Journal of Plankton Research Vol.18 no.6 pp.819-834. 1996
Development of Eodiaptomus japonicus Burckhardt (Copepoda,
Calanoida) reared on different sized fractions of natural plankton
Uszl6 G.T6thu and Kenji Kato1
'School of Allied Medical Sciences, Shinshu University, Matsumoto, 390, Japan
and 2Balaton Limnological Research Institute of the Hungarian Academy of
Sciences, H-8237 Tihany, Hungary
Abstract. The nutritional value of different sized fractions of natural plankton was investigated for the
growth of Eodiaptomus japonicus Burckhardt by comparing the development of its naupliar and copepodid stages fed on differentially fractionated planktonic assemblages of a eutrophic pond, at 20°C.
Water filtered through a 0.8 urn Nuclepore filter, containing mainly small coccoid bacteria (0.45-0.6 jim
in cell diameter), at a concentration of 82.7 p.g C I"1 could not support the development of E.japonicus.
The 3 p.m filtered water, containing bacteria and picoalgae. at a total concentration of 259 jig C I 1 ,
supported development but not egg production. The 20 jim filtered water, containing bacteria, picoalgae and large algae, at a total concentration of 2600 u.g C I 1 , supported rapid development of the
juveniles and continuous egg production by the adults. The separated 3-20 (j.m fraction, containing only
large algae, could not support the development at concentrations of 131 and 196 |xg C I 1 . However, the
same rapid development of the juveniles and continuous egg production by adults occurred at all of the
tested concentrations between 261 and 3920 M-g C I 1 of the large algae. The results suggest that E.japonicus favours algae larger than 3 \im during its complete lifespan, and that the threshold food concentration for its development varies between 200 and 250 |j.g C I '.
Introduction
Adult calanoids are usually characterized as macrofiltrators, preferentially selecting the 5-20 u-m size faction (65-800 u,m3 in biovolume), containing phytoplankton, heterotrophic nanoflagellates and protozoans (e.g. Gras etai, 1971; Meskova,
1975; Infante, 1981; Ferguson etai, 1982; Price etai, 1983; Paffenhofer, 1984; Horn
1985a,b; Hartmann, 1991; Gifford, 1993). Bacteria are considered to be food for
adult calanoids only if the cells are attached to larger particles or aggregated
(Pedr6s-AH6 and Brock, 1983a,b; Nagata and Okamoto, 1988).
Calanoids pass through six naupliar (Nl-NVI) and five copepodid (CI-CV)
developmental stages before they become adults. Since juveniles usually constitute 60-80% of the natural populations, they are supposed to play a significant role
in energy cycling in the plankton community (Fryer, 1987; Zinkai, 1991, 1994).
However, our knowledge of the feeding habits of these immature stages is very
limited and contradictory. Some authors demonstrated that NI-NII nauplii of
calanoids do not usually ingest external food (they use energy stored in the yolk)
and they start feeding in the NIII instar on phytoplankton larger than 4-5 u.m
(PaffenhOfer and Knowles, 1978; Infante, 1981; Sastry, 1983; Burns, 1985,1988). In
contrast to this, other experimental evidence has suggested that the early naupliar
development of some calanoids is limited by the external food supply, and nauplii
ingest bacteria- and picoalga-sized plastic beads (Dagg, 1977; Fernandez, 1979;
Hamburger and Boetius, 1987; Zdnkai, 1991, 1994; Santer, 1994). Thus, it is still
uncertain whether NI-NII nauplii do or do not require external food and whether
© Oxford University Press
819
UG.Tdth and K.Kato
feeding starts immediately on the larger algae. It is possible that juvenile stages of
some species ingest bacterial and picoalgal cells which thus function as a link in the
microbial resource chain.
Eodiaptomus japonicus is a common calanoid in lakes in Japan. The ontogenesis
and feeding of the species were studied by Kawabata (1989, 1993), Nagata and
Okamoto (1988), and Okamoto (1984a,b) in Lake Biwa. These authors observed
that naupliar development of E.japonicus became dependent on the food supply
from the NIII stage onwards. However, further details of the food requirements
remain unknown, because the quantity and composition of the food particles were
not determined in these former studies. We investigated the influence of the size
composition and concentration of the natural planktonic assemblage on the development of E.japonicus in a long-term, continuous experiment. The aim was particularly to focus on the nutritive value of the bacterio- and picoplankton.
Method
Collection and cultivation of E.japonicus
Eodiaptomus japonicus samples were collected from Lake Kizaki (Japan) in October of 1994 and kept in 1.51 beakers. The beakers contained eutrophic water of the
moat system of Matsumoto Castle (for details, see Kurasawa et ai, 1991), which
had been filtered through a 20 jun plankton net, and were replenished daily. The
breeding stock was illuminated with dim natural light and kept at 19.5-20.0°C.
