Relative contribution of bacterial and photosynthetic production to

Journal of Plankton Research Vol.18 no.l2pp.2269-2281, 1996

Relative contribution of bacterial and photosynthetic productionto metazooplankton as carbon sources

Hiroshi Koshikawa,1 Shigeki Harada,2 Masataka Watanabe,2 Kazumi Sato1 andKoji Akehata1

1 Department of Industrial Chemistry, Science University of Tokyo, 1-3Kagurazaka, Shinjuku-ku, Tokyo 162, and 2Laboratory of Marine Environment,National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki305, Japan

Abstract. Percentages of label transfer (PLT) from bacteria and autotrophs to metazooplanktonduring 4 h were determined in parallel using dissolved organic and inorganic I3C tracers, respectively,in in situ batch incubations, to estimate the relative contribution of each production to the metazoo-plankton food requirement. The batch incubation for each pathway was done eight times, respectively,during 20 days in a mesocosm where continuous species succession of organisms was observed. ThePLT in the two pathways, dependent on metazooplankton abundance and species composition ratherthan changes in the activities of producers (bacteria or autotrophs), showed a similar tendency ofchange and were of a similar magnitude: mean 1.0% (0.09-2.7%) in the photosynthetic pathway and0.5% (0.22-1.5%) in the bacterial pathway. This finding suggests that metazooplankton are supportedby entire microbial food webs including both the bacterial and photosynthetic pathways.

Introduction

It is widely accepted that free-living heterotrophic bacterial production in aquaticecosystems has a large (Fuhrman and Azam, 1980, 1982; Cole et ai, 1988) andimportant role in the microbial loop (Azam et al, 1983). Thus, there has beenincreasing interest in the fate of bacterial production in food webs. Some studieshave reported that a small proportion of radiotracer originally taken up by bac-teria enters the zooplankton size fraction (e.g. Ducklow et al, 1986; Pernie et al,1990), indicating that the microbial loop is a respiratory energy sink (e.g. Sandersand Wickham, 1993) and cannot support the metazooplankton food requirement.On the other hand, Sherr and Sherr (1988) have suggested that bacterial pro-duction can be supplied to metazooplankton as a component of the microbial foodweb including both autotrophic and heterotrophic organisms. In microbialfood webs, although both bacterial and primary production can be consumed byprotozoa with a loss of respiratory energy, the protozoa that have ingested it cansupport some portion of the metazooplankton food requirement. In fact, recentstudies have found significant grazing of both nano-size bacterivores and herbi-vores by metazooplankton in various environments (e.g. Weisse et al, 1990;Gifford and Dagg, 1991; Lessard, 1991; Kleppel, 1991). Hence, only the apparentsmall carbon transfer from bacteria to metazooplankton is insufficient as evidencethat bacterial production is unimportant for metazooplankton.

One approach for clarifying whether or not bacterial production can contributeto metazooplankton as a food resource, would be to compare the carbon transferfrom bacteria and autotrophs to metazooplankton concurrently. Sherr etal (1987)also pointed out the need for this comparison by citing the data of Davies (1984),who reported a low carbon transfer from autotrophs to metazooplankton in the

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same system as that used by Ducklow. To our knowledge, however, few studieshave addressed this issue except for that by Wylie and Currie (1991). Furthermore,there is little information about the degree to which a change in plankton struc-ture influences carbon transfer to metazooplankton.

