Preliminary Investigation of the Ecological Role of Microzooplankton in the Kariega Estuary, South Africa

Estuarine, Coastal and Shelf Science (1997) 45, 689–695

Preliminary Investigation of the Ecological Role of Microzooplankton in the Kariega Estuary, South Africa P. W. Froneman and C. D. McQuaid Southern Ocean Group, Department of Zoology and Entomology Rhodes University, Box 94, Grahamstown, 6140, South Africa Received 24 June 1996 and accepted in revised form 25 October 1996 A preliminary investigation of the ecological role of microzooplankton (20–200 ìm) was conducted in the Kariega Estuary along the south coast of South Africa during summer (November) 1994. Microzooplankton grazing impact on phytoplankton was estimated at 15 and 20 )C in the laboratory using the dilution technique. Size-fractionated chlorophyll studies showed that the nano- and picophytoplankton dominated phytoplankton standing stock, comprising between 53 and 78% of total chlorophyll. During the entire investigation, the microzooplankton were numerically dominated by protozooplankton with densities ranging between 900 and 2850 cells l "1. Based on biovolume: carbon estimates, microzooplankton contributed between 13 and 43% of the total biological seston in the size range 2–200 ìm. Amongst the protozoans, ciliates and dinoflagellates numerically dominated. Microzooplankton densities co-varied with chlorophyll concentrations (r2 =0·45; P<0·05). Analysis of variance indicated that the grazing impact of microzooplankton was not significantly different between the two temperatures (F=12·68; P>0·05). The instantaneous grazing rates of microzooplankton on nanophytoplankton varied from 0·010 to 0·105 day "1, and those on microphytoplankton from 0·007 to 0·091 day "1. This level of grazing corresponds to a daily loss of about 5 and 2% of the initial standing stock and about 50 and 65% of the potential production of the two size fractions, respectively. During this study, the grazing rates of microzooplankton correlated with the growth rates of nanophytoplankton (r2 =0·51; P<0·05) reflecting tight coupling between these two fractions. These data suggest that microzooplankton are important consumers of phytoplankton production in the Kariega Estuary. ? 1997 Academic Press Limited Keywords: microzooplankton; grazing; Kariega Estuary; South Africa coast

Introduction Microzooplankton (20–200 ìm) constitute a significant proportion of total zooplankton biomass in a variety of aquatic environments, and have consequently been the subject of numerous studies (Porter et al., 1985; Mazumder et al., 1990). Theoretical studies on aquatic food-web dynamics have suggested that microzooplankton are capable of consuming a significant proportion of primary production (Frost, 1991). Indeed, field experiments have demonstrated that microzooplankton consume between 10 and 75% of daily primary production (Garrison, 1991; Pierce & Turner, 1992). Furthermore, these studies have shown that microzooplankton grazing can play an important role in regulating bacterial populations (Gast, 1985; Andersen & Sorenson, 1986; Albright et al., 1987; McManus & Fuhrman, 1988; Reid & Karl, 1990) and regenerating nutrients (Goldman et al., 1987; Probyn, 1987). Microscopic examinations of consumer gut contents, feeding structures 0272-7714/97/050689+07 $25.00/0/ec960225

and faecal material reveal that a number of invertebrate and fish larvae consume microzooplankton (Stoecker & Capuzzo, 1990). Thus microzooplankton act as trophic intermediates between the small bacteria, nanoplankton and the larger meso- and macrozooplankton (Gifford, 1991). There are few studies of microzooplankton in estuaries in South Africa. Indeed, according to Jerling and Wooldridge (1995), nothing has been published on any of the ecological aspects of microzooplankton in South African estuaries. A recent quantitative study conducted in the Sundays River Estuary has shown that microzooplankton densities may be >5000 cells l "1 (Jerling & Wooldridge, 1995). This suggests that microzooplankton may form an important component of the plankton community of this system. Presently, however, the most compelling argument for the importance of microzooplankton in estuarine environments is their high abundances. The Kariega Estuary (33)41*S, 26)42*E) is situated on the south coast of South Africa. As a result of the ? 1997 Academic Press Limited

