K .W. Tang et al. J. Exp. Mar. Biol. Ecol. 256 2001 185 –198
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represent highly concentrated pockets of DMSP in the water column, and may also act as ‘hot spots’ for microbial DMSP consumption.
In the present study, we quantified DMSP-consuming bacteria DCB associated with the body and fecal pellets of the copepod A
. tonsa, using the most probable number MPN method. We also studied the growth of the DCB on DMSP in enrichment
cultures. Our results show that the copepod body and fecal pellets contained dense populations of DCB, whose activity may have important implications for the fate of
DMSP in the water column.
2. Material and methods
2.1. MPN determination The calanoid copepod Acartia tonsa Dana was collected from eastern Long Island
Sound 418189260N, 728039270W. Twenty individuals CV to female were placed into each of four 500-ml bottles: one ‘no food’ bottle contained only 0.2-mm filtered Instant
Ocean artificial seawater ASW of a salinity 30‰; three ‘with food’ bottles contained
4 21
2 3 10 ml of Tetraselmis impellucida McLachlan et Parke PLY429 prepared with
the same ASW used in the ‘no food’ bottle. The collection of copepods and the maintenance of algal culture are described in detail by Tang 2000a. All bottles were
fastened to a spinning plankton wheel 2 rpm and kept at 20 C in dark. After 24 h, five individuals from the ‘no food’ bottle were transferred to 1 ml of autoclaved ASW in a
sterile vial and homogenized with a sterile pestle. The homogenate was then washed with autoclaved ASW into a sterile test tube final volume 5 2 ml, and was labeled as
inoculum 4. Copepods from the three ‘with food’ bottles were pooled together. Five copepods were immediately removed, rinsed in autoclaved ASW and homogenized
inoculum 1. Another 25 copepods were rinsed and transferred to an alcohol-cleaned mini-trap with | 5 ml of autoclaved ASW for collecting fecal pellets. The mini-trap was
a pellet collection device after the design by Small et al. 1979. The use of a large number of copepods in a small volume of water enabled the collection of enough fecal
pellets in a short period of time. After 2 h, the copepods were removed from the mini-trap and five copepods were removed for homogenization inoculum 2. Fecal
pellets were allowed to settle inside the mini-trap; excess water was removed with the sterile pipet and all the fecal pellets were rinsed into a sterile test tube final volume 5 5
ml. The test tube was shaken vigorously on a vortex for several minutes to homogenize the fecal material inoculum 3. Aliquots for determination of MPN of suspended DCB
were taken from the upper portion of the homogenate. Even if the homogenization was not 100 efficient, this procedure would have likely resulted in an underestimation of
the bacterial abundance due to particle settlement in the homogenate.
The abundance of DMSP-consuming bacteria DCB associated with each homoge- nate was determined by the MPN method. Aliquots from each homogenate were
inoculated in 1:1 marine mineral medium Hines et al., 1997 and autoclaved 0.2-mm
188 K
.W. Tang et al. J. Exp. Mar. Biol. Ecol. 256 2001 185 –198
filtered seawater pH 7.0 with 1 mM DMSP University of Groningen, The Nether-
21
lands; . 98 purity as the organic carbon source. Nine dilution levels between 10
29
most concentrated and 10 most diluted were prepared. Each dilution level
consisted of three tubes 5-ml sterile snap-cap test tube, Fisher Scientific, with a final volume of 3 ml in each tube. The tubes were loosely capped and maintained at | 308C
in dark to prevent growth of phototrophs. The chosen pH and temperature are commonly used as optimal conditions for culturing marine mesophilic bacteria Austin, 1992;
Madigan et al., 1997. After 15 days, DMSP in the tubes was replenished by spiking with 50 mmol of DMSP final concentration 5 17 mM. After 7 weeks, all tubes were
examined, and the presence or absence of bacteria was confirmed with acridine orange staining Hobbie et al., 1977. MPNs of DCB in the original homogenates, with 95
confidence intervals, were determined according to DeMan 1975.
2.2. Growth kinetics of DMSP-consuming bacteria Since we were interested in DMSP consumption rates by the bacterial community
instead of individual strains, we prepared enrichment cultures with the MPN inocula of
21
the 10 dilution level. This dilution level conceivably represented the more diverse and
active DCB recovered from the copepod bodies and fecal pellets in the MPN experiment. It should be emphasized that the enrichment cultures provided merely an
estimate of the growth and DMSP-consumption characteristics of the DCB, since the DCB may behave differently when they are attached to the copepods and fecal pellets
than when they are in suspensions e.g. Bright and Fletcher, 1983; Diab and Shilo, 1988. One ml from each of the four MPN inocula was transferred to a sterile glass test
tube with 40 ml 50 marine mineral medium see above and 5 mM DMSP pH 7.0. The test tubes were loosely capped to allow gaseous exchange, and were maintained at
298C in the dark. After 1 week, enrichments were set up by transferring 5 ml from each test tube to a glass Erlenmeyer flask with 200 ml 50 marine mineral medium and 5
mM DMSP pH 7.6. The four enrichments were labelled corresponding to the original four MPN inocula see Section 2.1.. All enrichments were incubated aerobically in the
dark at 298C. Aliquots from each enrichment were taken for optical density OD measurements at 580 nm daily and acridine orange AO cell counts day 0, 1, 2 and
5. OD was converted to cell density based on the linear relationship between AO cell
2
counts and OD measurements r 5 0.97, 0.97, 0.94 and 0.99 for enrichment 1, 2, 3 and 4, respectively.
At the end of the growth experiment, three types of samples in duplicate were taken from each enrichment for DMSP and DMS measurements: 1 Total: 1-ml aliquot for
total DMSP 1 DMS; 2 Filtrate: 1-ml aliquot was filtered through a 0.2-mm cellulose ester membrane syringe filter, and the filtrate was collected for dissolved DMSP 1 DMS;
3 Purged filtrate: 1-ml filtrate was first purged with N for 5 min to remove DMS, then
2
collected for dissolved DMSP. All samples were hydrolyzed with 4 ml 1 N NaOH in closed serum vials overnight, and the resultant DMS was measured using the purge-and-
trap technique Kiene and Service, 1991 with a GC–FPD system Tang et al., 1999.
K .W. Tang et al. J. Exp. Mar. Biol. Ecol. 256 2001 185 –198
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The detection limit of the system was 0.4 pmol DMSP per ml injection, which would be equivalent to 2 nM DMSP per sample.
3. Results
