Journal of Fish Biology (1998) 53, 1145–1154
Article No. jb980782
Drinking rate, uptake of bacteria and microalgae in
turbot larvae
K. I. R*‡, C. M. N*  O. V†
*Norwegian University of Science and Technology, Brattøra Research Centre,
Department of Botany, N-7034 Trondheim, Norway and †Norwegian University of
Science and Technology, Trondhjem Biological Station, N-7034 Trondheim, Norway
(Received 10 January 1998, Accepted 11 July 1998)
The drinking rate of turbot larvae increased from 14 to 120 nl larva 1 h 1 from day 2 to 11
after hatching, which gave a slightly increased specific drinking rate (calculated per biomass)
from day 2 to 7 (0·8–1·9 nl ìg carbon 1 h 1). The clearance rate of both algae and bacteria
was 10–100 times higher than the drinking rate, which indicated that the larvae had an active
uptake of both algae and bacteria. On day 2 and 4 after hatching highest clearance rate was
observed for Tetraselmis sp. On day 6 about the same clearance rate was observed for bacteria,
Isochrysis galbana and Tetraselmis sp. Until day 4 the turbot larvae had a higher ingestion rate
of Tetraselmis sp. than of I. galbana, whereas on day 6 the rates were similar (28–41 ng carbon
larvae 1 h 1). The assimilation efficiency was somewhat higher for I. galbana than for
Tetraselmis sp., and on day 6 the assimilated algae constituted 1·5 and 0·9% of the larval
biomass for I. galbana and Tetraselmis sp., respectively. 1998 The Fisheries Society of the British Isles
Key words: Scophthalmus maximus L.; fish larvae; drinking; algae ingestion; bacterial
ingestion; clearance rate.
INTRODUCTION
Experience from aquaculture has shown that the presence of microalgae and the
composition of the bacterial flora in the water are essential for the growth,
survival and viability of marine fish larvae (Naas et al., 1992; Reitan et al., 1993;
Skjermo et al., 1997). In the early feeding period the larvae have the opportunity
to ingest both microalgae and bacteria, which are present together with the prey.
Both, the ingestion of microalgae by the larvae (Reitan et al., 1994a; Tytler et al.,
1997) and the microbial colonization of the young larvae (Bergh, 1995; Skjermo,
1996) are believed to be fundamental for the viability of the larvae. However, so
far little is known about the process determining the colonization of the intestine.
Different mechanisms for both microalgal and bacterial uptake in first feeding
marine fish larvae have been suggested, including passive drinking, and active
processes. Fish larvae drink water at an early developmental stage; cod Gadus
morhua L., larvae from day 1 after hatching (Mangor-Jensen & Adoff, 1987);
Atlantic halibut Hippoglossus hippoglossus L., larvae from day 2–3 (Tytler &
Blaxter, 1988; Reitan et al., 1994a); rainbow trout Oncorhynchus mykiss
(Walbaum), (Tytler et al., 1990) and killifish Fundulus bermudae from day 4
(Guggino, 1980).
‡Author to whom correspondence should be addressed. Tel.: 47 73 59 77 29; fax: 47 73 59 63 11; email:
[email protected]
1145
0022–1112/98/121145+10 $30.00/0
1998 The Fisheries Society of the British Isles
1146
. .    .
Marine larvae must drink in order to maintain their water balance, but
dissolved and particulate compounds may be taken up passively by the larvae
due to the drinking process. For species such as cod and Atlantic halibut,
observations have revealed selective feeding on microalgae in the very early
feeding period, suggesting size selection of the particles with filtering over the
gills (van der Meeren, 1991; Reitan et al., 1994a). The mechanisms of uptake of
bacteria are not known, but even newly hatched cod larvae have been shown to
take up bacteria (Olafsen, 1994).