To obtain newborn nauplii, after a 1 week acclimatization periodfiveegg-carrying females from the cultivation were repeatedly pipetted into particle-free (0.2
u.m membrane-filtered) moat water three times, then transferred into 150 ml
experimental beakers containing 100 ml of differentially fractionated moat water,
in triplicate. The beakers were kept at 19.5-20°C. After 14-16 h, females and nauplii older than NI were removed, and the experiment was started with 45-50 NI
instars in each beaker. The food suspension was renewed daily.
When changing the suspension, animals were concentrated to 2 ml volume using
20 n.m plankton mesh and carefully rinsed with particle-free (0.2 u.m membranefiJtered) moat water. They were then poured into a round-bottom evaporating
dish, diluted with 30 ml freshly fractionated experimental water and examined at
x80-x200 magnifications. The removed exuviae and corpses were subsequently
checked on a microscope slide, at x400-x600 magnifications. The checked animals
were poured back into the cleaned experimental beakers and filled up to 100 ml
with freshly prepared suspension.
Beakers were gently shaken several times daily in order to resuspend the food
particles. The incubation was continued until the animals became adults, ovigerous adults or died.
Fractionation and control of the food suspensions
First we prepared four different grades of water for use in the experiment by filtration of the moat water through a 20 p.m plankton mesh and Nuclepore filters
with pore diameters of 3.0,0.8 and 0.2 |xm (the latter was done in order to prepare
820
Development on different plankton size fractions
control water with the smallest possible number of particles). However, the size
fractionation resulted in confusion of the size of food particles with the amount of
food; the 20 u,m filtered water contained larger food with respect to particles and
also much larger amounts of food than the 3.0 and the 0.8 ^m filtered water.
Therefore, we conducted an additional experiment employing a series of food
concentrations of the 3-20 u,m plankton fraction (large algae), to test the
nutritional value of large algae if presented in amounts similar to the natural biomass of the bacterioplankton and picoalgae. For this, we separated the 3-20 u,m
phytoplankton fraction by gentle refiltration of the 20 u.m filtered water through a
3.0 u.m Nuclepore filter, then washed the particles on the filter surface three times
with 0.2 |xmfilteredwater and resuspended this in the same volume as the original.
The suspension was then diluted 5, 10, 15, 20 and 30 times with 0.2 u.m filtered
water.
At the beginning of the experiment, and whenever the incubation water was
exchanged, we checked the density and the size composition of the food plankton
in every fraction. To count the plankton, 1 ml of each fraction was preserved with
glutaraldehyde (2.6% final concentration), stained with DAPI (4,6-diamino-2phenylindole; Porter and Feig, 1980) and passed through a 0.2 p,m Nuclepore filter
pre-dyed with Sudan Black. The filters were examined with an Olympus BH-2
epifluorescence microscope equipped with an image analyser (Olympus-Avis,
XL-500) and a personal computer (NEC, PC9801RA), at x600 and xlOOO
magnifications.
We grouped bacteria into four different morphological categories, such as cocci,
dividing cocci (two cells), rod shaped and curved. Within the group 'cocci', we
distinguished three further subdivisions according to cell size. Altogether we
counted >600 bacterial cells on each filter. We estimated the size (diameter,
breadth and length) of 100-120 cells in each morphological group, on four filters.
Cell biovolume (BV) was estimated using the most appropriate geometric formula
for the differently shaped cells. BVs were converted to cell carbon (C) by assuming
0.2 or 0.35 pg C u.irr3 B V, according to the B V of the cells (Lee and Fuhrman, 1987;
Simon etal, 1992).
We distinguished small picoalgal cells from bacteria by their autofluorescence
under the light of a 100 W mercury vapour lamp passed through a BP 490 nm
excitation filter and a 515 nm barrierfilter.Larger algae were taxonomically determined. More than 500 algal cells (individuals) were counted on each filter. BVs
were estimated by the shapes and the major dimensions of the algal individuals
using the appropriate geometric formulae. For diatoms, 20% of the estimated volume of the silica box was considered as the effective biovolume (K.Kato, unpublished observation). Algal BVs were converted to carbon per cell (per individual)
using the formula C (pg) = 0A33BW*63 (C is in picograms and BV is in cubic
micrometers; Verity et al., 1992; Table I).
In order to evaluate the calculated organic carbon content, two times three parallel subsamples of the 20 |im filtered water fractions were filtered through precombusted GF/Ffilters(pore size 1 jim) and analysed with a Yanaco CHN corder
MT-5.
821
L.G.Tolh and K.Kato
Table I. Size (|xm). biovolume (BV. u,m'). carbon content (pgCind. ') and occurrence (+) of the food
particles in the differentially fractionated experimental waters
Food particle
Bacteria
Cocci 1
Cocci 2
Cocci 3
Diplococci
Rod
Curved
Fungi
Short
Long
Picoalgae
Cocci 1
Cocci 2
Cocci 3
Centric
Rod
Size
(jim)
BV
(p.m')
0.46
0.6
1.76
1 2
0.98
4
12
45
0.6
2
3
2.55
3.5
0.05
0.11
2.86
0.22
0.14
1.378
pg C
ind.'