The purpose of this study was to determine (i) the magnitude of the differencein transfer of bacterial and photosynthetic production to metazooplankton(>100 u.m) in a eutrophic coastal environment, and (ii) how the carbon transfervaries with changes in plankton structure. Our approach was to employ[13C]glucose and [13C]bicarbonate tracers in parallel, instead of natural labile dis-solved organic carbon (DOC) and dissolved inorganic carbon (DIC), for tracingthe bacterial and photosynthetic carbon pathways. The experiment was carriedout within a large-scale mesocosm, which enabled us to observe rapid planktonspecies succession. We carried out short-term (4 h) in situ incubations frequently(every few days) using 4.5-1 bottles, to avoid adding the tracers to the whole meso-cosm. The use of frequent short-term incubations makes it possible to evaluate therelative influence of change in plankton structure on carbon transfer. The aim ofthe short-term incubation was to confirm the unidirectional transfer of the tracersinto metazooplankton: i.e. DOC —» bacteria —> bacterivores —> metazooplanktonin the bacterial carbon pathway and DIC -* autotrophs —> (protistan herbivores)—» metazooplankton in the photosynthetic carbon pathway.

Method

The mesocosm (5 m diameter and 18 m deep; -350 m3 volume) (Watanabe et al.,1995) was deployed in the Seto Inland Sea, Japan, from 24 July (Day 0) to 12August (Day 19) 1991. A vertical circulation system was installed, which provideda well-mixed surface layer (0-5 m depth), and prevented immobile species such asdiatoms from sinking rapidly (Watanabe et al, 1995). Nutrients (NO3-N, PO4-P,Si(OH)4-Si) were added to the mesocosm in order to promote a clear successionof plankton species (Harada et al., 19%). Seawater temperature, dissolved oxygen(DO) and pH (0,1,2.5 and 5 m depth) were measured almost every day at 9:00(Surveyer II, Hydrolab. Co.). Seawater samples were collected almost every daybetween 9:00 and 10:00 in 5-1 glass bottles through a silicone-tubing samplingsystem from 0, 2.5 and 5 m depth. Particles were collected on precombusted(450°C for 4 h) Whatman GF/F glass fiber filters and stored at -20°C until chloro-phyll (Chi a) analysis by HPLC (Kohata et ai, 1991) at a laboratory. The filtrateswere also stored at -20°C until determination of nutrients (Technicon AutoAna-lyzer). Phytoplankton samples were collected with the tubing system every otherday, and the species and abundance were determined by microscopy. Metazoo-plankton were collected from 0,2.5 and 5 m depth with a plankton net (100 jtmmesh size, 10-1 filtration) and fixed with 6-10% formalin for microscopic determi-nation of species and abundance. The above experimental procedures have beendescribed in more detail by Watanabe et al (1995).

Seawater for the tracer experiment was collected from 0.5 m inside themesocosm at 9:00 using a 10-1 Van Dorn sampler and transferred to an acid-cleaned 4.5-1 transparent polycarbonate bottle. As tracers for the bacterial and

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photosynthetic carbon pathway experiments, [13Q]glucose (ISOTEC Inc. USA,22.5 mg per bottle) and [13C]NaHCO3 (ISOTEC Inc. USA, 91.5 mg per bottle)were used, respectively, since the use of radio isotope in the field is strictly pro-hibited in Japan. After addition of the organic or inorganic 13C tracer, the bottlewas suspended for 4 h, until 13:00, at the depth from which the seawater had beencollected (0.5 m). The in s/Tu-incubated sample was fractionated into different sizefractions by sequential filtration using plankton nets (200,100,20 jim mesh size),precombusted glass fiber filters (Whatman GF/C and GF/F) and precombustedinorganic membrane filters (0.2 ^m Whatman Anodisc). The filtration order was200 p-m —> 100 jim —> 20 .̂m —> GF/C —> 0.2 jxm for the bacterial pathway, and200 (xm -» 100 jtm -» 20 p,m —> GF/F for the photosynthetic pathway experiment.Particles collected on each plankton net were washed with filtered seawater(GF/F) to remove particles smaller than each mesh size. After washing, the par-ticles were resuspended in filtered seawater (GF/F) and collected again on theprecombusted GF/C filter. The original seawater was also filtered with a WhatmanGF/F filter at the starting point of each incubation to determine the naturalcarbon isotope ratio of POC. The first tracer experiments for the bacterial andphotosynthetic pathways were done on 25 July (Day 1) and 26 July (Day 2),respectively. Subsequently, each experiment was carried out almost every otherday and each was conducted eight times in total. All filters were stored at -20°Cuntil analysis. The POC and 13C abundance (Atom%) were determined using asystem comprising an Elemental Analyzer (Carlo Erba NA1500) and an IsotopeRatio Mass Spectrometer (VG Isotech Prism Series II) (Thompson and Calvert,1994).