The aim of this investigation has to quantify the drinking activity and
the clearance rate of two microalgal species and a bacterium isolated from
turbot Scophthalmus maximus L. In addition, the ingestion rate and the food
assimilation efficiency of algae in turbot larvae was investigated. The uptake
mechanism of bacteria and microalgae was evaluated by comparing the clearance
rate of algae and bacteria with the drinking rate.
MATERIAL AND METHODS
LARVAL CONDITIONS
One batch of turbot eggs was incubated in black 70 l flat bottomed tanks at an initial
stocking density of 20 larvae l 1. The water used had a salinity of 34‰. It was sand- and
successively membrane-filtered and microbially matured (Skjermo et al., 1997). The
water exchange in the larval tanks was initiated at day 6 after hatching. The tanks were
aerated gently throughout the period. The temperature was kept at 12 C at hatching,
gradually increased to 18 C at day 4 after hatching and kept constant thereafter.
A mixture of the microalgae Isochrysis galbana, clone T. Iso, and Tetraselmis sp. was
added to the tanks from days 2 to 12, at a density of 1 mg C l 1. The larvae were fed
with n-3 HUFA-enriched rotifers as described in Reitan et al. (1993). The specific growth
rate of the larvae was calculated as increase in carbon content of individual larvae. The
carbon content was analysed in single larvae by use of a Carlo Erba Elemental Analyzer
as described in Reitan et al. (1993).
DRINKING ACTIVITY
The drinking rate was measured in turbot larvae at days 2, 4, 7 and 11 after hatching
by incubation of larvae in sea water to which 3H-labelled dextran (250 ìCi, TIA 382,
Amersham) had been added. The short-term exposure to 3H-labelled dextran was
performed by transferring larvae to a 5-cm diameter Petri dish. Thereafter most of the
sea water was removed carefully and replaced by 15 ml sea water to which 3H-labelled
dextran had been added (activity of about 5 ìCi ml 1). The larvae were kept at the same
temperature as in the rearing tanks, and samples of the larvae (three replicates) were
taken after 0, 20 and 40 min of incubation. After incubation, the larvae were washed
three times with micro filtered sea water, anaesthetized (1% methomidate) and transferred
individually to scintillation vials. Excess water was removed by use of a Pasteur pipette
before tissue solubilizer (0·5 ml Soluene-350, Packard) was added. The vials were
incubated for 16 h at 60 C and thereafter scintillation cocktail (4 ml Hionic-fluor,
Packard) was added. Three samples of 100 ìl sea water were taken from the Petri dish
immediately after the first sampling of larvae at time 0 to determine the total activity
(cpm ìl 1, equation 1). In addition, rinsing water, tissue solubilizer and scintillation
liquid were analysed for 3H-activity. The samples were counted twice for 10 min.
The drinking rate (RD) was calculated from the accumulation of 3H-dextran in
individual larvae [cpm larva 1, equation (1)], divided by the total activity of the
incubation media [cpm ìl 1, equation (1)], using linear regression of data from 0-, 20and 40-min incubations. The accumulation of labelled dextran in fish larvae by drinking
       
1147
has been shown to follow a hyperbolic model, and the drinking rate was calculated from
the first period when the increase was linear (cod, Mangor-Jensen & Adoff, 1987).
RD =[(cpm larva 1)/(cpm ìl 1)] exposure time 1
(1)
INGESTION AND CLEARANCE RATE OF ALGAE
The ingestion and assimilation rates of I. galbana and Tetraselmis sp. in turbot larvae
were measured on days 2, 4 and 6 after hatching by use of 14C-labelled algae.
The algal cells were labelled in 50 ml F/2 algae medium (Guillard & Ryther, 1962) to
which 14C labelled inorganic carbon (NaH14CO3, 20 ìCi) was added. Starting concentrations of the algal cultures were c. 105 cells ml 1, and the cells were harvested in the
stationary phase. The algal suspension, in which the larvae were incubated, was prepared
from labelled algae which were washed twice by centrifugation and diluted in filtered sea
water to give a concentration of 5 mg C l 1 (Nielsen & Olsen, 1989). Samples for specific
activity (14C/12C) of the algal suspension (5 mg C l 1) were taken in triplicate every
sampling day, and the specific activities were 12·1–15·3 cpm ng C 1 for I. galbana and
6·2–10·6 cpm ng C 1 for Tetraselmis sp. [equation (2)].