0.017 +
0.02
0.57
0.044 +
0.028 +
0.274
4.62
20
0 924
4
0.11
4.1
14.1
8.7
0.44
0.064
1.463
4.248
2.801
0.213
Large algae
Cyanophyta detected and counted
Chroococcus sp. 1
6.1
Chroococcus limneticus 4.47
Microcystis aeniginosa 26
Anabacna spiroides
35
Anabacnopsis sp. (?)
40
119
56
189
336
200
291.3
65.2
41.9
Euglenophyta detected and counted
Euglena sp.
(pistiformis?)
25
788
134.8
26.7
20
Cyanophyta detected
Chroococcus sp. 2
Cloeoihece sp.
Aphanothece sp.
Aphanocapsa sp.
Pyrrophyta detected and counted
Rhodomonas sp. 1
Rhodomonas sp. 2
4
8
22.4
42
6J37
10.88
17
36.2
250
555
555
800
7.2
141
20
4.99
1.91
10.16
20.22
20.22
22.72
0.476
6.21
1.15
Chrysophyta detected and counted
Chrysidalis (?) sp.
Cyclotclla sp. 1
Cyclotella sp. 2
Stephanodiscus sp.
Melosira granulata
Cocconeis puella (?)
Achnanta sp.
Nitzschia sp. 1
Nitzschia sp. 2 (palea?)
822
4
5
12
16
52
23
10
35
8
0.2 jim 0.8 p.m 3 u.m
20 jim 3-20 urn
fraction fraction fraction fraction fraction
Development on different plankton size fractions
Table I. (com.)
Food particle
Size
(urn)
BV
((imJ)
pg C
ind."1
0.2 jim 0.8 \im 3 \im
20 jim 3-20 (tm
fraction fraction fraction fraction fraction
Chrysophyta detected
Fragillaria constntens
Navicula sp.
Nitzschia sigmoidea
Nitzschia sp. 3
Rhizosolenia sp.
Surirella sp.
Chlorophyta detected and counted
Ankistrodesmus falcalus 40
Dictyosphaenum
pulchellum
4(x4)
Oocystis sp. (lacustris?) 12
Planktonema lauterbornil\6
Golenkima radiata
4
5
6
6.5
7
7.5
8
Scenedesmus acumtnatus 16
Sccnedcsmus
quadncauda
15
Selenastrum gracile
8
Tetraedron minimum
4
Tetrastrum glabrum
6
120
26.97
56.2
135
12
33.5
65.4
113
144
176
221
268
34
14.01
29.85
3.7
9.08
16.3
25.8
31.3
38.6
45
53.6
9.08
192
18
10.6
34
40.45
5.246
3.32
9.078
Chlorophyta detected
Ankistrodesmus falcatus
var. mirabilis
Crucigaenia quadrala
Pediaslrum duplex
Schroedena setigera
Scenedesmus sp. 1
Scenedesmus sp. 2
Scenedesmus sp. 3
Telraedron irigonum
Tetraedron trigonum
var. gracile
Results
Quantity and composition of the food in the different water fractions
We detected small-sized cocci (cocci 1) and small rod-shaped bacteria at an average concentration of 10.8 p,g C I"1 in the 0.2 jim filtered water (Tables I and II).
Thus, it was not absolutely free from particles, but 'particle minimal'.
In the 0.8 (im filtered fraction, we found three different sized cocci, diplococci,
rod-shaped and curved bacteria at a total average concentration of 68.2 y.g C 1"'.
We also detected a small spherical picoalga (0.6-0.7 p.m cell diameter) at an
823
L.G.T61H and K.Kato
Table II. Q u a n t i t y of the p a n i c u l a t e organic carbon c o n t e n t (jj.g C I ' ) of the differentially fractionated
e x p e r i m e n t a l waters (the 3-20 (tm fraction was diluted further 5 . 1 0 . 1 5 . 20 and 30 times)
Bacteria
Picoalga
Large alga
Total
0.2 u.m
fraction
0.8 Jim
fraction
3 jim
fraction
fraction
3-20 p.m fraction
(basic, undiluted)
10.8 ± 7.2
0
0
10.8 ± 7.2
68.2 ± 25.6
14.5 ± 3.8
0
82.7 ± 29.4
114 ± 54.5
146 ± 4 3
0
259 ± 97.5
167 ± 73.2
236 ± 46.7
2197 ± 744
2600 ±864
17.4 ± 11
11.2 ± 7.7
3891 ± 1886
3920 ± 1905
average concentration of 14.5 ^g C 1 ' , and sometimes the presence of a filamentous water fungus (Tables I and II).
The 3 n-m filtered fraction contained 1.2 times more bacteria and 10 times more
picoalgae on average than the 0.8 ^.m fraction (Table II). We detected on average
259 (xg C 1 ' total particulate organic carbon in this fraction (Table II).
