Effect of turbidity, prey density and culture history on prey consumption by greenback flounder Rhombosolea tapirina larvae

Aquaculture 253 (2006) 447 – 460 www.elsevier.com/locate/aqua-online

Effect of turbidity, prey density and culture history on prey consumption by greenback flounder Rhombosolea tapirina larvae G.W. Shaw a,b,⁎, P.M. Pankhurst a,1 , S.C. Battaglene b a

b

School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute and Cooperative Research Centre for Sustainable Aquaculture of Finfish, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia Marine Research Laboratories, Tasmanian Aquaculture and Fisheries Institute and Cooperative Research Centre for Sustainable Aquaculture of Finfish, University of Tasmania, Nubeena Crescent, Taroona, Tasmania 7053, Australia Received 31 March 2005; received in revised form 14 September 2005; accepted 16 September 2005

Abstract Fish larvae cultured in turbid “green water” conditions commonly show improved feeding, growth and survival, however, the underlying mechanisms remain unclear. Greenback flounder Rhombosolea tapirina (Günther) larvae, reared in either green water (Tetraselmis suecica, 5 NTU) or clear water tanks were used in short duration feeding trials to investigate the effect of larval culture history, live prey density (0.01–5 prey ml− 1), and turbidity level (0–40 NTU) on feeding performance. Prey consumption was density-dependent at prey densities below 0.1 ml− 1 and 0.05 ml− 1 for feeding on rotifers and Artemia, respectively. Green water reared larvae fed in green water consumed more rotifers across the range of prey densities tested compared with clear water reared larvae fed in clear water. At low prey density, where absolute performance capabilities of the larvae are tested, green water provided immediate improvement to rotifer intake at turbidity levels of 5–20 NTU for larvae with experience of either a clear or green water environment. However, larvae with experience of a green water environment outperformed larvae with experience of a clear water environment. Thus mechanisms that operate over the short term, such as contrast enhancement and chemical stimulation of feeding, as well as mechanisms that operate over the longer term, such as possible differences in retinal development, improvements in handling times and experience, are likely responsible for improved early larval performance in green water. © 2005 Elsevier B.V. All rights reserved. Keywords: Greenwater; Turbidity; Prey density; Experience; Feeding; Consumption

1. Introduction

⁎ Corresponding author. School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute and Cooperative Research Centre for Sustainable Aquaculture of Finfish, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia. Tel.: +61 3 6227 7263; fax: +61 3 6227 8035. E-mail address: [email protected] (G.W. Shaw). 1 Current address: School of Marine Biology and Aquaculture and Cooperative Research Centre for Sustainable Aquaculture of Finfish, James Cook University, Townsville, Queensland 4811, Australia. 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.09.025

Improved growth and survival of many larval fishes grown in turbid environments induced by algal cell addition (green water) or suspended inorganic particles has been reported (Boehlert and Morgan, 1985; Naas et al., 1992; Reitan et al., 1993; Tamaru et al., 1994; Rieger and Summerfelt, 1997; Utne-Palm, 1999; Lazo et al., 2000; Cobcroft et al., 2001). However, not all species benefit from turbidity [e.g., Bluegill Lepomis macrochirus (Rafinesque) (Miner and Stein, 1993)] and others