690 P. W. Froneman & C. D. McQuaid

low annual freshwater influx, the system is regarded as a homogenous oligotrophic marine system (Grange & Allanson, 1995). Generally, chlorophyll concentrations in the Estuary are <2·0 ìg l "1 and are dominated by small nano (2–20 ìm) and picophytoplankton (0·45–2·0 ìm) cells (Allanson & Read, 1987; Grange & Allanson, 1995). The low chlorophyll concentrations in the estuary are thought to reflect low primary production rates due to nutrient limitation associated with low freshwater influx (Allanson & Read, 1987; Grange & Allanson, 1995). Although considerable information is available on hydrodynamic structure and the processes controlling phytoplankton production in the estuary (Allanson & Read, 1995; Grange & Allanson, 1995), the influence of biological factors such as grazing by zooplankton in determining chlorophyll concentrations has been poorly studied. Studies of the biota in the Kariega Estuary have largely focused on examining the distribution and biology of both invertebrates (de Villiers, 1988; Hodgson, 1988; Grange, 1988,1992) and vertebrates (Whitfield, 1994). In situ grazing studies conducted with the dominant zooplankton (copepods) have shown that they are able to clear up to 25% of the water column per day (Grange, 1992). Recent studies have, however, shown that the bulk of the seston (detritus and chlorophyll) is <5 ìm (Grange & Allanson, 1995), below the optimum particle size for the dominant zooplankton in the Kariega Estuary (Grange, 1992). These facts suggest that copepods are not the most important grazers of phytoplankton production in the estuary. Several studies in a variety numerous aquatic environments have shown that where small phytoplankton cells dominate (<20 ìm), microzooplankton are often the most important consumers of phytoplankton (Kivi & Kuosa, 1994; Froneman & Perissinotto, 1997). These facts suggest that microzooplankton may represent the most important grazers of phytoplankton production in the Kariega Estuary. The aim of this study was to estimate the grazing impact of microzooplankton on phytoplankton in the Kariega Estuary during Summer 1994. Materials and methods Microzooplankton grazing impact on phytoplankton in the Kariega Estuary along the south coast of South Africa (Figure 1) was estimated in austral summer (November) 1994 using the dilution technique (Landry & Hassett, 1982). Water was collected from the Kariega Estuary in 20-l polyethylene carboys and transported to the

A 32°

East London

Port Elizabeth

Kariega River Estuary N

34° S

Indian Ocean

26°

27°

28° E

F 1. Location of study area with inset illustrating the position (A) from which water samples were obtained for the microzooplankton herbivory studies.

laboratory within 1 h. The water was then gently filtered through a 200 ìm mesh to isolate the microzooplankton fraction from larger predators. Particlefree water was obtained by gently filtering (<5 cm Hg) half the water collected through a 0·2 µm serial filtration unit. Dilution series of 1:0, 3:1, 1:1, 1:3 filtered to particle-free water were then prepared in 2-l acid-washed polyethylene bottles. Three replicas were prepared for each dilution series. The three sets of dilution series were then incubated for 24 h in a constant environment room (CE) at 15 )C, while another three were incubated at 20 )C. The experiments were conducted with a 14: 10 h light dark phase. These temperatures represent average summer conditions along the south coast of South Africa (unpubl. data). Before incubation was begun, a water sample (250 ml) was taken from each bottle of the dilution series to provide a measure of initial chlorophyll a concentration. The corresponding bottles were sampled again (250 ml) at the end of the incubation period to determine final chlorophyll a concentration. Chlorophyll a was fractionated into the <20 ìm (nano- and picophytoplankton) and microphytoplankton (>20 ìm) using serial filtration. Chlorophyll a concentrations were determined fluorometrically (Turner 111 fluorometer) after extraction in 100% methanol (Holm-Hansen & Riemann, 1978). A chlorophyll:carbon ratio of 34·8 (Cushing,

Ecological role of microzooplankton 691 T 1. Rate estimates with regression coefficients and confidence limits (r2) of microzooplankton grazing studies on (a) the <20 ìm and (b) the >20 ìm chlorophyll fraction conducted in austral summer (November) 1994. Experiment Chl a number (ìg l "1)

r2

Growth coeff. Grazing coeff. % Initial stock % Potential prod. (k) (day "1) (g) (day "1) removed (day "1) removed (day "1)