In the 20 jim filtered fraction, we detected 1.2 times more bacteria and 1.8 times
more picoalgae than in the 3 (im fraction, at a total concentration of 403 (ig C I"1. In
addition, we found a diverse large phytoplankton, represented by 45 taxa, at an
average concentration of 2197 u-g C I"1, dominated by Cyclotella, Euglena and
Golenkinia (Figures 1 and 2; Tables I and II).
Dilution series of the large algal fraction (the separated 3-20 ^.m fraction which,
however, also contained individuals larger than 20 (xm, e.g. elongated cells, filamentous algae, which passed the 20 ^m mesh) contained 131,196,261,392,784 and
3920 (xg C I"1. Bacterial and picoalgal contamination of this fraction was minimal
(< 30 n-g C I"1 in the undiluted stock suspension; Table II).
Figures 1-3 describe the main distribution of carbon in the different fractions,
according to the diameter (length) and the biovolume (BV) of the food particles.
-100 -
0. 3um fraction
3 urn fraction
20 urn fraction
350 -
C/Li
300 250 -
a>
Carbon
200 150 100 -
50 0 -
1
1
^_J
to
-
f
i
1
10
10
Diameter (length, tog, um)
Fig. 1. Distribution of the particulate organic carbon in three differentially fractionated experimental
waters according to the diameter (length) of the bacterial and algal cells (individuals).
824
Development on different plankton size fractions
10
100
1000
Biovolume (log,
Fig. 2. Distribution of the particulate organic carbon in three differentially fractionated experimental
waters according to the biovolume of the bacterial and algal cells (individuals).
The amount of food plankton fluctuated within ±20% of the mean during the
experiments. Carbon analyses showed that the measured carbon was 15 ± 12%
higher than that calculated, summarized for the 1-20 ^m fraction.
800
800
600-
600-
-400-
4O0-
200-
200-
I
CD
u
0.1
1
10
100
Diameter (length, log. urn)
1
10
100
1000
Biovolume (log. um3)
Fig. 3. Distribution of the particulate organic carbon in the dilution experiment for the 3-20 ujn phytoplankton fraction according to the diameter (length) and the biovolume of the algal and bacterial
cells (individuals).
825
I~G.T6th and K-Kato
Development of the nauplii
Almost all of the nauplii examined reached the NIII stage in this experiment. After
that, the animals in the fractions of 0.2 and 0.8 n.m (at concentrations of 10.8 and
82.7 jxg C 1"' respectively) and in the two lowest concentrations of the separated
large algae (3-20 jim fraction, concentrations of 131 and 196 ng C I"1, respectively)
suddenly died. All the nauplii died by the fifth day and only one of the initial 146
reached stage NV before death in the 0.2 |im fraction. In the 0.8 \LXX\ fraction,
3-18% of NIV nauplii transformed successfully to NV, but they also died before
transforming to NVI, judging from the corpses and the exuviae (Figures 4-6).
Nauplii did not develop further than the NIV stage in the two lowest densities of
the large algae and they also perished within 5 days of the incubation (Figures 7
and 8).
In contrast to these, 67% of the nauplii reached NV and 26% transformed to
NVI by the eighth day in the 3 (im fraction (at a concentration of 259 u.g C I 1 )After 3 days of apparent inactivity, 21 % of the nauplii successively moulted to CI
by the 12th day (Figures 4-6).
A total of 60-95% of the nauplii reached stage CI by the sixth and seventh days
of incubation in the 20 u.m fraction (2600 u,g C I"1) and at all of the concentrations of
the separated large algae between 261 and 3920 u,g C I"1 (Figures 4-8; Table III).
0.2 |im fraction
(10.8|igC/L;
n = 146)
60-
60-
40-
40-
20-
20-
*
i
0.8 nm fraction
(82.7 Mg C/L;
n= 142)
O-Lf
z
z z
z
z ° C O
12
40
3 |im fraction
(259»igC/L;n
150)
20 urn
20-
fraction
9- (2600 pg C/L
n - 148)
6-
10-
3-
30-
0
u
M-
0
Instar
Fig. 4. Percentage of the instars of E.japonicus reaching successive developmental stages before death
in four differentially fractionated experimental waters (NI-N VI: nauplius stages I-VI; CI-CV: copepodid stages I-V; A: adult; SOs of the three parallel incubations with 45-50 initial animals in each are
indicated).
826
Development on different plankton size fractions
0.8 pm fraction
(82.7 Mg C/L)
0
4
8
12 16 20 24 28 32
0
4
8
12 16 20 24 28 32
3|im fraction
(259 ,ig C/L)
20 tun fraction
(2600 ng C/L)
0
4
8
12 16 20 24 28 32
0
4
8
12 16 20 24 28 32
Time (days)
Fig. 5. Development of E japonicus in four differentially fractionated experimental waters (areas represent the populations, if more than one developmental stage had been detected in the same time. 1-6:
nauplius stages I—VI: 7-11: copepodid stages I-V; 12: adult; 13: propagating adult).