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such as gobies Gobiusculus flavescens (Fabricius) (Utne, 1997) and Pacific herring, Clupea harengus pallasi (Boehlert and Morgan, 1985) show enhanced feeding at moderate turbidity but decreased feeding at higher turbidity levels. In addition, age-specific differences in feeding in green and clear water environments have been shown for striped trumpeter, Latris lineata (Forster), larvae (Cobcroft et al., 2001). In this case it was suggested that experience of feeding in one environment may have affected subsequent feeding in another. Differences in performance in relation to prior culture history may be associated with the retinal morphology of teleost fishes being relatively ‘plastic’ such that retinal cell type and density may vary in relation to the ambient light environment (Pankhurst, 1984; Raymond et al., 1988; Judge, 1990; Pankhurst, 1992), possibly predisposing larvae for visual prey detection in the familiar environment. However, whether larvae that are grown in a green water environment are better adapted to feed in such environments is unknown. Many mechanisms for enhanced feeding, growth and survival of larvae in turbid environments are given in the literature. First, larval rearing improvements have been attributed to direct nutritional input from algal cells in the water (Moffatt, 1981; Vasquez-Yeomans et al., 1990). Many fish species are known to ingest algal cells in higher concentrations than drinking rates would suggest (Meeren, 1991); these algal cells are thought to provide essential nutrients, macro-nutrients and enzymes (Munilla-Moran et al., 1990; Reitan et al., 1993; Lazo et al., 2000). However, many algal cells are poorly digested by larval fish (Reitan et al., 1993) with benefit likely to be derived from the stimulation of enzyme secretion (Hjelmeland et al., 1988; Cahu et al., 1998) which may enhance digestion at first-feeding. Furthermore, Nass et al. (1992) states that while algae must provide some nutrients it is not sufficient to explain the pronounced earlier onset of feeding in green water treatments by halibut larvae, Hippoglossus hippoglossus L. Algae also likely plays an indirect role in improving growth and survival by improving the maintenance of the nutritional quality and health of live prey within culture tanks (Reitan et al., 1993; Tamaru et al., 1994). Second, algal cell induced turbidity may affect larval gut and/or tank microflora (Bergh et al., 1994) and it has been proposed that algae afford probiotic functions, with Tetraselmis suecica shown to exhibit anti-microbial properties (Austin et al., 1992). Third, enhanced feeding has been attributed to improved vision and reaction distances in moderately turbid waters (Naas et al., 1992; Utne-Palm, 2001). Miner and Stein (1993) and Boehlert

and Morgan (1985) suggest that in the short visual field of fish larvae, turbidity may provide greater contrast between prey and background, thereby increasing the rate of prey detection and thus facilitating greater feeding proficiency. If this is the case then enhancement of prey detection in turbid conditions may provide greatest benefit under conditions of low prey density, or in young larvae, when visual function is most highly constrained (Kotrschal et al., 1990). The altered light environment in green water tanks has also been reported to affect larval distribution in tanks (Vandenbyllaardt et al., 1991; Naas et al., 1992; Rieger and Summerfelt, 1997), possibly through a reduction in light reflecting from tank walls (Bristow et al., 1996). It is also likely that larvae are able to actively choose an optimal light intensity for visual feeding due to increased light attenuation with depth in green water tanks (Mathews, 1984; Huse, 1994; Naas et al., 1996; Cobcroft et al., 2001). Finally, sibling cannibalism is a major problem in the culture of some species and green water can reduce the incidence of predation and thus significantly increase the survival and growth of cannibalistic species (Hecht and Pienaar, 1993). Greenback flounder, R. tapirina, is a candidate aquaculture species under investigation at the University of Tasmania, School of Aquaculture. Conditions for successful culture of this species have been investigated (Crawford, 1984; Hart and Purser, 1995; Hart et al., 1996; Hart and Purser, 1996; Shaw et al., 2003) as well as sensory development and feeding behaviour (Pankhurst and Butler, 1996; Cox and Pankhurst, 2000; Shaw et al., 2003). Preliminary evidence also suggests a benefit to feeding in a green-water environment. The current study tests four hypotheses: 1) Green water will enhance prey consumption of larvae, but benefits will diminish as larvae age; 2) At low prey densities green water will enhance prey intake; 3) Prey consumption will be increased at moderate turbidities in comparison to clear and higher turbidity water and; 4) Larvae reared in either a green or clear water environment will consume more prey items in the environment of which they are experienced. 2. Materials and methods 2.1. Larval rearing Broodstock greenback flounder (R. tapirina) were housed at the School of Aquaculture Aquatic Centre in two recirculation systems. Each system comprised a 4000 l culture tank and a 300 l sump containing a trickle biofilter. Broodstock flounder, maintained at 10.5 °C

