T . Moens, M. Vincx J. Exp. Mar. Biol. Ecol. 243 2000 137 –154
139
Findlay and Tenore, 1982; Alkemade et al., 1992a,b. Data on ecological and physiological responses of marine and estuarine Monhysteridae and Rhabditida to
changes in food or abiotic conditions include studies of growth and fecundity as a function of temperature 11 species, of salinity five species, of food quality and of
food density two species see Heip et al., 1985, 1995; Vranken, 1985; Vranken et al., 1988, for reviews. These studies include P
. marina as the sole marine rhabditid nematode, and several species of Monhysteridae. Although respiration has been
investigated in some marine nematodes, the respiratory response to temperature and salinity has been established for but a single monhysterid species Warwick, 1981b. No
studies have linked marine or estuarine monhysterid or rhabditid feeding rates to food density.
This paper focuses on one rhabditid and one monhysterid nematode species from an estuarine, intertidal ‘Aufwuchs’ community. It tries to define biotic food supply and
abiotic temperature, salinity ranges and optima a as a means of clarifying zones and periods of occurrence and abundance of these nematodes, and b to get an indication of
the relative importance of food and abiotic variables in determining the community structure in these highly unstable environments.
2. Materials and methods
2.1. Culture of experimental organisms Details on the isolation and agnotobiotic cultivation of the bacterivorous nematodes
Pellioditis marina Bastian, 1865 Andrassy, 1983 and Diplolaimelloides meyli Timm, 1967 are given elsewhere Moens and Vincx, 1998. Briefly, nematodes were isolated
from macrophyte detritus by the use of spot plates, and cultivated on a 1 agar prepared with artificial seawater ASW, Dietrich and Kalle, 1957 with a salinity of 20‰, and
with bacto- and nutrient agar in a weight weight ratio of 4 1. Bacteria cotransferred from the spot plates served as food to the nematodes. Our nematode cultures originated
from the mesohaline reach of the Westerschelde Estuary, SW Netherlands. They were kept at 208C in the dark. By the time the present experiments were run, both species had
been in permanent culture for several years Moens and Vincx, 1998.
Separate bacterial batch cultures, established with isolates from the respective nematode cultures see Moens and Vincx, 1999, for more details on isolation and
enumeration of bacteria, were grown on Luria–Bertani-medium LB-medium, Sam- brook et al., 1989 with a salinity of 20‰. Judging from colony morphology, batch
cultures BPM1 and BDM1, originating from the P . marina and D. meyli cultures,
respectively, each contained at least four strains, two of which together comprised approximately 90 of total bacterial numbers.
2.2. Respiration measurements O -consumption of groups of 100 for P
. marina to 1000 for D. meyli nematodes
2
was determined using a polarographic electrode technique described elsewhere Moens
140 T
. Moens, M. Vincx J. Exp. Mar. Biol. Ecol. 243 2000 137 –154
et al., 1996a. In the case of P . marina, only adult and fourth instar J4 individuals were
used in our experiments. They were handpicked from cultures, transferred once through sterile ASW, and resuspended in 1 ml of an antibiotics solution 5000 U benzylpenicillin
21
and 1000 mg ml streptomycin sulphate in ASW. Rhabditid nematodes are tolerant of
high levels of these antibiotics Gochnauer and McCoy, 1954, and preliminary experiments showed that their respiration rates were unaffected by the concentrations
used. They were then introduced into a closed, 1-ml respiration chamber, and their O -consumption was determined over a 20–30 min interval while gently stirring the
2
medium. The procedure for respiration measurements with D . meyli was similar, except
that nematodes, including all ages, were collected from the surface of cultures, and surface-cleaned by washing with sucrose Moens et al., 1996a. In view of their lower
respiration rates, high numbers of D. meyli were needed for reliable measurements, and hand-picking proved inefficient at obtaining such numbers. Lower levels of antibiotics
21
1000 U benzylpenicillin and 1000 mg ml streptomycin sulphate were used for this
species. Nematodes were allowed to acclimate for 2–3 h at the experimental temperature or salinity prior to respiration measurements Dusenbery et al., 1978. Preliminary
experiments showed that longer acclimation periods strongly reduced respiration rates as a result of starvation, but yielded similar patterns of respiration vs. salinity and
temperature Moens, unpublished.