Development of the copepodids
In the 3 (xm water fraction, in which 21 % of the nauplii transformed to CI, 8.3% of
the population become adults by the 24th day (Figures 4-6; Table III). Adults in
this fraction were continuously incubated for a further 3 weeks, to check whether
the females produce eggs. However, pregnancy was not observed. In order to
stimulate egg production, on the 35th day of the experiment we transferred three
healthy males from the continuously propagating population (from the 20 n-m filtered water fraction) into the sterile one. Fertilization did not occur and the introduced males died within 4 days.
In the 20 jtm filtered fraction and in the large algal fractions at and above the
concentration of 261 jig C I"1, copepodids reached adulthood by the 12th-13th day
and females started producing eggs on the 13th-14th day of incubation. Although
we detected some mortality of CII-CIV instars, copepodid survival was high and
>55% of the initial nauplii became adults in these fractions (Figures 5-8; Table
III).
Discussion
In their study, Marshall and Orr (1955, 1956) presumed that filter feeding was
adopted by the nauplii of the marine Calanusfinmarchicus.Later, Gauld (1958,
827
L.G.T6th and K.Kato
Table III. Development of E.japonicus on different size fractions of natural plankton
Food
Bacteria
82.7
259
Bacteria, picoalga
and large alga
Large alga
Naupliar
Copepodid
12-13
11-12
No
10.8
Bacteria and picoalga
Development time
(days)
Development
Quantity of
the food
OigCl-')
No
Yes, but no egg production
2600
Yes and egg production
6
6
131
196
261
392
784
3921
No
No
Yes and
Yes and
Yes and
Yes and
egg
egg
egg
egg
6-7
7
6-7
6-7
7-8
5-7
6-7
6-7
12
16
production
production
production
production
100-
0
4
8
20
Time (days)
Fig. 6. Survival of E.japonicus during the development in four differentially fractionated experimental
waters (CI: copepodid stage I; A: adult; A+eggs: propagating adult; SDs of the three parallel incubations with 45-50 initial animals in each are indicated).
828
to
oo
1
3
A
5
9
9
7
7
Diluted x 30
(131ngC/L)
11 13 15
11 13 15
13-
543210
6-
1110987-
12-
1
7
9
1
1
11 13 15
11 13 15
Time (days)
5
Dflutedx 20
(196ngCVL)
3
3
5
5
7
7
9
9
11 13 15
11 13 15
Fig. 7. Development of E.japomcus in the dilution experiment for the diluted food concentrations of 3-20 u,m phytoplankton (stages of
development: 1-6: nauplius stages I—VI; 7-11: copepodid stages I-V: 12: adult; 13: propagating adult).
543210
6-
13121110987-
L.G.T<Jth and K.Kato
100-
80-
60-
20-
6
8
10
12
14
16
Time (days)
Fig. 8. Survival of E.japonicus in the dilution experiment for the diluted concentrations of the 3-20 (j.m
phytoplankton (CI: copepodid stage I; A: adult; A+eggs. propagating adult).
1966) claimed that the nauplii collect food actively. Fernandez (1979) showed that
the distance between setules on the mandibles of the nauplii of Calanus pacificus
was too great to retain bacterial-sized particles. Paffenhoffer (1971) and Paffenhofer and Knowles (1978) demonstrated that the marine species Temora stylifera,
T.turbidata, Eucalanus pilateus and Calanus helgolandicus start to feed by selecting algae of 4.5-25 u,m in diameter (0.06-5 x 104 u,m3 in BV) in stage NIII. It was
shown by Allan etal. (1977) that NIII and NIV nauplii of Eurytemora affinis,Acartia tonsa and A.clausi also select algae larger than 6.3-9.0 u^m (130-393 u.m3 in BV).
The earlier studies mentioned above did not suggest possible feeding on bacteria
and picoalgae by the nauplii of marine calanoids.
In the freshwater environment, Infante (1981) and Burns (1985,1988) showed
that Notodiaptomus venezolanus, Boeckella triarticulata and B.dilatata grow to
NIII using energy stored in the yolk, and start to feed on large algae in stages
NIII-NIV, like the marine calanoids. On the other hand, development of young
nauplii of Eudiaptomus graciloides proved to be food limited (Hamburger and
Boetius, 1987). Moreover, young nauplii of E.gracilis positively selected the 1.2
M-m particles from a mixed suspension of plastic beads in the experiments by
Zankai (1991,1994), indicating their capability to ingest picoplankton.