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and a photoperiod of 10 : 14 LD (lights on at 0700 h), were either allowed to court and spawn naturally (cohorts 1 and 3), in which case eggs were collected on a submerged 280 μm sieve attached to the outlet drain above the biofilter, or eggs and sperm were stripped from ovulated and spermiated broodstock and fertilised artificially (cohort 2). Eggs were transferred to the larval rearing room, allowed to acclimate over 2–4 h to the water temperature of 12 ± 0.5 °C, and transferred to a 100 l egg incubation upweller. Eggs were incubated with vigorous aeration and eggs hatched 4 (cohort 3) or 5 (cohort 1 and 2) days post-fertilisation. Immediately after hatching larvae were harvested from the egg upweller, divided equally and stocked into two larval rearing recirculation systems, each comprising two 270 l blue gel-coated cylindro-conical larval rearing tanks and a 200 l sump containing a trickle biofilter. Larvae were stocked at an approximate density of 100 l− 1 (cohort 1 and 2) and at 72 l− 1 (cohort 3). One recirculation system was maintained with green water (Tetraselmis suecica) at a turbidity ranging from 3–5 Nephelometric Turbidity Units (NTU) throughout the larval rearing period and the other was clear water with turbidity b 0.05 NTU. Only one tank from each system was used for cohorts 1 and 2, whereas both tanks were used for cohort 3. Larval rearing systems were maintained with low aeration and water flow of 1 l min− 1, until 19 days post-hatching (dph) at which stage water flow was increased to 3 l min− 1. Air conditioning in the larval rearing room maintained tank water temperatures at 12.5 °C (cohort 1 and 3) and 12.0 °C (cohorts 2) at hatching, but was increased stepwise to 14 ± 0.5 °C by 7 dph (cohort 1), 14 dph (cohort 2) and, 28 dph (cohort 3) where it remained for the duration of experiments. Salinity on experiment days was constant between treatments but varied across days from 32 to 35, pH ranged from 7.98 to 8.14 and water changes were made using 1 μm filtered seawater to maintain total ammonia levels below 0.2 mg l − 1 . Larvae were maintained at a photoperiod of 16 : 8 L : D (lights on at 0830 h) and light intensity at the tank water surface ranged from 6.72–8.54 μmol s− 1 m− 2 for all cohorts. First feeding occurred 5 dph for all cohorts in both green and clear water tanks. Cohort 2 was fed the rotifers Brachionus plicatilis (70%) and Brachionus rotundiformis (30%) twice daily (0900 h and 1700 h) at a total density of 5 ml− 1 from 4 to 20 dph and a density of 2.5 ml− 1 from 21 to 29 dph. Artemia nauplii were fed from 12 to 23 dph (Artemia consumption did not occur until 16 dph) and enriched Artemia from 23 dph onwards at a density of 2 ml− 1. Similarly, cohorts 1