For the bacterial carbon pathway, it was assumed that the glucose would be takenup extensively by the bacterioplankton (Azam and Hodson, 1977). Because of thelow sensitivity of 13C analysis, a relatively high glucose concentration (~2 mg C I"1)in comparison with the natural DOC concentration inside the mesocosm(mean: 1.4 mg C I"1, Harada et al, in preparation) was added. Addition of glucose atthis concentration enabled us to neglect any tracer dilution effect by natural labileDOC. A proportion of the labeled glucose utilized by bacteria would be mineral-ized and might be consumed by autotrophs. However, transfer of the label from bac-terial production to autotrophs would be negligible in our incubations, since,assuming growth efficiency to be 50%, the amount of mineralized 13C would beequivalent to that of 13C fixed by bacteria, which was of the order of a few micro-grams 13C per hour per liter (see below for calculation). Hence, the regeneratedlabeled DIC might be diluted by a large amount of natural DIC (mean:21 mg C H).

In order to evaluate the influence of added glucose on bacterial production,another incubation was done supplemented by mesocosm seawater (0.5 m depth).This showed no obvious difference in the apparent increase of bacterial biovol-ume (jim3 ml"1) determined by image-analyzed fluorescence microscopy (Vilesand Sieracki, 1992) during the short incubation (6 h), with or without glucose(2 mg C I"1) (Koshikawa et al, in preparation). It seems that there were originallyenough carbon sources to support the potential production of bacteria in theeutrophic mesocosm during a short period, and that the added glucose might betaken up as an alternative.

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For 13C analysis it is necessary to use combustible filters such as those made ofglass fiber, in which no carbon is contained. However, a significant proportion ofthe bacterioplankton often passes through a normal glass fiber filter (GF/F) (e.g.Nagata, 1986), and therefore we used Whatman Anodiscs (0.2 u,m) in the bacterialpathway experiment, which allowed higher retention of bacteria (Jones et al,1989) and were combustible (Altabet, 1990).

For the photosynthetic carbon pathway, the added [13C]bicarbonate corre-sponded to -10% of total DIC, which would not be expected to increase photo-synthetic activity (Hama era/., 1983). A proportion of the added label which mightbe converted to dissolved organic substances through extracellular release byphytoplankton etc., would be used rapidly by bacteria (e.g. Cole et al, 1982).However, the transfer of label from phytoplankton to the bacterial pathway wouldbe very small in comparison with that in the photosynthetic pathway for thefollowing reasons, (i) Only a small amount of extracellular release that can beused in the bacterial pathway, for example, -13% of photosynthetic total fixation(Baines and Pace, 1991), can be followed in the photosynthetic pathway, (ii) Theadded 13C was -10% of total DIC, and so the extracellular release of labeledcarbon was <10% of the total available exudation at most, (iii) Originally existinglabile DOC, including that derived from non-extracellular release, would dilutethe labeled extracellular release, (iv) Primary production is generally severaltimes greater than bacterial production. Thus, it was considered that the transferof label to the bacterial pathway was insignificant, and that the added label wasused mostly in the photosynthetic pathway.

The GF/F filter was used to obtain the lowest size fraction in the photosyn-thetic pathway. A proportion of pico-sized autotrophs might pass through thefilter. However, it was determined separately that their photosynthetic activity(passing through the GF/F but retained on the 0.2 (xm Anodisc) was <5% of thetotal.

