1. Introduction
Irrespective of the feeding mode adopted in adult life, most early life history stages of fish are selective predatory planktivores. This involves sensory detection of individual
Ž prey by the predator, prior to initiation of the predator strike Arnold and Holford, 1990;
. Browman and O’Brien, 1992; Miller et al., 1993 . Most first-feeding fish larvae are
Ž .
dependent upon vision for prey detection Blaxter, 1986 , although non-visual senses Ž
have also been implicated in prey detection by selective planktivorous fish larvae Batty .
and Hoyt, 1995; Salgado and Hoyt, 1996 . This is in contrast to the non-selective particulate mode of feeding displayed by juveniles and adults of some fish species, in
which gill rakers are used to filter prey from the environment without prior discrimina- Ž
. tion of individual prey Janssen, 1980; Gibson and Ezzi, 1985; Batty et al., 1986 .
Selective planktivory and non-selective particulate feeding are both active feeding behaviours and are not to be confused with passive prey engulfment, which may occur at
low rates, especially in marine species that must drink seawater for osmotic control Ž
. Tytler and Blaxter, 1988 .
The success of prey capture by planktivorous fish larvae depends upon larval age, size, and motor and physiological competence, all of which increase during ontogeny
Ž .
Blaxter, 1986 . Detection of a prey organism does not always result in a predator strike, implying a prey selection process is involved. Studies that compare the prey spectra
available in the environment with prey ingested by fish larvae, confirm that prey Ž
characteristics besides size strongly affect patterns of prey selectivity Checkley, 1982; .
Ž .
Magnhagen, 1985; Govoni et al., 1986; Meng and Orsi, 1991 . Jenkins 1987 suggested that some fish larvae display innate prey preferences at an early age, whereas others
Ž .
suggest that learning plays an important role in prey selection Werner et al., 1981 ; such that fish positively select for and are more effective at capturing prey that are
Ž .
familiar Checkley, 1982; Meyer, 1986; Coughlin, 1991; Wahl et al., 1995 . In this study, feeding performance of larvae of the greenback flounder Rhombosolea
tapirina, was assessed in two light regimes, irradiance of 5–6 mmol s
y1
m
y2
and total darkness, to confirm that greenback flounder larvae are primarily visual feeders. Feeding
response of two groups of larvae, one with prior exposure to rotifers only and one with Ž
prior exposure to both Artemia and rotifers, was then examined in the light 5–6 mmol
y1 y2
. s
m to determine the effect of prior prey exposure on subsequent prey selection.
2. Materials and methods
2.1. Source of larÕae Ž
. Two cohorts of greenback flounder larvae cohorts 1 and 2 , each from separate
groups of broodstock, were reared during August 1996. Wild female flounder that were Ž
. Ž
. sexually mature n s 15 were caught during June and August 1996 , from Waubs Bay,
Bicheno, Tasmania, by SCUBA divers using hand nets. Sexually mature male F1 broodstock were held at the School of Aquaculture, University of Tasmania. Upon
capture, wild-caught females were treated with an intraperitoneal injection of LHRHa at a dose of 100 mg kg
y1
body weight. Forty eight hours after injection, and daily thereafter, females were anaesthetised in a solution of 0.02 2-phenoxyethanol, and
checked for the presence of hydrated ovulated eggs. For each cohort of larvae, eggs from 2–3 ovulated females were stripped by the application of gentle pressure to the
abdominal area; and, in a similar fashion, milt was expressed from 2–3 naturally spermiated males and it was collected in a syringe. Eggs were fertilised by adding 1 ml
of milt per 100 ml of eggs, plus a small volume of seawater to activate the sperm. After 2–3 minutes, the eggs were transferred to a bucket of seawater from which fertilised
eggs were skimmed from the surface and stocked into 200 l larval rearing tanks at a density of 50 l
y1
. Eggs and larvae were incubated in a recirculating seawater system in which 25–50 of the tank volume was exchanged per day. Temperature was maintained
at 12 18C, photoperiod at 13 h light: 11 h dark, and light intensity at the water surface was 5–6 mmol s
y1
m
y2
during the photophase. Live feed organisms, large strain rotifers, Brachionus plicatilis, andror Artemia
Ž .
nauplii and metanauplii INVE — Artemia Systems, Belgium , were enriched with Nutripake and introduced into the tanks from day 4 post-hatching. Live feed regimes
varied according to experimental protocol and are described below.
2.2. Examination of feeding behaÕiour in light and dark Greenback flounder larvae from cohort 1 were reared in a single 200 l culture tank.
Ž
y1
. Ž
Rotifers were introduced twice daily 5 ml from the time of first feeding day 4
. Ž
y1
. post-hatching . Artemia nauplii were added once daily 1–2 ml
in addition to the rotifers from day 12 post-hatching. Feeding behaviour trials were conducted on days 12,
Ž .