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and 3 received rotifers B. plicatilis (60%) and B. rotundiformis (40%) from 4 to 20 dph at 5 ml− 1 and from 21 to 23 dph at 2.5 ml− 1. Artemia nauplii were fed from 18 dph (cohort 3) and 19 dph (cohort 2) to 24 dph and enriched Artemia from 23 dph onwards at a density of 2 ml− 1. Live feeds were enriched with a mixture of T. suecica, Isochrysis galbana and Super Selco (INVE) for 8 to 12 h. 2.2. Feeding experiments–general methods All short duration feeding experiments were conducted in the larval culture room during the photophase, under a constant air temperature of 14 °C and light intensity at the surface of test aquaria ranging from 6.06 to 7.40 μmol s− 1 m− 2. This light intensity ensured that light levels at the bottom of each aquarium, despite light attenuation with depth, were within the optimal range for first feeding greenback flounder larvae (Taylor, 1998; Cobcroft et al., 2001). The night before each experiment, larvae were transferred either by wide-bore (3–8 mm) pipette (9 dph) or by 50 ml beakers (10–39 dph), from each 270 l culture tank into a corresponding 30 l ‘gut evacuation’ tank without food. Each gut evacuation tank was provided with aeration and larvae were left undisturbed overnight to digest prey already consumed. The following morning ten larvae from each gut evacuation tank were sampled as “gut evacuation controls”, placed on a frozen glass slide to induce hypothermic anesthesia, and the digestive tract of each larva examined under a compound microscope. Mastax/ trophi structures of rotifers were still evident in the digestive tract of larvae, thus, in subsequent feeding trials only rotifers that still contained both a lorica and mastax structure (recently ingested prey) were counted. A further sample of approximately 30 larvae was taken from each tank for length determination. Hemispherical plastic test aquaria (3-l), painted blue to match the culture tanks were then prepared; each containing 2-l of seawater of a specific turbidity. Turbidity for all experiments was provided by T. suecica and measured daily using a Hach 2100P portable turbidimeter. Turbidity measurements were made using water that did not contain rotifers or Artemia. Once gut evacuation assessment was completed, the remaining larvae were transferred to the prepared test aquaria and left undisturbed for 30 min in order to acclimate to the new environments. The number of larvae transferred to each aquarium differed according to larval age and the prey density used in each experiment. Prey consumption rates increased with larval age, thus, for turbidity experiments (3–5) the

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number of larvae added to each aquarium was reduced with increasing larval age to ensure that the final prey density at the end of each feeding period was at least 80% of the initial prey stocking density. For prey density experiments (Experiments 1 and 2) three larvae were added to each aquarium to ensure that some prey remained even at the lowest prey density tested. During acclimation of larvae, rotifers (B. plicatilis or B. rotundiformis) or Artemia nauplii that were enriched overnight with the alga T. suecica, were harvested, prey density measured in triplicate using a Sedgwick–Rafter counter and the required volume to be delivered to each test aquaria calculated. The order of addition of prey to each aquarium was randomly allocated with an interval between the addition of prey to one treatment and the next. This interval allowed for microscopic assessment of larval gut contents to occur immediately following the feeding period. Gut contents of larvae 9–20 dph were assessed by first pipetting larvae from each test aquarium onto a frozen glass slide, covering them with a cover slip to disclose the gut contents, then counting the number of prey consumed by each larva. Older larvae (25 to 39 dph) were euthanased in cold (4 °C) 10% neutral buffered formalin, left for 2 h, washed, and transferred into 70% ethanol. A preliminary trial confirmed that fixation did not induce gut evacuation in larvae of this age. Larvae were then measured (total length), their digestive tract removed under a dissecting microscope, and the number of Artemia in the digestive tract counted. 2.3. Experiment 1: the effect of rotifer density on the feeding performance of larvae from different culture environments Larvae 12 and 18 dph from cohort 3 were fed the rotifer B. plicatilis at one of 6 different densities, 0.025, 0.05, 0.1, 0.5, 1 and 5 ml− 1, for 60 min (Table 1). There were 5 replicate aquaria per treatment each containing 3 larvae. Larvae were fed in the same environment as their original culture tank; i.e., green-water (5 NTU) or clear water (0 NTU).

Table 1 Experiment parameters used in Experiments 1 and 2 Experiment Number of replicate aquaria

Number of larvae per aquarium

Age of larvae tested (dph)

Density Turbidity of prey (NTU) fed (ml− 1)