The threshold food concentration (the lower limit) for the development of nauplii and copepodid stages of different calanoids varies between 25 and 65 \x.g C1 ',
and between 25 and 120 \x.g C I 1 , respectively, if easily ingestible and digestible
algal food is offered (Mullin and Brooks, 1970; Paffenhofer and Harris, 1976; Fernandez, 1979; Vidal, 1980; Landry, 1983; Richman and Dodson, 1983; Muck and
830
Development on different plankton size Tractions
Lampert, 1984; Lampert and Muck, 1985; Schiemer, 1985; Hamburger and Boetius, 1987; Santer, 1994). In our experiment, nauplii of E.japonicus suddenly died
when they reached the NIII and NIV stages, if fed on the plankton fraction <0.8 |xm
containing mainly bacteria at an average concentration of 82.7 jxg C I"1, which was
higher than thresholds for nauplii cited in the literature. These results suggest that
E.japonicus start to feed in stage NIII-NIV, like the majority of calanoids, and that
they immediately favour larger food than natural bacteria.
It is very remarkable, however, that we did not observe development even if
trying to raise nauplii at concentrations of 131 and 196 jig C I*1 of large algae
(the separated 3-20 \im fraction), concentrations which were above the range in
the literature for the threshold of the development of copepods. This suggests that
the question is not only the mechanical ingestibility of the smallest bacterial cells,
but also that E.japonicus is characterized by such a high threshold food concentration, which does not meet with the amount and nutritional value of natural bacterioplankton even in eutrophic lakes.
Some nauplii in the 3 ^m filtered fraction (259 u.g C1') reached stage CI, and a
few copepodids became adults by the 24th day. The main difference between
this fraction and those containing smaller particles was the abundance (>10-fold)
of picoalgae. In all probability, nauplii and copepodids developed mostly on
this picoalgal fraction dominated by spherical cells of 0.6-2.3 ^m in diameter.
The high mortality of juveniles, the period of inactivity during stage NVI before
moulting to CI, and the sterility of the adults suggest that we had roughly determined the threshold bacterial and picoalgal food concentration for the development of E.japonicus by incubating animals in the 3 u.m filtered fraction of moat
water. The quick death of the subsequently introduced well-fed males, which had
previously been adapted to a surplus of food, offers further support for this
assumption.
Development in the 20 p.m filtered fraction containing 1300 u.g C 1"' of supplementary food comprising 45 taxa of larger algae and at all the tested concentrations of the separated large algae between 261 and 3920 n-g C I"1, was fast (12
days), the mortality was low, and females quickly started egg production. It is also
very remarkable that in comparison with the slow development (generation time
24 days) of the juveniles and the sterile adults on bacteria and picoalgal food (concentration of 259 p-g C I"1), the separated large algae at the same concentration (261
\y.g C I"1) supported rapid development (generation time 12-14 days) and continuous egg production (Table III). The parallel measurement of the carbon content of
the 1-20 jim fraction showed the amount to be —15% higher than the calculated
value. This probably originated from the non-living detritus which might also have
been used as food. However, as was mentioned, it amounted at most to 20% of the
total carbon in our experiment and the nutritional value of the detritus is generally
considered to be low, since detritus mostly consists of stabilized, undigestible carbon (e.g. Rodina, 1963; Oldh, 1972; Wetzel, 1983). Although the differential
acceptability and the nutritional value of the different large algae are also uncertain, there is no doubt that E.japonicus favoured this fraction of the phytoplankton
during its complete lifespan.
831
LG.Toth and K.Kato
Bacteria are known to be the main food source of small heterotrophic nanoflagellates and ciliates (Porter etai, 1979; Sherr etai, 1991;Simek and Straskrabovd,
1992). However, the populations of crustacean zooplankton consist of small j uvenile stages in nature, the trophic relationships of which have not yet been sufficiently
investigated. Our results with E.japonicus support the opinion that juvenile calanoids are not directly linked to the bacterioplankton.
Acknowledgements
This study was carried out during the tenure of a scholarship from the Japan
Society for Promotion of Science (JSPS No. 94088) to L.G.T. and was supported by
the Mambusho's Grant-in-Aid for JSPS Fellows in 1994 and 1995. Many thanks to
Prof. Y.Saijo, who kindly provided research facilities in Lake Kizaki. Organic carbon was kindly measured by T.Nakane, and S.Ajisawa helped in identification of
the nauplii of E.japonicus.
References
AllanJ.D., Richman.S., Heinle,D.R. and Huff.R. (1977) Grazing m juvenile stages of some estuarine
calanoid copepods. Mar. Bioi, 43,317-331.
Burns.C. W. (1985) The effects of starvation on naupliar development and survivorship of three species
of Boeckella (Copepoda: Calanoida). ,4/-c/i. Hydrobiol. Beth. Ergcbn. Limnoi, 21, 297-309.
Burns.C.W. (1988) Starvation resistance among copepod nauplii and adults. Verh. Int. Ver. Limnoi.. 23,
2087-2091.
Daggjvl. (1977) Some effects of patchy food environment on copepods. Limnoi. Oceanogr, 22,
334-340.
Ferguson,A.J.D., Thompson J.M. and Reynolds.C.A. (1982) Structure and dynamics of zooplankton
communities maintained in closed system, with special reference to the algal food supply./ Plankton
Res.. 4,523-543.
Fernandez.F. (1979) Nutritional studies in the nauplius larva of Calanus pacificus (Copepoda: Calanoida). Mar. Bioi, 53, 131-147.
