely operated submersible
Gitai Yahel, Frank Whitney, Henry M. Reiswig, Dafne I. Eerkes-Medrano, and Sally P. Leys
Web Appendix 1. Details of SIP Sampler design, water
processing, and statistical analysis of ‘In-Ex’ samples from
glass sponges.
Water processing
Total organic carbon (TOC) analysis—Analysis of TOC
was carried out using high-temperature catalytic oxidation
on a TOC-V total organic carbon analyzer with oxygen as
the carrier gas. Six repeated 100-mL injections were
analyzed for each sample using the multi-injection method
after 2-min sparging inside the analyzer syringe pump.
The instrument was slightly modified, as suggested by
J. H. Sharp (pers. comm.), by removing the cooling coil and
pure water trap, replacing the top layer of quartz wool in
the combustion tube with a layer of platinum mesh
cushions, and eliminating the purging of the detector light
source. A five-point calibration curve was run at least
once a day using potassium hydrogen phthalate in
DDW. At least one deep-sea (43.2 6 1.7 mmol L21) and
one low-carbon (C) (1.8 6 0.7 mmol L21) International
Consensus Standard (Batch 3) was analyzed for every 10
samples (Sharp et al. 2002). TOC values exceeding 62
standard deviations (SDs) of the mean were considered
outliers and were removed from subsequent analysis. The
error associated with TOC measurements was 1–2 mmol C
L21 (coefficients of variation of repeated injection were
,4%).
SIP sampler design
The basic components of the SIP water sampler
(numbers pertain to Fig. A1.1) are (1) stainless-steel bleed
valve (Swagelok PN SS-BVM4); (2) double-ended sample
cylinder (Swagelok PN 316L-HDF4-150-T) with internal
Teflon coating and internal pressure rating of 125 bars;
(3) street elbow (Swagelok PN SS-4-SE); (4) quarter-turn
plug valve (Swagelok PN SS-4P4T1); (5) reducer (Swagelok
PN SS-100-R-4); and (6) inlet PEEK tube (Upchurch
Scientific 1532) with external diameter of ,1.59 mm and
internal diameter of ,510 mm, which allowed a simple
control of 4the suction rate according to the equation
DP:p:r
F~ : : :
where F 5 flow rate (cm3 min21), DP 5
8K LV
differential pressure (bar), r 5 inlet tubing internal
radius (cm), K 5 2.417 3 1029 (s22), L 5 tube length
(cm), and V 5 water viscosity (g cm 21 s 21). Dye
visualization indicated that oscular flow velocity was
.1 cm s21, resulting in a pumping rate .50 mL s21 for
an osculum 8 cm in diameter. To ensure that only exhaled
water was sucked into the SIP, we used 10-cm inlet tubing with a 508-mm internal diameter that delivered an
initial suction rate of ,1 mL s21 at working conditions
(145 m in depth, 8uC, 33%). Since DP and sampling rate
decreased exponentially with sampling time, we sampled
for a minimum of 5 min. The total sample volume was
,120 mL.
An attempt to control for possible bacterial activity in
the samples was made by fitting every second SIP pair with
a 2-mm prefilter (Upchurch A-330) and an inline 0.25-mm
stainless-steel frit filter (Swagelok SS-400-6-1LV-S8). However, microscopy and flow cytometry analysis indicated
that particles larger than 2 mm were absent in the filtered
samples, and no significant difference was found between
the bacterial counts or bacteria properties of filtered and
unfiltered samples (paired t-test, p . 0.1, n 5 22),
indicating that the Swagelok 0.25-mm frits were not
functioning. Therefore, prefiltered samples were considered
to be 2-mm filtered, and it was acknowledged that our
experiment lacks a control for bacterial effect on the
sampled waters during retrieval. Nevertheless, no trend in
sampled water properties as a function of time since
collection could be detected, indicating that limited, if any,
bacterial activity occurred in the samples prior to water
processing.