1. Effect of 5 rotifer density 2. Effect of 5 Artemia density

3

12, 18

0.025–5

3

25, 31, 39

Clear–0 Green–5

0.01–2.5 Clear–0 Green–5

were fed in the same environment as their original culture tank; i.e., green-water or clear water. 2.5. Experiment 3: the effect of prior culture history on feeding performance of larvae fed rotifers at 1 ml− 1 in a range of algal cell induced turbidities Experiment 3 examined the effect of culture history (green or clear water culture environment) on the feeding performance of 9, 16 and 20 dph larvae from cohort 1 in a range of algal cell induced turbidities (0, 10, 20 and 40 NTU). Larvae were fed B. plicatilis at a density of 1 ml− 1. There were three replicate aquaria per treatment, each containing 20 larvae. Prey ingestion and digestion rates increased with increasing age of larvae and so as to ensure the accurate counting of rotifers it was necessary to sequentially reduce the feeding period with increasing larval age (Table 2). 2.6. Experiment 4: the effect of prior culture history on feeding performance of larvae fed Artemia at 1 ml− 1 in a range of algal cell induced turbidities Experiment 4 examined the effect of culture history on the feeding performance of 31 and 38 dph larvae from cohort 1 in a range of turbidities from 0 to 40 NTU fed Artemia nauplii at a density of 1 ml− 1. The feeding period and the number of larvae per test aquarium were sequentially reduced with increasing age of larvae for the reasons described in Experiment 3 above (Table 2).

2.4. Experiment 2: the effect of Artemia density on the feeding performance of larvae from different culture environments

2.7. Experiment 5: the effect of prior culture history on feeding performance of larvae fed rotifers at a prey density of 0.1 ml− 1 in a range of algal cell induced turbidities

Larvae 25, 31 and 39 dph from cohort 3 were fed Artemia nauplii at 0.01, 0.025, 0.05, 0.1, 0.5, and 2.5 ml− 1 for 10 min (Table 1). There were 5 replicate aquaria per treatment each containing 3 larvae. Larvae

Experiment 5 examined the effect of culture history on the feeding performance of 10, 14 and 18 dph larvae from cohort 2 in a range of turbidities (0, 5, 10, 15 and 20 NTU) fed B. plicatilis or B.

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Table 2 Experiment parameters used in Experiments 3, 4 and 5 and the actual turbidity values (mean ± se; n = 3) of each treatment Experiment

Experiment Prey day (dph)

Prey No. of Feeding Actual turbidity of treatment NTU (S.E.) density larvae per duration 0 10 20 30 aquarium (min)

40

3. Effect of culture 9 history during rotifer 16 period (high density) 20

B. plicatilis B. plicatilis B. plicatilis

1 1 1

20 60 20 30 20 or 15 * 20

0.00 9.92 (0.16) 19.35 (0.30) 0.00 9.50 (0.07) 20.86 (0.33) 0.00 11.09 (0.30) 21.85 (0.15)

4. Effect of culture history during Artemia period

Artemia Artemia

1 1

15 10

0.00 9.77 (0.09) 22.02 (0.21) 38.81 (0.30) 0.00 11.62 (0.42) 30.51 (0.98)

31 38

10 3

0 5. Effect of culture 10 history during rotifer 14 period (low density) 18

B. rotundiformis 0.1 B. plicatilis 0.1 B. plicatilis 0.1

3 3 3

60 60 30

0.00 0.00 0.00

5

10

43.40 (0.41) 37.09 (0.19) 43.32 (0.25)

15

20

4.74 (0.04) 9.78 (0.09) 15.23 (0.20) 20.83 (0.41) 4.97 (0.26) 9.92 (0.05) 14.84 (0.22) 19.82 (0.72) 4.62 (0.30) 10.36 (0.02) 14.79 (0.42) 20.83 (0.93)

* Aquaria with larvae from green water had n = 20, those from clear water had n = 15, due to an overnight mortality event in the 20 l ‘gut evacuation’ tank.

rotundiformis (10 dph) at a lower density of 0.1 prey ml− 1. At a prey density of 0.1 ml− 1, significantly higher feeding in a treatment could significantly reduce prey density and therefore affect subsequent feeding within that treatment. Thus, it was decided that the number of larvae added to each aquarium would be reduced to three to ensure that the prey density remained above 0.08 ml− 1 (80% of initial stocking density) for the duration of the feeding period. Accordingly, the number of replicates in each combination of culture history and turbidity treatment was increased to five (Table 2).