Fryer.G. (1987) Quantitative and qualitative numbers and reality in the study of living organisms.
Freshwater BioL. 17,177-189.
Gauld.D.T. (1958) Swimming and feeding in crustacean larvae: the nauplius larva. Proc. Zool. Soc.
London, 132, 21-50.
Gauld.D.T. (1966) The swimming and feeding of planktonic copepods. In Barnes.H. (ed.). Some Contemporary Studies in Marine Science. Allen & Unwin. London, pp. 313—334.
Gifford,DJ. (1993) Consumption of protozoa by copepods feeding on natural microplankton assemblages. In Kemp,P.F., Sherr.E.B. and ColeJ J. (eds). Handbook of Methods in Aquatic Mtcrobtol
Ecology. Lewis Publishers, Boca Raton, FL, pp. 723-729.
Gras.R., Iltis^A. and Saint-Jean.L. (1971) Biologic des crustaces du Lac Tchad. II. Regime alimentaire
des Entomostraces planktoniques. Can, O.R.S.T.O.M. Ser. HydrobioL, 5.285-296.
Hamburger.K. and Boetius.F. (1987) Ontogeny of growth, respiration and feeding rate of the freshwater calanoid copepod Eudiaptomusgradloides. J. Plankton Res., 9,589-606.
Hartmann.HJ. (1991) In situ predation on planktonic ciliates by copepods, measured with diffusion
exclosures. Verh. Int. Ver. Limnoi, 24, 2821.
Horn.W. (1985a) Investigations into the food selectivity of the planktonic crustaceans Daphnia
hyalina, Eudiaptomus gracilis and Cyclops vicinus. Int. Rev. Ces. Hydrobiol, 70,603-612.
Horn,W. (1985b) Results regarding the food of the planktonic crustaceans: Daphnia hyalina, and
Eudiaptomus gracilis. Int. Rev. Ges. HydrobioL, 70. 703-709.
Infante J\.G. de (1981) Natural food of copepod larvae from Lake Valencia, Venezuela. Verh. Int. Ver.
Limnoi, 2L 709-714.
Kawabata.K. (1989) Natural development time of Eod'uiptomus japonicus (Copepoda. Calanoida) in
Lake Biwa./ Plankton Res., 11,131-136.
Kawabata.K. (1993) Mortality rate of Eodiaptomus japonicus (Copepoda: Calanoida) in Lake Biwa.
Jpn. J. Limnoi, 54, 131-136.
832
Development on different plankton size fractions
Kurasawa.H., Okino.T. and Kato.K. (1991) Ecological Studies on Five Moats around the Castle of Matsumoto and Counterplans for Restoration of their Landscape. Matsumoto City Board of Education,
Matsumoto, Japan, pp. 1-255.
Landry,M.R. (1983) The development of marine calanoid copepods with comment on the isochronal
rule. Limnol. Oceanogr., 28,614—624.
Lampert.W. and Muck.P. (1985) Multiple aspects of food limitation in zooplankton communities: the
Daphnia—Eudiaptomas example. Arch. Hydrobiol. Beih. Ergebn. Limnol. 21, 311-322.
Lee.S. and FuhrmanJ.A. (1987) Relationships between biovolume and biomass of naturally derived
marine bacterioplankton. Appl. Environ, Microbiol, 53,1298-1303.
Marshall.S.M, and Orrj\.P. (1955) The Biology of a Marine Copepod. Springer-Veriag, New York, p.
188.
Marshall.S.M. and Orr,A.P. (1956) On the biology ol Calanusfinmarchicus.IX. Feeding and digestion
in the young stages. /. Mar. Biol. Assoc UK, 35, 587-603.
Meskova,T.M. (1975) Zhakonomernosty razvitiya zooplanktona v Ozere Sevan (Rules of the development of the zooplankton of Lake Sevan). ltd. Armanskoy A.N.. Yerevan, pp. 1-265.
Muck.P. and Lampert.W. (1984) An experimental study on the importance of food conditions for the
relative abundance of calanoid copepods and cladocerans. 1. Comparative feeding studies with
Eudiaptomus gracilis and Daphnia longispina. Arch. Hydrobiol, 66 (Suppl.), 157-179.
Mullin,M.M. and Brooks.E.R. (1970) The effect of concentration of food on body size, cumulative
ingestion, and rate of growth of the marine copepod, Calanus helgolandicus. Limnol Oceanogr., IS.
748-755.
Nagata,T. and Okamoto.K. (1988) Filtering rates on natural bactena by Daphnia longispina and
Eodiaptomus japonicus in Lake Biwa. J. Plankton Res., 10,835-850.
Okamoto.K. (1984a) Size selective feeding of Daphnia longispina hyalina and Eodiaptomus japonicus
on a natural phytoplankton assemblage with the fractionizing method. Mem. Fac. Sci. Kyoto Univ.
(Ser. Biol), 9, 23-40.