The amount of net tracer transformation from dissolved to particulate carbonfor each size fraction was calculated as the excess 13C (13Cex) against natural abun-dance as follows (equation 1).

13Cex (Mg »C I"1) = (as - aa) X POC (1)

where a, and aa are the 13C atom% in an incubated sample in a given size-fraction,and in a natural sample, respectively, and POC is the particulate organic carbon[mg C I*1] in a given size fraction. The sum total of all 13C excess in each pathwaywas used to estimate bacterial or photosynthetic activity. These activities wereassumed to indicate the difference in bacterial or photosynthetic productionactivities in the eight experiments

Carbon transfer from producers to metazooplankton was evaluated using theproportion of 13CeX in the sum of the 100-200 u-m and >200 n-m fractions (I3Cex

>i<»|im) m relation to the sum of all the 13C excess (13Cex M) (equation 2). Wedefined this value as the percentage of label transfer (PLT).

PLT(%) = (13Ccx>>1(XVm/13Cex,aU) X 100 (2)

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Free-living bacterioplankton would not exist in the >100 nm fractions. Any con-tamination with attached bacterial cells on the >100 u.m fractions was alsoassumed to be non-significant relative to the whole, because attached bacteriawould always be less numerous than free-living bacteria (e.g. Iriberri et al, 1987)and a large portion of attached bacteria would be found on small suspendedparticles (e.g. Almeida and Alcantara, 1992). On the other hand, some large-sizedphytoplankton might be retained on the >100 u,m fractions. It was confirmed sep-arately that the overestimation due to phytoplankton contamination was <10% tothe total label on the >100 u,m fractions in the photosynthetic pathway experiment(Harada et al, in preparation). Thus, the label on the >100 u.m fractions would bedefined as resulting from the feeding activity of metazooplankton on smaller par-ticles produced initially by bacteria or phytoplankton.

The PLT defined in the present study would only represent the proportion ofparticulate carbon produced by bacteria or phytoplankton during 4 h of incuba-tions that could be transferred and retained in metazooplankton by their feedingactivities during the same period. The addition of 13C label was done directlyinstead of pre-labeling the cells (phytoplankton or bacteria) prior to incubation,and so it was assumed that 13C uptake by phytoplankton and bacteria was a linearfunction of time and that 13C uptake by zooplankton was a parabolic function (orprobably n-dimensional [n = 2,3,4,...] function according to numbers of trophicsteps between producers and metazooplankton) of time during incubation forseveral hours (Roman and Rublee, 1981). Hence, the PLT is a time-dependentvalue, and different from the energetic transfer efficiency proposed by Lindeman(1942). However, a difference in the magnitude of the PLT would indicate a rela-tive difference in magnitude of the transfer efficiencies. Our aim in this study wasto compare the carbon transfer in the bacterial pathway with that in the photo-synthetic pathway under closely similar conditions to each other, and thus the PLTwas considered to be sufficient for this purpose.

Results

Environmental variables were almost homogeneous at 0-5 m depth (Harada et al.,1996) due to the circulation system installed in the mesocosm (Watanabe et al.,1995). The changes in the major variables are shown in Figure 1 as the average forthe 0-5 m layer.

In the well-mixed surface layer, the plankton structure showed obviouschanges with time. Phytoplankton species showed a shift from centric diatoms topennate diatoms and to dinoflagellates, and the Chi a concentration varied con-comitantly (Figure 2a). Photosynthetic activity (Figure 2b) defined by[13C]bicarbonate uptake (|xg 13C I"1 4 h"1) also changed concurrently. However,the bacterial activity (Figure 2c) defined by [13C]glucose uptake (u,g13C I"1 4 h"1)was steady in comparison with the large variation in photosynthetic activity.Metazooplankton showed continuous species succession (Figure 3). At thebeginning (Day 0), the zooplankton community comprised mainly Copepoda (65individuals I"1), but these were the dominant species for only a few days. AfterDay 6, Copepoda decreased rapidly and the total abundance of zooplankton

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26

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(a) Saawater temp.

mean: 25.3°C

(b)DO

mean: 7.8mgl_-1

i • • i i i i i i i . •_

(c)pH

mean: 8.1

(d) Nutrients cone.