(nested within prior culture history and turbidity) as a random factor and number of prey consumed as the dependent variable. The percentage of larvae feeding in each experiment was analysed using a two factor ANOVA with arcsin√ transformed ratio data. Where significant differences were found (P b 0.05) a Tukey post-hoc test was performed to separate treatments. Linear regressions of larval length with age for larvae reared in clear or green water were compared using ANCOVA. 3. Results 3.1. Larval growth

2.8. Data analysis All data were tested for homogeneity of variance using Levene's test of equality of error variances and use of residual plots; where necessary data was square root transformed. Some mortality occurred in test aquaria during experiments, thus, sample size between treatments was not always equal. Feed intake data from all surviving larvae, including non-feeding larvae, from each test aquarium was used in analyses. Prey intake data from Experiments 1 and 2 were analysed using a three factor ANOVA with prey density and culture environment as fixed factors and aquarium as a random factor (nested within prey density and culture environment). The hypothesis for Experiments 1 and 2 was that green water reared larvae would have an improved ability to feed at low prey densities, and the data were grouped into two blocks; the three low prey density treatments and the three high prey density treatments. Data from 3–5 were analysed using a three factor ANOVA with prior culture history and turbidity as fixed factors, test aquarium

Growth in length of larvae from cohort 1 was significantly greater in green water compared with clear water (ANCOVA, F = 6.234; df 1, 24; P = 0.020). However, there was no significant difference in the growth of larvae from green or clear water in cohort 2 (ANCOVA, F = 1.694; df 1, 20; P = 0.203) or cohort 3 (ANCOVA, F = 1.309; df 1, 8; P = 0.286) (Fig. 1). In cohort three, the single cohort in which larval survival was determined at the end of the experiment (32 dph), mean survival (n = 2 replicate culture tanks) was 38.1 ± 15.1% and 20.0 ± 1.9% in green water and clear water tanks, respectively. 3.2. Experiment 1: the effect of rotifer density on the feeding performance of larvae from different culture environments Green water reared larvae fed at low prey densities (0.025, 0.05 and 0.1 rotifers ml− 1) consumed significantly more rotifers than did clear water reared larvae

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A

10

3.3. Experiment 2: the effect of Artemia density on the feeding performance of larvae from different culture environments

8 6

Green water reared larvae 25 dph consumed significantly more Artemia at low prey densities (0.01, 0.025, and 0.05 ml− 1), compared to clear water reared larvae, and Artemia intake increased with increasing prey density (Fig. 3A). At higher prey densities (0.1, 0.5, 2 ml− 1), neither culture environment nor prey density affected Artemia intake (Fig. 3A, Table 3). On average 64.4 ± 13.2% of larvae were feeding in each treatment and this was not affected by prior culture history (2 factor ANOVA, F = 0.757; df 1, 48; P = 0.389) or density (2 factor ANOVA; F = 0.993; df 5, 48; P = 0.432). Older larvae 31 dph, at the start of metamorphosis, fed at low prey densities were not affected by culture environment but did show increased feeding with increased prey density. At higher prey densities

4 2 0

Total length (mm)

10

B

8 6 4 2 0 10

C

8 6

30

4

A

12 dph

25

0 0

5

10

15

20

25

30

35

40

Age (days post-hatching)

Fig. 1. Growth (mean ± S.E. total length, n = 10) of larval greenback flounder of A cohort 1; green water (●, r2 = 0.987) and clear water (○, r2 = 0.969) and B cohort 2; green water (●, r2 = 0.933) and clear water (○, r2= 0.916) and C cohort 3; green water (●, r2= 0.982) and clear water (○, r2 = 0.975). Growth in clear and green water was significantly different in cohort 1 only.

on both 12 and 18 dph. There was also a significant increase in rotifer intake with increasing prey density. At higher prey densities (0.5, 1 and 5 rotifers ml− 1) green water reared larvae 12 and 18 dph still consumed significantly more prey than did clear water reared larvae; however, prey density no longer significantly affected prey intake (Fig. 2 and Table 3). Feeding at the lowest prey density (0.025 ml− 1) was similar for both 12 dph and 18 dph larvae; however, 18 dph larvae consumed far more prey at higher rotifer densities (Fig. 2). Prey density and prior culture history had no effect on the percentage of larvae feeding 12 dph with 81.9 ± 3.3% of larvae feeding across all treatments. Density did affect the percentage of larvae feeding 18 dph with 58.3 ± 11.4% of larvae feeding at 0.025 rotifers ml− 1 whereas the percentage of larvae feeding in all other treatments was greater than 85% (2 factor ANOVA, F = 2.860; d.f. 5, 48; P b 0.001).