The algal ingestion rate was determined by transferring carefully 40 larvae from the
rearing tanks into 100-ml beakers containing 14C-labelled algae (5 mg C l 1). After
20 min incubation [exposure time, equation (2)], 20 larvae were washed, anaesthetized
(1% methodmidate) and transferred to scintillation vials (two larvae per vial). Tissue
solubilizer (0·25 ml Soluene-350, Packard) was added to the vials, incubated at 60 C for
16 h, and then 4 ml scintillation liquid (Hionic-fluor, Packard) were added. The samples
were counted twice for 10 min [cpm larva 1, equation (2)]. Zero controls of larvae and
scintillation liquids were sampled regularly and counted as described above. The algal
ingestion rate (RI) was calculated as:
RI =[(cpm larva 1)/(cpm ng C 1)] exposure time 1
(2)
The remaining 20 larvae were rinsed carefully in sea water and transferred to a beaker
containing 200 ml unlabelled algal culture (5 mg C l 1). After 3 h incubation, which
allowed complete gut evacuation of radioactive algae, the larvae were washed again,
transferred to scintillation vials, and treated as described above.
The assimilation rate (RA) of algal carbon was calculated as:
RA =[(cpmACCUM larva 1)/(cpm ng C 1)] exposure time 1
(3)
where cpmACCUM larva 1 is the algal carbon accumulated in the larvae after gut
evacuation of ratioactive algae and cpm ng C 1 is the specific activity of the ingested
algae. The exposure time was 20 min. The respiratory loss of carbon was assumed to the
zero during the short-term incubation.
The clearance rate of algae (RCA) is defined as the volume of water removed for algae
per unit of time. The algal cells might have been cleared from the water by drinking,
filtering or other processes.
RCA =RI Cc 1
(4)
where Cc was the concentration (mg C l 1) of algae in the suspension.
CLEARANCE RATE OF BACTERIA
Clearance rates of bacteria cells in turbot larvae were investigated using a clone
isolated from the intestine of turbot (isolate no. 4:44, Westerdahl et al., 1991). This
isolate has shown an antagonistic effect to Vibrio anguillarum (Westerdahl et al., 1991)
and an ability to colonize the gut (Olsson et al., 1991).
The bacterial cells were grown on marine agar [Marin Broth, added 15 g Bacto agar
(Difco) l 1, at 15 C] and then grown in TSB (Difco) medium (tryptic soy broth with 50%
sea water and 1 g glucose l 1).
1148
. .    .
The cells were labelled by adding 14C-glucose (60 ìl Amersham D-[U-14C] glucose,
268 mCi mmol 1) to 10 ml TSB medium, inoculated, and incubated at 18 C for 24 h
14
C-glucose was removed by centrifuging for 3000 rpm for 30 min (Sigma 3K-2 centrifuge), and the bacterial pellet was resuspended in 80% sterile sea water. This treatment
was repeated twice. (At day 2 the culture was centrifuged only twice.) Any aggregates of
bacterial cells in the suspension were removed by filtering through a 3-ìm polycarbonate
filter by gravity. The activity of the bacterial suspension was 3·95–15·98 cpm ìl 1 [cpm
ìl 1, equation (5)].