15, 18, 21, 24 and 27 post-hatching in a stable temperature environment 128C 18C ,
y1 y2
Ž .
at two light intensity treatments: 0 mmol s m
absolute darkness and 5–6 mmol
y1 y2
Ž .
s m
the light intensity at which larvae fed actively in the culture tank . Light was Ž
. provided by broad-spectrum 400–700 nm Osram Daylight fluorescent tubes. The night
before each experiment, 30 larvae were transferred from the 200 l larval culture tank into each of eleven 2.5-l black chambers, where larvae were maintained in static
seawater culture. The chambers were then covered with blackout cloth and left undis- turbed overnight. There were 5 replicates per treatment and an additional chamber from
which twenty larvae were sampled the next morning and examined under a dissecting microscope to confirm gut evacuation prior to commencement of the feeding trial. Large
strain rotifers were then added sequentially at ten minute intervals to each of the remaining ten chambers at a density of 2 ml
y1
. The order of addition of rotifers was randomly allocated between the treatments. Rotifers were added to the dark treatment
chambers under cover of a blackout cloth so larvae were never exposed to light. Lights were turned on and covers removed from the light treatment chambers as rotifers were
added. The larvae were then left undisturbed for 1 h at which time 20 larvae from each chamber were pipetted onto a microscope slide, examined under a dissecting microscope
and the number of fish that had fed was recorded. Prey presence or absence criteria were used to assess feeding behaviour because individual rotifers could not be distinguished.
2.3. Determination of the effect of preÕious exposure to prey Fertilised greenback flounder eggs from cohort 2 were divided and stocked into two
200-l culture tanks, to form two treatments: treatment 1, in which larvae were subse- Ž
y1
. quently exposed only to rotifers 5 ml
; and treatment 2, in which larvae were Ž
y1
. Ž
y1
. subsequently exposed to both rotifers 5 ml
and Artemia 1–2 ml . Prey selection
Ž .
by larvae from treatment 1 prior exposure to rotifers only s R-treatment larvae and Ž
. treatment 2 prior exposure to rotifers and Artemia s A R-treatment larvae was then
examined in feeding trials in which larvae were offered a mixed diet of both rotifers and Artemia. In this fashion, Artemia nauplii were a novel prey for R-treatment larvae.
Feeding behaviour trials were conducted in 3-l chambers on days 11, 14, 17, 20, 23, 26 and 29 post-hatching at 12 18C and with light intensity at the test chamber water
surface of 5–6 mmol s
y1
m
y2
. There were five replicates per treatment, plus an additional chamber to confirm gut evacuation of larvae. The night before each experi-
ment 30 larvae from the appropriate 200-l culture tank were stocked into each of the 11 test chambers, which were covered and left overnight in the dark. The next morning, gut
evacuation was confirmed in larvae from a single chamber and a mixed diet of rotifers Ž
y1
. Ž
y1
. 1 ml
and newly hatched Artemia nauplii 1 ml were added sequentially into the
test chambers, in a random order, at ten minute intervals, at a total density of 2 ml
y1
. The larvae were left undisturbed to feed for 1 h before 20 larvae from each chamber
were examined for presence of rotifers andror Artemia in the gastro-intestinal tract. All feeding responses were recorded using a presence or absence criterion.
Rotifers and Artemia used in the feeding trials were first washed through a 200 mm screen and collected on a 100 mm screen to produce a discrete size fraction of prey.
Ž .
Mean lorica width and length of rotifers n s 50 and mean width and length of
Ž .
Artemia n s 50 , were measured to determine absolute dimensions of prey. Ten greenback flounder larvae from each of the R- and A R-treatments, were
randomly sampled from the culture tanks on each of the days tested. Larvae were anaesthetised in 0.02 2-phenoxyethanol and examined under a dissecting microscope
Ž fitted with an eyepiece graticule. Standard length SL — distance from the rostral tip of
. the larva to the posterior end of the notochord was measured to confirm that any
differences in feeding responses of larvae in the two treatments were not due to differential growth rates of larvae.
2.4. Statistical analysis Ž
. A two-way analysis of variance ANOVA and a Tukey–Kramer multiple compari-
son of means test were used to analyse the effect of increasing age on feeding response Ž
of larvae in the light and dark. Residual values replicate means subtracted from
. Ž
treatment means of arcsin6 transformed data were normally distributed Shapiro–Wilk .
test, Prob - W s 0.139 for data from the light treatments, but data from the dark Ž
. treatments were not normally distributed
Shapiro–Wilk test, Prob - W s 0.000 . Cochran’s test for homogeneity of variance was used to test that variances were equal.
On days 21 and 24 post-hatching, Artemia were accidentally introduced into the dark treatment chambers. While the difference in feeding response will be discussed, for the
purpose of analysis, only data for rotifer consumption was used.
In the prior experience feeding trial, larvae were offered two prey species, which Ž
resulted in three possible feeding responses selection of Artemia only, rotifers only or .
both Artemia and rotifers . These data were analysed using a multiple analysis of Ž
. variance MANOVA , in conjunction with a canonical distribution analysis, to test for
the treatment effect on the three possible larval feeding responses with increasing larval age.
3. Results
Kategori