•NO -N

• NH4-N

-PO4-P

mean: 4.9uM

mean: 1.4|jM

mean: 2.1uM

Si(OH)4-Si mean: 3.0uM

12 15 18 21

Days

Fig. L Environment variables (seawater temperature, DO, pH, nutrients) averaged by data sets within0-5 m depth. The initial concentrations of NO3-N, NH4-N, PO4-P and Si(OH)4-Si after nutrientaddition were 15.8,0.7,6.3 and 7.8 [u.M], respectively.

reached a minimum on Day 10. Subsequently, Doliolida (Dolioletta gegenbauri)emerged and increased gradually. The zooplankton community thus showed ashift from Copepoda to Doliolida dominance. Finally the abundance of Doliol-ida reached 24 individuals I"1 at Day 17 and accounted for a significant portionof the total zooplankton.

The PLT in both the photosynthetic and bacterial pathways (Figure 4) did notcorrespond to the variation in bacterial or photosynthetic production (Figure2b,c) but to that of metazooplankton abundance and species composition (Figure3). The PLT in the photosynthetic pathway declined from 1.3% on Day 2 to 0.09%on Day 10, but subsequently it increased and reached 2.7% on Day 17 (Figure 4a).In the bacterial pathway, PLT declined similarly from 0.43% on Day 1 to 0.22%on Day 11, and then increased gradually, finally reaching 1.5% on Day 18 (Figure4b). In both pathways, the gradual decrease in PLT (Days 0-4) was apparently dueto the decrease in Copepoda. The low PLT on Days 8-10 was apparently due tothe minimum abundance of total zooplankton. The marked increase thereafter

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Bacterial and photosynthetk production as carbon sources

(a) phytoplankton centric diatomspennate diatomsdinoflagellates

iers

(b)- - photosynthetk: activity

(c)- - bacterial activity

9

Days

I ' l l12 15 18

Fig. 2.(«) Changes in phytoplankton cell density for dominant categories within the surface layer(0-5 m depth), (b, c) Photosynthetic and bacterial activities defined as amounts of [13C]bicaibonate or[l3C]glucose uptake.

(Day 12-19) was apparently due to the emergence of Doliolida. The mean PLT inthe photosynthetic and bacterial pathways were -1.0% and 0.5%, respectively(Table I), the former being somewhat larger than the latter. However, consider-ing the overestimate of PLT in the photosynthetic pathway due to contaminatingautotrophs in the >100 \im fractions and lack of data for pico-autotrophs passingthrough the GF/F filters, the PLT in the two pathways seem to be almost of thesame magnitude. Furthermore, the PLT in the bacterial pathway was sometimeslarger than that in the photosynthetic one.

These results indicate that the PLT in the two pathways were dependent onmetazooplankton abundance and species composition rather than changes inthe activities of producers (bacteria or autotrophs), and were of a similar magni-tude. This suggests that both bacterial and primary production can be trans-ferred to metazooplankton through their feeding activities with a similar overallefficiency.

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100-

^ 80

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§ 60

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Copepoda phase Dolioiida phase

15 18

30

25 g.

2 0 C.15 |

10 |CO

0 "^

Fig. 3. Changes in total raetazooplankton abundance within the surface layer (0-5 m depth) and abun-dance of two dominant species.

Discussion

It seems to be generally accepted that a large proportion of bacterial productionis lost as respiration before transfer to metazooplankton (e.g. Sanders andWickham, 1993), since there are many trophic steps between bacteria andmetazooplankton (e.g. Wikner and HagstrOm, 1988). In our experiment, on theother hand, the PLT in the bacterial pathway was of a similar magnitude to that inthe photosynthetic pathway (Table I), and the tendencies of change of both thePLTs, which appeared to be affected by metazooplankton species succession(Copepoda to Doliolida), were similar (Table I). This indicates that bacterial pro-duction is not insignificant if primary production contributes to metazooplankton.Below, we discuss the effect of zooplankton species composition on carbon flowin an aquatic food system, in terms of metazooplankton feeding strategies and theobserved 13C tracer distribution in the prey fractions.