(a) 1 2 3 4 5 6

1·139 1·213 0·772 0·845 0·967 1·236

0·85** 0·64* 0·87* 0·72* 0·76** 0·54**

0·139 0·108 0·168 0·009 0·121 0·151

(0·007) (0·009) (0·006) (0·008) (0·013) (0·013)

0·088 0·078 0·105 0·010 0·025 0·037

(0·002) (0·007) (0·011) (0·003) (0·008) (0·009)

8·51 7·50 6·61 1·06 2·48 3·64

8·50 58·10 67·90 101·01 21·13 25·87

(b) 1 2 3 4 5 6

0·881 0·833 0·496 0·312 0·431 0·433

0·87* 0·53** 0·61* 0·71* 0·51* 0·57*

0·019 0·027 0·020 0·014 0·027 0·017

(0·002) (0·006) (0·005) (0·004) (0·003) (0·001)

0·014 0·016 0·008 0·007 0·009 0·009

(0·002) (0·007) (0·006) (0·002) (—) (0·001)

1·36 1·59 0·81 0·32 0·93 1·15

52·85 84·33 42·34 50·01 33·89 54·85

**=P<0·01; *=P<0·05). Values in parentheses are standard errors.

1957) was then employed to estimate the autotrophic carbon component of the biological seston. To identify and enumerate the various components of the microzooplankton communities during each experiment, a 250-ml sample of natural sea water was passed gently through a 200 ìm mesh and fixed with 10% Lugols’ solution (Leakey et al., 1994). Microzooplankton species composition and densities were then estimated using the Utermohl settling technique after sedimentation in a 10-ml settling chamber (Reid, 1983). A Nikon-TMS inverted microscope operated at X400 magnification was used for this analysis (Reid, 1983). A minimum of 500 cells or 100 fields were counted for each sample. The total carbon of the microzooplankton fraction was then estimated by calculating the mean biovolume of 50 ciliates and 50 dinoflagellates, assuming that 1 ìm3 =0·19pg C (Putt & Stoecker, 1989; Sime-Ngando et al., 1992). The microzooplankton species were identified using the works of Wood (1954) and Boltovskoy (1981). The apparent growth rate of chlorophyll a in each bottle was calculated using the exponential model of Landry and Hassett (1982): Pt =Poe

(k"f)t

where Pt is chlorophyll a concentration at time t; Po is initial chlorophyll a concentration; and k and f are the instantaneous algal growth and microzooplankton grazing coefficients, respectively. The coefficients

were determined from linear regression analysis (95% confidence limits) between dilution factor and apparent growth rate of chlorophyll a in each bottle using the computer program Statgraphics-Version 5.0 (Statistical Graphics Corporation, 1992). Both g and k were used to calculate the grazing loss of potential production, while only the grazing mortality coef ficient (g) was employed to calculate the daily loss of initial standing stock. Pearson’s correlation analysis was used to identify possible relationships between grazing rate, temperature and chlorophyll. Grazing rate data, expressed as percentage initial phytoplankton stock and potential primary production removed per day, were transformed using arcsin transformation (Sokal & Rolhf, 1969), while chlorophyll concentration values were transformed using the factor: log (x+1) (Legendre & Legendre, 1983). The computer package, Statgraphics, Version 5.0, was again used for this analysis. Results Fractionated chlorophyll The results of the size-fractionated chlorophyll study are shown in Figure 2. During the investigation, the <20 ìm chlorophyll fraction dominated, comprising between 53 and 78% of the total (Figure 2). Chlorophyll concentrations of the <20 ìm fraction during

2.5

3000 15°C

20°C

2 1.5 1 0.5 0

1

2

3 4 Experiment no.