Cell counts and microscopy—Total counts of bacteria
were obtained by flow cytometry (FACSCalibur, Becton
Dickinson) following staining with the nucleic acid dye
SYBR Green I at a concentration of 1024 and 25-min dark
incubation at room temperature (Marie et al. 1999). Flow
rate was set to low (6–8 mL min21, measured daily), and
inhaled and exhaled samples were analyzed sequentially for
2 min each using the inhaled sample settings. Fluorescent
beads (Flow-Check High Intensity Green Alignment
1.0 mm; 23517-10, Polysciences) were introduced to each
sample as an internal standard, and all cellular parameters
were normalized to the bead values. Since the same settings
were used for both inhaled and exhaled samples, the
number of bacteria cells counted was usually .10,000 for
inhaled samples but as low as a few hundred for exhaled
samples. A subset of the inhaled samples was also analyzed
using a high flow rate (60 mL min21) and a low Fl3 (red
fluorescence) threshold to search for chlorophyll-containing (phytoplankton) cells; none were found.
To quantify the number of heterotrophic protists and
larger phytoplankton (Sherr and Sherr 1993), 10–14 mL of
the sample water was incubated with 49,6-diamidino-2phenylindole dilactate (DAPI; D-3571, Molecular Probes)
at a final concentration of 4.4 mmol L21 for ,15 min in
the dark and filtered under low vacuum (,0.05 bar)
onto 0.8-mm black polycarbonate membrane (110659,
Whatman Black Nuclepore). Each filter was scanned using
1
Yahel et al.
Fig. 1. Change in ammonium concentration measured in situ
during a single pass of the water through the glass sponge. Paired
water samples are represented by a solid circle for the inhaled
water and an open circle for the exhaled water connected by
a vertical line, indicating the magnitude of removal/excretion per
liter pumped. Specimen numbers are indicated on the abscissa: A,
the cloud sponge Aphrocallistes vastus; R, the boot sponge
Rhabdocalyptus dawsoni. Duplicate samples were obtained whenever possible. A small fish was residing in the osculum of
sponge A9.
a 460–490-nm excitation filter at 3400 magnification for
the presence of chlorophyll-containing cells. Bacteria
and heterotrophic protists were quantified under ultraviolet excitation using epifluorescence and a highresolution monochrome digital camera. Thirty randomly
selected images representing 2.4% of the entire filtration
area were captured from each filter. Close examination
of 10 fields at 31,000 magnification was also used. In
some cases we also used scanning electron microscopy
(SEM; Hitachi S-3500N) to examine cell properties. SEM
samples were filtered with no staining on 0.45-mm
2
membrane filter and were air-dried and gold coated prior
to analysis.
The dimensions of all cells with a minor axis of .1 mm
were measured using an automated script written for
Image-Pro (Version 4.5) and were converted to biovolume
assuming an ellipsoid shape (V 5 4/3 ? p ? 0.5a ? 0.25b2,
where V 5 cell volume and a and b are the cell’s major and
minor axes, respectively). To convert the protists’ cell
volumes to carbon and nitrogen (N) values, we followed the
recommendations of Menden-Deuer and Lessard (2000),
assuming C 5 0.216V0.939, where C is the cellular carbon
content (pgC cell), V is the cell volume (mm3), and a C : N
ratio of 8. Because the preservation method we used for
protists may have resulted in cell loss and deformation, the
direction or magnitude of the bias cannot be determined
(Menden-Deuer et al. 2001). For bacteria, a subset of the
inhaled samples was also analyzed as above using 0.2-mm
membrane filters. Bacterial carbon content was estimated
using a constant conversion factor of 0.148 pgC mL21
(Gundersen et al. 2002). The resulting average cell content
estimate (Yahel et al. in press, table 1) was used to convert
the flow cytometer bacterial counts to carbon and nitrogen
(C : N ratio 5 5) estimates.
Statistical analysis—Data were analyzed using SPSS
(version 12) and SigmaStat (version 3). Throughout the
text, means and regression slopes are reported with 61 SD,
unless otherwise stated. Paired t-test or two-way repeatedmeasures analysis was used to test for differences between
inhaled and exhaled water constituents (Yahel et al. 2005).
Percent data were arcsine square root–transformed when
necessary to meet the requirements of normality and
homogeneity of variance. For repeated-measures analysis
of variance (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’
l (a multivariate criterion). The equivalent nonparametric
tests were used whenever data did not meet the ANOVA
assumptions. In cases in which no significant difference was
found between inhaled and exhaled concentrations, the
detection limit (sensitivity) of the test is given for
a statistical power of .0.9 and a confidence limit of 95%.