The feeding strategies of the observed metazooplankton in the bacterialpathway are as follows: Copepoda are inefficient at capturing pico-sized particlessuch as free-living bacteria, and so they would utilize bacterial carbon by preyingon protozoans which had ingested bacteria previously (e.g. Sherr et aL, 1986), oringesting detritus (e.g. Roman, 1984) to which bacteria would adhere. On the other

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g

3.0

2.5

2.0

1.5

1.0

0.5

0.0

1.5

1 0-

0.5-

0.0-

(a) —•— photosynthetic pathway( mean: 1.0 % )

(b) —o— bacterial pathway( mean: 0.5 % )

9

Days

12 15 18

Fig. 4. Changes in PLT in photosynthetic carbon and bacterial pathway.

hand, Doliolida have a mucus net which enables them to collect both bacteria-sizedsmall (<5 Jim) and large (>100 u,m) particles equally (Crocker et aL, 1991), andtherefore they would ingest free-living bacteria directly in addition. Thus, the trans-fer of carbon to metazooplankton may be strongly affected by temporal variationsin 13C abundance in bacteria (free-living and attached) and protozoa, which wouldbe reflected in the 13C distribution in the <100 |xm fractions (Figure 5).

In the bacterial pathway experiment, the GF/C-20 jim fractions had higherdistribution ratios than the 0.2 ^m-GF/C fractions from the beginning to Day 11,and afterwards this tendency was reversed until the end (Figure 5). Considering

Table L Average PLT for the three phases defined by dominant zooplankton species (Copepodaphase, transition phase from Copepoda to Doliolida and Doliolida phase)

Period

Day 0-7Day 8-11Day 12-19

Average

PLT [%]

Bacterialpathway

0.420.320.76

0.5

Photosyntheticpathway

1.00.511.4

1.0

Dominantzooplankton

Copepoda

Doliolida

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100-0.2Mm-GF/CGF/C-20pm20jjm-100pm

Flf. 5. Changes in tracer size distribution in the <100(j.m size fractions in bacterial flow.

that free-living bacteria might pass through the GF/C filter (e.g. -80% of totalbacterial abundance: Nagata, 1986), the higher distribution ratios of the GF/C-20 urn fractions during the former period were probably due to bacterivorous pro-tozoa which ingested bacteria, bacteria attached to small suspended detritus, andflocculated bacteria. Although we could not distinguish which factor (protozoa,attached or flocculated bacteria) had a large effect in the change of the 13C distri-bution in the <100 u.m fractions, the reversal of the ratios between the GF/C-20 n,m and 0.2 (x,m-GF/C fraction appeared to suggest the presence of ecologicalinteractions: i.e. a decrease of bacterivorous protozoa resulting in an increase offree-living bacteria, rather than a rapid change in the relative proportion of free-living and non-free-living bacterial activities. Thus, it was presumed that the tracerin the prey fractions was distributed more richly in bacterivorous protozoa duringthe former period and in bacteria during the latter. Accordingly, Copepoda (in theformer period) and Doliolida (in the latter) would each have ingested the 13C-richprey sufficiently, explaining our observation of carbon transfer in the bacterialpathway.

In the Doliolida phase, the mean PLT of the bacterial pathway was 0.76% (TableI), which was higher than that in the Copepoda phase (0.42%). This was due pri-marily to the higher feeding activity of Doliolida (e.g. Alldredge and Madin, 1982),and was a direct link between bacteria and Doliolida as described above.