5

6

F 2. Initial size fractionated chlorophyll during the microzooplankton herbivory studies. Open bars, <20 ìm fraction; solid bars, <20 ìm fraction.

this investigation ranged between 0·772 and 1·236 ìg l "1. Analysis of the species composition showed that micro-sized diatoms (Nitzschia sp.) and unidentified flagellates dominated the nanophytoplankton. The chlorophyll concentration of microphytoplankton (>20 ìm) during the investigation ranged between 0·321 and 0·881 ìg l "1. The microphytoplankton were numerically dominated by large marine diatom species such as Grammatophora marina, Ditylum brightwelli and Thalassiosira sp. Also well represented amongst the microphytoplankton cell counts were species belonging to the genera Rhizosolenia and Nitzschia. Microzooplankton community structure During the entire study, the microzooplankton community was numerically dominated by protozoans (Figure 3), the densities ranging between 900 and 2850 ind.l "1. The ciliates, comprising aloricate ciliates (Oligotrichs) and tintinnids generally dominated, accounting for 40–65% of the total. An exception was Experiment 5 when dinoflagellates constituted the most numerous component (Figure 3). Aloricate forms constituted the main component of the ciliate group, with densities ranging between 450 and 1950 ind.l "1 (Figure 3). Tintinnid densities were always <100 ind.l "1 (Figure 3). The second most abundant group among the microzooplankton assemblage were dinoflagellates with densities ranging from 350 to 750 cells l "1 (Figure 3). Among the flagellates, members of the genus Amphisolenia were the most abundant species with densities ranging from 300 to 750 ind.l "1. Also well represented were unidentified species of the genus Ceratium (densities of between 125 and 400 ind.l "1) and Gymnodinium spp. (densi-

Microzooplankton densities (cells l–1)

Chlorophyll concentration (µg l –1)

692 P. W. Froneman & C. D. McQuaid

Ebriids 2500

Dinoflagellates Tintinnids

2000

Aloricates

1500 1000 500 0

1

2

3 4 Experiment no.

5

6

F 3. Microzooplankton taxonomic composition during the herbivory studies.

ties range from 25 to 275 ind.l "1). The least abundant component of the microzooplankton assemblages during the investigation were the ebriids (Figure 3). Densities of these flagellates were always <100 cells l "1. No metazoan larvae were found in the samples. Grazing studies Instantaneous growth and grazing coefficients with confidence limits derived from the herbivory experiments are shown in Table 1(a,b). In all the dilution experiments, there was a significant relationship between the apparent growth rate and dilution (P<0·05). Analysis of variance indicates that the grazing impact of microzooplankton was not significantly different between the two temperatures (F=12·68; P>0·05). Consequently, the results of the experiments have been combined. Instantaneous growth rates (k) of the nanophytoplankton ranged between 0·009 and 0·168 day "1 [Table 1(a)]. This level of growth is equivalent to between 0·012 and 0·242 chlorophyll doublings day "1. Instantaneous grazing rates (g) of microzooplankton on nanophytoplankton ranged from 0·010 to 0·105 day "1 These correspond to daily losses of between 1·06 and 8·51% (x¯ =4·96%) of the initial standing stock and 8·50–101·01% (x¯ =48·87%) of the potential primary production of the nanophytoplankton fraction [Table 1(a)]. Instantaneous growth rates in the microphytoplankton fraction were lower and ranged between 0·014 and 0·027 day "1 [Table 1(b)]. These rates correspond to chlorophyll doubling rates ranging between 0·020 and 0·039 day "1. The instantaneous grazing