Okamoto.K. (1984b) Diurnal changing patterns of the in situ size-selective feeding activities of Daphnia hyalina longispina and Eodiaptomus japonicus in a pelagic area of Lake Biwa. Mem. Fac Sci.
Kyoto Univ. (Ser. Biol), 9,107-239.
Olaii J. (1972) Leaching, colonization and stabilization during detritus formation. Mem. Inst. Hal IdroWo/., 29.105-127.
Paffenhofer,G.-A. (1971) Grazing and ingestion rates of nauplii, copepodites and adults of the marine
planktonic copepod Calanus helgolandicus. Mar. BioL, 11, 286-298.
Paffenh6fer,G.-A. (1984) Food ingestion by the marine planktonic copepod Paracalanus in relation to
abundance and size distribution of food. Mar. BioL, 80,323-333.
Paffenhofer,G.-A. and Harris.R.P. (1976) Feeding, growth and reproduction of the marine planktonic
copepod Pseudocalanus elongatus Boeck. J Mar. Biol. Assoc. UK, 56, 327-334.
Paffenhofer,G.-A. and Knowles.S.C. (1978) Feeding of marine planktonic copepods on mixed phytoplankton. Mar. Biol, 48,143-152.
Pedr6s-AH6,C. and Brock,T.D. (1983a) The impact of zooplankton feeding on the epilimnetic bacteria
of a eutrophic lake. Freshwater Biol, 13, 227-239.
Pedros-Ali6,C. and Brock.T.D. (1983b) The importance of attachment to particles for planktonic bacteria. Arch. Hydrobiol, 98, 354-379.
Porter.K.G. and Feig.Y.S. (1980) The use of DAPI for identifying and counting aquatic microflora.
Limnol. Oceanogr, 25,943-948.
Porter.K.G., Pace,M.L. and BatteyJ.F. (1979) Ciliate protozoans as links in freshwater planktonic food
chains. Nature, 277,563-565.
Price,HJ., Paffenhofer,G.-A. and StricklerJ.R. (1983) Models of cell capture in calanoid copepods.
Limnol. Oceanogr., 28,116-123.
Richman.S. and Dodson,S.I. (1983) The effect of food quantity on feeding and respiration by Daphnia
and Diaptomus. Limnol Oceanogr., 28,948-956.
Rodina,A.G. (1963) Microbiology of detritus of lakes. Limnol. Oceanogr., 8,388-393.
Santer3- (1994) Influences of food type and concentration on the development of Eudiaptomus gracilis and implications for interactions between calanoid and cyclopoid copepods. Arch. Hydrobiol,
131,141-159.
Sastry.A.N. (1983) Pelagic larval ecology and development. In Vernberg.FJ. and Vernberg,W.B.
(eds). The Biology of Crustacea. Behavior and Ecology. Academic Press, New York, Vol. 7, pp.
213-282.
Schiemer ,F. (1985) Bioenergetic niche differentiation of aquatic invertebrates. Verh. Int. Ver. Limnol,
22, 3014-3018.
833
L-G.Toth and K.Kato
Sherr.E.B., Sherr.B.F., Berman.T. and Hadas.O (1991) High abundance of picoplankton-ingesting
ciliates during late fall in Lake Kinneret. Israel. J Plankton Res.. 13, 789-799
Simek.K. and Straskrabova\V. (1992) Bacterioplankton production and protozoan bacterivory in a
mesotrophic reservoir./ Plankton Res., 14, 773-787.
Simon.M., Cho.C.B. and Azam.F. (1992) Significance of bacterial biomass in lakes and the ocean: comparison to phytoplankton biomass and biogeochemical implications. Mar. Ecol. Prog. Ser. 86.
103-110.
Verity.P.C, Robertson.Ch.Y., Tronzo.G.R., Andrews.M.C, NelsonJ.R. and Sieracki.M.E. (1992)
Relationships between cell volume and the carbon and nitrogen content of marine photosynthetic
nanoplankton. Limnol. Oceanogr.. 37,1434-1446.
,
Vidal J. (1980) Physioecology of zooplankton. I. Effects of phytoplankton concentration, temperature,
and body size on the growth rate of Calanus pacificus and Pseudocalanus sp. Mar. Biol., 56,195-201.
Wetzel.R.G. (1983) Limnology, 2nd edn. Saunders College Publishing, Philadelphia, New York, Montreal, Chicago, San Francisco, Toronto, London. Sydney, Tokyo, Mexico City, Rio de Janeiro,
Madrid, pp. 667-706.
Zinkai.N.P. (1991) Feeding of nauplius stages of Eudiaptomus gracilis on mixed plastic beads, y. Plankton Res., 13,437-453.
Zinkai.N.P. (1994) Feeding of copepodite and adult stages of Euduiptomus gracilis (G.O.Sars, 1863)
(Copepoda, Calanoida) on mixed plastic beads. Crustaceana, 66,90-109.
Received on August 17, 1995; accepted on January 4, 1996
834