In the Copepoda phase, the fact that 0.42% of the label originally taken up bybacteria was transferred to Copepoda within 4 h, despite the absence of a directlink between bacteria and Copepoda, suggests that protozoa could mediate thetransfer of bacterial carbon to Copepoda rapidly. For transfer of carbon frombacteria to Copepoda within such a short period, the following process can beenvisaged: protozoa ingest bacterioplankton (a dozen bacteria captured per hourper individual, e.g. Weisse and Scheffel-M6ser, 1991), and the ingested bacteriaare left inside the protozoan vacuole during their processing time (-20-60 min,

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e.g. McManus and Fuhrman, 1986; Sherr et ai, 1988). Then, the protozoa retain-ing the bacteria inside their cells are ingested by Copepoda. Furthermore, theCopepoda retain the protozoa in their gut during their gut passage time (-60-90min, e.g. Dagg and Walser, 1987). Our incubation period of 4 h might have beentoo short to allow accumulation into metazooplankton body tissue, but longenough to observe the transfer of the gut content, since the order of incubationtime was very close to that of gut passage time. Therefore, in our experiment, thegut and vacuole content might have accounted for a major proportion of theobserved carbon transfer to metazoa and also protozoa. Carbon transfer throughthis 'gut content transfer' process would occur more efficiently than that with atypical 'step by step transfer', leading to an increase of PLT in the bacterialpathway in our experiment.

On the other hand, herbivorous protists, if they were present at our site, wouldhave been one of factors decreasing the PLT in the photosynthetic pathway (her-bivorous protists often consume a large proportion of primary production; Burkillet ai, 1993; Dam et ai, 1993). Although a direct pathway of carbon transfer fromautotrophs to Copepoda must have been present, herbivorous protists mightingest autotrophs and mediate the transfer of carbon to Copepoda as well as bac-terivory (e.g. Sherr and Sherr, 1994). The role of both bacterivorous and herbivo-rous protists may explain the similarity of PLT in both the bacterial andphotosynthetic pathways.

Ducklow et ai (1986) found a smaller proportion of carbon transfer [e.g.0.03% (>100 n-m) after 24 h, calculated from data in their Table I] in the hetero-trophic pathway than we did in our study (0.5% on average for 4 h). They main-tained that bacterial production was not important for metazooplankton.However, the small carbon transfer they observed might have been due to fewermetazooplankton or lower feeding activity at their site (cold, 8-10°C, Scottishsea lochs). At the same time, the carbon transfer in the photosynthetic pathwaymight also have been low. A further difference between their results and ourswas their smaller distribution ratio of the protozoa size fraction [e.g. - 5 %(1-10 u,m) after 24 h, calculated from data in their Table I], indicating that thefeeding activity of Protozoa on bacteria was also small at their low-temperatureenvironmental site.

In conclusion, the PLT from producers to metazooplankton in the bacterial andphotosynthetic pathways was affected by metazooplankton abundance andspecies composition. One of the reasons was the emergence of Doliolida, whichwould ingest both bacteria and phytoplankton, and another was that intermediateheterotrophs could not sufficiently hinder bacterial carbon transfer to Copepodaand/or that protists could mediate the transfer of both bacterial and photosyn-thetic carbon to Copepoda equally. Consequently, the percentages of bacterialand photosynthetic carbon transfer to metazooplankton were of a similar magni-tude in our mesocosm. Our result indicates that microbial loop is not necessarilya sink, but possibly a link of magnitude similar to that of the photosynthetic foodchain, suggesting that metazooplankton are potentially supported by both bac-teria and autotrophs according to their production, namely, by entire microbialfood webs (Sherr and Sherr, 1988).

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Acknowledgement

We thank T.Kimura for technical support, and K.Kogure, T.Nagata, K.Kato,F. Azam and three anonymous reviewers for valuable comments on the manuscript.

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Received on February 9,1996; accepted on July 11,1996

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Relative contribution of bacterial and photosynthetic production to