Ecological role of microzooplankton 693

rates of microzooplankton on microphytoplankton ranged between 0·007 and 0·091 day "1. This level of grazing corresponds to a daily loss of initial standing stock <1·50% (range 0·32–1·35%) or between 33·89 and 84·33% (x¯ =53·05%) of the potential production [Table 1(b)]. Pearson’s correlation analysis indicated that microzooplankton densities were significantly related to total chlorophyll concentrations (r2 =0·43; P<0·05). The grazing impact of microzooplankton was significantly correlated to the concentration of the <20 ìm chlorophyll fraction (r2 =0·53; P<0·05). Also, the relationship between the microzooplankton grazing coefficient and growth rate of the <20 ìm chlorophyll fraction was significant (r2 =0·47, P<0·05). To assess whether this relationship was due to auto-correlation, a Box Jenkins test was applied to the data set. A significant correlation was again obtained between the grazing of microzooplankton and growth rate of phytoplankton (r2 =0·44; P<0·05) indicating that the relationship between grazing and growth was real. Discussion The dilution technique is widely employed to estimate microzooplankton herbivory, and provides a simultaneous estimation of algal growth and mortality with the minimal manipulation of the natural assemblage (Landry & Hassett, 1982; Gifford, 1988; Paranjape, 1990). A potential source of error with the technique may result from nutrient enrichment associated with cell breakage during the production of the particlefree water. This is of particular importance since primary production in the Kariega Estuary has been shown to be limited by nutrient availability (Grange & Allanson, 1995). In the absence of similar studies, it is not possible to determine whether artificially elevated growth had occurred during the incubations. One is, however, able to compare the present growth rates with primary production data reported in the literature by converting photosynthetic rates to doublings day "1 (ì) by using for the formula ì=log 2(PC+ÄPC/PC) where PC is the phytoplankton carbon and ÄPC is the daily photosynthetic rate (Paranjape, 1987). The chlorophyll doubling rate was estimated from the dilution technique data using the formula ì=k/ln2 where k is the instantaneous phytoplankton growth coefficient (Paranjape, 1987). Since the Kariega Estuary is a homogenous marine system (Grange & Allanson, 1995), one can employ the primary production values presented in Allanson and Read (1995) for comparative results. Using their data, the chlorophyll doubling rate was estimated at ~2·18 day "1, compared to the authors’ estimated

doubling rate of between 0·02 and 0·24 day "1. This suggests that the growth of phytoplankton was not enhanced through nutrient enrichment during the grazing studies. The lower doubling rates recorded during this study may reflect the variability in primary production rates or nutrient limitation during the incubations. Although some investigations have added nutrients to incubation bottles, Gifford (1988) reported that such additions can damage the delicate microzooplankton. During this investigation, the microzooplankton densities ranged between 900 and 2850 cells l "1 (Figure 3). The authors’ estimates of microzooplankton abundance are substantially lower that those recorded in the Sundays River Estuary (Jerling & Wooldridge, 1995). The higher densities in the Sundays River Estuary can probably be related to higher chlorophyll concentrations recorded there. It is evident from the present data that microzooplankton are an important component of the biological seston in the Kariega Estuary contributing between 13 and 43% (x¯ =24%) of the total biological seston 2–200 ìm. These estimates can, however, be regarded as conservative since these estimates were derived from the biovolume:carbon estimates of microzooplankton cells fixed with Lugol’s solution. Recent studies have shown that cell shrinkage in samples fixed with Lugol’s solution may be as high as 25% (Leakey et al., 1994). The high abundance and contribution of the microzooplankton to the total biological seston 2–200 ìm, suggests that they constitute an important component of the planktonic food web in the Kariega Estuary. During this investigation, the microzooplankton grazing impact was highest in the <20 ìm chlorophyll fraction [Table 1(a,b)]. This result is consistent with previous studies which have shown that the <20 ìm size fraction is the most suitable food for microzooplankton (Hansen et al., 1994; Peters, 1994; Froneman et al., 1996). In particular, ciliates, the dominant component of the microzooplankton during this investigation (Figure 3), show a strong preference for nanoplankton (Hansen et al., 1994). During this investigation, the grazing impact of microzooplankton was significantly correlated to the concentration of the nanophytoplankton (P<0·05). Although temperature has been implicated as a key factor controlling the grazing rates of protozoans (Choi & Peters, 1992; Peters, 1994), during the present study, the grazing impact of microzooplankton did not appear to be temperature dependant over the 5 )C temperature range considered. Thus, food particle size appears the most important factor controlling microzooplankton grazing impact. It is evident, however, that large