The uptake of bacteria in the turbot larvae was investigated on days 2, 4, 7 and 11 after
hatching. At each time 30 larvae were transferred carefully to a 200-ml beaker containing
14
C-labelled bacteria. After 0, 20 and 40 min of exposure, 10 larvae were washed,
anaesthetized (1% methomidate) and transferred to individual scintillation vials. After
adding tissue solubilizer (0·2 ml Soluene-350, Packard, 45 C in 24 h) to the vials, 4 ml
scintillation cocktail (Hionic-fluor, Packard) were added, and the vials were counted for
10 min twice [cpm larva 1, equation (5)]. The specific activity of the bacterial cells was
analysed at every sampling time (triplicates).
The clearance rate (RCB) of bacteria was calculated as the volume of the culture cleared
by the larvae:
RCB =[(cpm larva 1)/(cpm ìl 1)] exposure time 1
(5)
The experimental data were tested for statistical significance using the Student’s t-test
and the level of statistical significance was given as P<0·05. The accuracy of measurements is given as standard error.
RESULTS
The growth rate calculated by increase in carbon content of individual larvae
during the experimental period corresponded with earlier results obtained at our
laboratory (Reitan et al., 1993, 1994b; Øie et al., 1997). The specific growth rate
over days 2–4 was 0·057 day 1, for days 4–7 was 0·383 day 1 and for days 7–11
was 0·275 day 1 (Fig. 1).
In 2-day-old turbot larvae, when the mouth had just opened, the ingestion of
Tetraselmis sp. was significantly higher (P<0·05) than that of I. galbana (Fig. 2).
These two algal species have different cell sizes; I. galbana at a mean of 4·0 ìm
diameter and Tetraselmis sp. at 8·0 ìm width and 11·0 ìm length. At day 4
(58 day) after hatching the ingestion rate of Tetraselmis sp. declined by 40% and
was relatively constant until day 6. The ingestion rate of I. galbana increased
during the investigated period, and at day 6 there was no significant difference
between the two algae.
The uptake of I. galbana corresponded to 0·07 and 2·0% per day of larval
carbon biomass on days 2 and 6, respectively. The daily ingestion rate of
Tetraselmis sp. decreased rom 5·6 to 1·4% of larval carbon from day 2 to day 6.
There was almost no assimilation of either species of algal cells on days 2 and 4
after hatching. On day 6 the assimilation efficiency of the ingested algal cells had
increased to 74 and 65%, for I. galbana and Tetraselmis sp., respectively. At this
age the assimilated algae constituted to 1·5 and 0·9% of the larval biomass per
day, for I. galbana and Tetraselmis sp., respectively.
The drinking rate increased during the larval period from 14 to 120 nl larva 1
1
h
(day 2–11, Fig. 3). The clearance rate of bacteria was >10 times the
drinking rate. The high uptake of bacteria on day 2 may have been due to
non-quantitative removal of excess dissolved 14C (cf. Material and Methods).
       
1149
180
140
–1
Biomass (µg carbon larva )
160
120
100
80
60
40
20
0
20
40
2
60
4
80
100
120
140
Day° after hatching
6
8
Day after hatching
160
10
180
200
12
F. 1. The average carbon content of individual turbot larvae used in this experiment, from day 2 to day
11 after hatching. Day is calculated as days times the actual temperature of those days. Mean
values, bars indicate standard errors (n=10).
On days 4 and 6 in the clearance rates of bacteria and algae were of the same
magnitude, 10–100 times higher than the drinking rates.
During the early feeding period there was no correlation between clearance
rates and particle sizes.
DISCUSSION
The ingestion of I. galbana was significantly lower than for Tetraselmis sp. on
days 2 and 4 after hatching, but there was no significant difference (P>0·05) on
day 6 (29–41 ng carbon larvae 1 h 1, corresponding to 1·4–2·0% of the larval
biomass per day). Studies with tilapia Oreochromis spp., have shown that
filter-feeding on algae cannot support growth for these species (Dempster et al.,
1995). The assimilation efficiency of the ingested Tetraselmis sp. increased from
2·6% on day 2 to 65% on day 6 after hatching. Low assimilation of ingested
Tetraselmis sp. has been reported for yolk-sac larvae of Atlantic halibut (1–5% of
the ingested carbon, Reitan et al., 1994a). Tytler et al. (1997) reported lysis of
ingested I. galbana by 2-day-old turbot larvae, and from day 3 after hatching
vacuoles containing chlorophyll were observed in the enterocytes of all larvae
examined. However, earlier observations on turbot larvae have suggested only
limited digestion of ingested I. galbana (Howell, 1979).