Measurements of respiration at different salinities were performed at 208C; measure-
ments at different temperatures used ASW with a salinity of 20‰. The same abiotic conditions were used in the food density dependence experiment and in the feeding
experiments see below. There were three replicates plus two controls consisting of 0.22-mm millipore filtered aliquots of the antibiotics solution in experiments with P
. marina, and two replicates plus two controls in tests with D
. meyli. 2.3. Feeding experiments
Feeding of groups of 25–35 J4 and adults of P. marina or 50 J4 and adults of D. meyli
3
was determined using H-labelled bacterial batch cultures. The higher D . meyli numbers
were meant to compensate for their lower feeding rates compared to P . marina. The
procedure for these experiments is detailed elsewhere Moens et al., 1999b. Briefly, nematodes were handpicked from cultures, rinsed once in sterile ASW, and transferred to
a 450-ml ASW drop in a 3.5-cm diameter Petri dish. Bacteria were grown in LB-medium
3
to which H-adenine was added in a final concentration of ca. 400 nmolar. Bacteria were harvested by centrifugation, washed five times with sterile ASW to remove non-
incorporated label, and diluted to the desired density in ASW. Bacterial density was determined via epifluorescence microscopy using acridine orange modified after Daley
and Hobbie, 1975; Hobbie et al., 1977. A volume of 150 ml of bacterial suspension was added to the nematodes, and the total 600 ml gently spread to give a water film. As such,
the nematodes were not suspended in the water but crawled on the bottom of the Petri dish, where they actively grazed bacteria. Nematodes were allowed to graze for 1 h in
the dark at 21628C or at a range of temperatures in the temperature dependence experiment. The experiment was stopped by the addition of 1 ml of a 4 buffered
formaldehyde solution. Nematodes were manually transferred twice through sterile ASW
T . Moens, M. Vincx J. Exp. Mar. Biol. Ecol. 243 2000 137 –154
141
to remove most adhering bacteria, and dissolved for 48 h in Lumasolve Lumac tissue solubiliser. It has been argued that feeding rates determined in this way represent a
measure of assimilation rather than ingestion Schiemer, 1987; Moens et al., 1999b. Radioactivity was determined via liquid scintillation counting on a Beckmann LS6000
after addition of the scintillation cocktail Lumasafe plus Lumac. Quenching was corrected for by external standards method. Controls consisted of nematodes which were
killed beforehand with formaldehyde, rinsed in ASW, and incubated with labelled bacteria under the same experimental conditions as the live ones.
2.4. Calculation of scope for production Based on our measurements of food assimilation and respiration at different
temperatures and salinities, a scope for production in J4 and adults of P . marina and D.
meyli was calculated as a function of these two abiotic variables, production equalling assimilation–respiration. For these calculations, the following assumptions were made:
3
1 the amount of H incorporated was proportional to the amount of C incorporated; 2 70 of the assimilated label was lost from the nematodes upon preservation with
213 21
formaldehyde Moens et al., 1999b; 3 bacterial cell weight was 2 3 10 g C cell
in both batch cultures; 4 1 l O consumed corresponded to 0.4 g C respired Heip et
2
al., 1985. 5 The influence of temperature and salinity on the respiration of D . meyli
was independent of stage, an assumption that is probably biased. However, this assumption allowed an estimate of O -consumption per individual adult nematode. To
2
this end, the O -consumption of samples incubated at 258C was divided by 2.1, i.e. the
2 21
O -consumption in nl h of adult D
. meyli at that temperature Moens et al., 1996b;
2
this calculation yielded a number of individuals which a sample would have contained if it had consisted of adult nematodes only. This average number of adults per sample
samples for all respiration measurements were taken from one homogeneous pool was subsequently used to calculate respiration rates per individual at the other temperatures.
3 4
6 Finally, 1 dpm disintegrations per min corresponded to approximately 10 and 10 bacteria in cultures of BDM1 and BPM1, respectively determined from a combination
of liquid scintillation measurements on aliquots of bacterial culture and of cell counts of serial dilutions of such aliquots. Evidently, the obtained assimilation rates should not be
treated as absolute, but as relative values, the use of which is justifiable only to illustrate the pattern of temperature and salinity dependence.
3. Results and discussion