694 P. W. Froneman & C. D. McQuaid

diatoms were also readily accepted as food [Table 1(b)]. This can probably be related to the presence of dinoflagellates in the microzooplankton assemblages which have been shown to consume particles larger than themselves (Peters, 1994). Although the microzooplankton grazing impact on initial standing stock was highest in the nanophytoplankton fraction, the percentage of potential primary production removed by microzooplankton was higher for the microphytoplankton [Table 1(a,b)]. During this investigation, the phytoplankton growth coefficients were lowest for the microphytoplankton [Table 1(b)]. The Kariega Estuary is regarded as an oligotrophic system (Allanson & Grange, 1995). Ecophysiological studies have shown that growth rates of algae in oligotrophic environments are largely determined by size (Fogg, 1988). Small cells are less likely to be nutrient limited than larger cells because of the favourable surface area to volume ratio (Fogg, 1988). Since the grazing impact of microzooplankton on potential phytoplankton production reflects the relationship between grazing (g) and the growth of phytoplankton (k), reductions in growth rate of the algae will result in a higher grazing impact (Banse, 1991). Thus, the high grazing impact of microzooplankton on the microphytoplankton potential production reflects the lower growth rates of the algae in a nutrient limited environment. The present results, however, suggest that microzooplankton may also be able to control the growth of faster growing nanophytoplankton. The high grazing impact of microzooplankton on the nanophytoplankton can probably be related to the fact that microzooplankton preferentially graze on particles of <20 ìm (Hansen et al., 1994; Peters, 1994). It should be noted, however, that only the grazing impact of microzooplankton on phytoplankton cells containing chlorophyll a was estimated during this study. The use of this pigment as an index of microzooplankton grazing excludes other potential food sources such as non-photosynthetic bacteria and detritus. This is of particular importance in the Kariega Estuary as recent studies suggest that rooted macroalgal production in the Kariega Estuary may account for a significant proportion of the total production (Allanson & Read, 1987, 1995). Since macroalgae are not directly consumed, much of this production enters the food web as detritus. The role of microzooplankton as detritivores requires further investigation. Microzooplankton played an important role in controlling the growth of phytoplankton during this investigation [Table 1(a,b)]. These grazing experiments were, however, conducted in the absence of potential

predators of microzooplankton. Predation by larger zooplankton and larval fish on microzooplankton is well documented (Pierce & Turner, 1992). In particular, copepods (e.g. Acartia spp.) have been shown to consume microzooplankton readily (Gifford, 1991; Pierce & Turner, 1992). According to Stoecker and Capuzzo (1990), microzooplankton appear to be the dominant food source for larger zooplankton in estuarine environments. Zooplankton biomass in the Kariega Estuary is dominated by two co-existing copepod species, Acartia longipatella and Pseudodiaptomus hessei, which comprise >95% of total zooplankton biomass (Grange, 1992). Predation by copepods on microzooplankton may, therefore, as a consequence substantially reduce the local grazing impact of microzooplankton on phytoplankton in the Kariega Estuary. There are few quantitative estimates of microzooplankton abundance, and their ecological role in South African estuaries is poorly documented (Jerling & Wooldridge, 1995). Consequently, the authors are unable to compare these grazing results with studies conducted in South Africa. The present results, however, compare well with similar studies conducted in coastal and estuarine habitats in the northern hemisphere (Gifford, 1988; McManus & EderingtonCantrell, 1992). A recent study conducted in the Sundays River Estuary along the south coast of South Africa showed that nanoflagellates dominate total chlorophyll and that microzooplankton densities may be as high as 6000 cells l "1 (Jerling & Wooldridge, 1995). On the basis of the main research findings during this investigation, microzooplankton appear to represent a key component of the planktonic food webs in this system and possibly coastal environments as a whole. Acknowledgements The authors would like to thank Rhodes University for providing funds and facilities for this investigation. Thanks also go to Val Meaton for identifying and enumerating the microzooplankton cell counts. References Albright L. J., Sherr E. G., Sherr B. F. & Fallon R. D. 1987 Grazing of ciliated protozoa on free and particle attached bacteria. Marine Ecology Progress Series 38, 125–129. Allanson, B. R. & Read G. H. L. 1987 The response of estuaries along the south eastern coast of southern Africa to marked variation in freshwater. Institute for Freshwater Research, Rhodes University, Special report 2/87. 40 pp. Allanson, B. R. & Read G. H. L. 1995 Further comment on the response of eastern Cape province estuaries to variable freshwater inflows. South African Journal of Aquatic Science 21, 56–70.

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