In spite of the low assimilation efficiency of the ingested algal cells, the algae
might be nutritionally important for the development of the larvae. Critical
. .    .
1150
50
ng carbon larva–1 h–1
40
30
20
10
0
30
2
40
50
60
Day° after hatching
70
3
4
Day after hatching
5
80
90
6
F. 2. Ingestion (RI) and assimilation (RA) rate of the microalgae Isochrysis galbana and Tetraselmis sp.
in turbot larvae in the period day 2–6 after hatching. Mean values, bars indicate standard errors
(n=20). ––, RI Isochrysis galbana; ––, RA Isochrysis galbana; ––, RI Tetraselmis sp.;
––, RA Tetraselmis sp.
micro nutrients for the larvae might have been sequestered from the ingested
algae. At the onset of feeding most marine fish larvae have an immature
digestive tract with a poor digestive capacity (Hjelmeland et al., 1988). In
seabass Dicentrarchus labrax L. larvae microalgal ingestion has been shown to
enhance the activity of several digestive enzymes compared with absence of algae
(Cahu et al., 1998).
The drinking rate per larva increased during development from days 2 to 11.
Also a small increase in specific drinking rate (normalized to body mass; carbon
content) was observed from days 2 to 7, being 0·82–1·9 nl ìg 1 carbon h 1,
whereas it decreased to 0·8 nl ìg 1 carbon h 1 on day 11 (Figs 1 and 3).
Assuming that the carbon content is 8–9% of the wet weight of the larvae
(unpubl. res.), the drinking rate was 0·007–0·02% of the larval wet weight h 1.
This is lower than the drinking rate per wet weight for yolk-sac larvae of cod
(Mangor-Jensen & Adoff, 1987) and Atlantic halibut (Tytler & Blaxter, 1988;
Reitan et al., 1994a).
In this experiment the larvae showed high clearance rate of both bacterial and
algal cells (I. galbana and Tetraselmis sp.), being >10 times the drinking rate.
The clearance rate of bacteria was 35–160 times the drinking rate, and <3% of
the bacterial uptake could be accounted for by drinking. Active uptake of
suspended bacteria has been shown in tilapia Oreochromis niloticus L. (Beveridge
et al., 1989), in carp Cyprinus carpio L. (Beveridge et al., 1991), and in newly
       
1151
nl larva–1 h–1
10 000
1000
100
10
20
40
2
3
60
4
80
120
140
100
Day° after hatching
5
6
7
8
Day after hatching
160
9
10
180
200
11
12
F. 3. Drinking rate (RD) of sea water and clearance rate of bacteria (RCB), the microalgae (RCA) I.
galbana and Tetraselmis sp. in turbot larvae day 2–11 after hatching. Mean values, bars indicate
standard error (n=20). ––, RCB; ––, RCA Isochrysis galbana; ––, RCA Tetraselmis sp.;
· · · ·, RD.
hatched cod larvae (Olafsen, 1994). The uptake of suspended small bacteria
might have occurred via aggregates of bacteria to the mucous layer of the gills or
the skin as mucuous-bacteria aggregates (Drenner et al., 1987; Northcott &
Beveridge, 1988). Histological studies of the gills have shown that the mucous
cells produce negatively charged mucus (Northcott & Beveridge, 1988) which
may lead to clustering of small particles and enhancement of uptake.
In this experiment the gills of the turbot larvae developed from smooth gill
arches on day 2 to well-developed gills with primary and secondary gill lamellae
on day 11 (Natvik, 1996). As a result the total area of the gills with mucosa cells
increases during the development, which may explain the increased clearance
rate of bacteria from day 4 onwards. Reitan et al. (1994a) suggested that
yolk-sac larvae of Atlantic halibut filtered small algal cells from the water by the
gills. Also the high clearance rate of bacteria in this experiment may be
explained partly by filtering by the gills, especially if some bacteria were
clustered, although aggregates of bacteria were removed by filtering through a
3-ìm sieve before being given to the larvae.
The clearance rate of the alga Tetraselmis sp. was higher than that of I. galbana
on days 2 and 4 after hatching, which is in accordance with earlier findings
showing increased clearance rate with increasing algal cell size in both cod and
halibut larvae (van der Meeren, 1991; Reitan et al., 1994a). For zooplankton
filter feeders such as cladocera, a correlation between filter mesh size and the
ability to retain small particles is well documented (Gophen & Geller, 1984;
1152
. .    .
T I. Clearance rate of bacteria (RCB, Fig. 3), direct ingestion (RIB) calculated from
RCB and the average bacterial levels of the larval tanks in this experiment
(47302700 CFU ml 1, Natvik, 1996), ingestion of rotifers (RIR, data from Reitan
et al., 1994b), and ingestion of bacteria associated wth the rotifers (RI-Bacteria-Rotifers)
calculated from RIR and the average bacterial levels of commonly enriched rotifers used
in first feeding of turbot (1771 CFU rotifer1, Øie et al., 1997
RCB (nl larva 1 h 1)
RIB (CFU larva 1 day 1)
RIR (Rotifers larva 1 day 1)
RI-Bacteria-Rotifers (CFU larva 1 day 1)
Day 2
Day 4
Day 7
2193536
2487719
—
—
921254
1044288
192 31
33 984 19
11 5871293
13 1391466
364 27
64 428 37
Brendelberger, 1985). The size of particles collected by cladocera can be
predicted by the size of meshes of the thoracic feeding limbs. But it is possible
that mechanisms other than pure sieving may be involved, resulting in capture of
smaller particles than predicted by the sieve meshes (Brendelberger, 1991).
However, on day 6 after hatching no significant difference in clearance rate
between the two microalgal species was observed, and the rates were at the same
level as for bacteria. This may be explained by an increase in the development of
the gill structure of the turbot larvae (Natvik, 1996), or that other factors than
mechanical sieving become increasingly important.
The gut colonization of the larvae might be due to either direct ingestion of
bacteria (RIB, Table I) or through bacteria associated with the live feed
(RI-Bacteria-Rotifers, Table I) (Skjermo, 1996). The direct ingestion of bacteria by
the turbot larvae, calculated from the clearance rate (Fig. 3) and the average
bacterial levels of the larval tanks in this experiment [47302700 CFU (colonyforming units) ml 1; Natvik, 1996], was 1044–2487 CFU larvae 1 day 1 on
days 2–4 after hatching (Table I). At day 4, when the larvae had started to feed
on rotifers the bacterial contribution through the rotifers was significantly higher
(P<0·05) than the direct ingestion of bacteria. On day 7 the ingested bacteria
were obtained mainly through the ingestion of the rotifers. Our result clearly
demonstrated that after the larvae had started feeding on rotifers the main
bacterial source for the colonization of the larvae was the live food, which is
in accordance with earlier findings (Skjermo, 1996). However, the bacterial flora
of the water used in the larval tanks and the cultivation of rotifers probably
will affect the flora of the larvae (Vadstein et al., 1993). In addition, ingestion
of microalgae has been shown to affect the establishment of the early gut flora of
halibut larvae (Skjermo & Vadstein, 1993).
The experiments were performed at SINTEF Centre of Aquaculture. The authors
thank C. Olsson for providing the bacteria used in the investigation, J. Korstad and M.
Schei for skilful assistance during the experiments. The work was supported financially
by EEC project AIR2-CT93-1449.
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