Mean number of B. plicatilis consumed (larvae-1 h-1)

2

20 High densities

15 Low densities

10 a

b

0 30

∗

b

∗

5

B

18 dph High densities

25 20

Low densities a

b

∗

c

15 10 5

∗

0 0.025 0.05 0.1

0.5

1

5

Rotifer density (ml-1)

Fig. 2. Mean number of B. plicatilis consumed (± se) by greenback flounder larvae (n = 15) of cohort 3 reared and fed in either a green water (●) or clear water (○) environment A) 12 dph and B) 18 dph. Larvae were fed at low (0.025, 0.05, 0.1 ml− 1) or high (0.5, 1 and 5 ml− 1) prey density. Subscripts within low or high prey densities indicate significant differences in feed intake. Large asterisks indicate there were significant differences between green and clear water reared larvae. Note log scale of density axis.

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Table 3 Results of ANOVAs of number of prey consumed by larvae from either a green or clear water environment fed prey at a range of densities Experiment

Factor

1. 12 dph

Environment Prey density Interaction Environment Prey density Interaction Environment Prey density Interaction Environment Prey density Interaction Environment Prey density Interaction

18 dph

2. 25 dph

31 dph

39 dph

Low densities

High densities

F value

d.f.

P value

F value

d.f.

P value

4.109 3.589 0.624 34.445 18.021 1.152 5.341 3.446 2.624 0.140 5.142 0.298 0.011 4.795 5.085

1, 67 2, 67 2, 67 1, 69 2, 69 2, 69 1, 84 2, 84 2, 84 1, 84 2, 84 2, 84 1, 83 1, 83 1, 83

0.047 * 0.033 * 0.539 0.000 * 0.000 * 0.322 0.023 * 0.048 * 0.078 0.709 0.008 * 0.743 0.916 0.011 * 0.008 *

26.184 2.455 0.011 4.895 0.494 0.703 0.076 1.830 0.262 0.965 1.717 6.290 0.094 8.477 0.652

1, 67 2, 67 2, 67 1, 81 2, 81 2, 81 1, 84 2, 84 2, 84 1, 84 2, 84 2, 84 1, 84 2, 84 2, 84

0.001 * 0.094 0.989 0.030 * 0.612 0.498 0.783 0.167 0.770 0.329 0.186 0.003 * 0.760 0.000 * 0.523

Prey density and culture environment were fixed factors and prey densities were grouped into the three low density and three high density treatments. * Significant effect.

there was a significant interaction between culture environment and prey density, resulting from a decrease in Artemia intake by green water reared larvae feeding at the highest prey density (Fig. 3B, Table 3). The percentage of fish feeding also significantly increased with prey density (two factor ANOVA, F = 4.143; df 5, 48; P = 0.003) with 23.3 ± 12.9% feeding at 0.01 Artemia ml− 1 and 70.0 ± 8.2% feeding at 2.5 Artemia ml− 1. Artemia intake by metamorphosed larvae 39 dph fed low prey densities was also affected by the interaction between culture environment and prey density. Green water reared larvae had a greater increase in Artemia intake with increasing prey density compared with clear water reared larvae. At higher prey densities larvae showed significant increases in Artemia intake with increasing Artemia density but culture environment had no effect on consumption (Fig. 3C, Table 3). Increasing prey density also significantly increased the percentage of larvae feeding, with 60.0 ± 10.0% feeding at 0.01 Artemia ml− 1 and 96.7 ± 3.3% feeding at 2.5 Artemia ml− 1 (two factor ANOVA, F = 6.346; df 5, 48; P b 0.000). Larvae 39 dph had greatly increased overall consumption compared with larvae 25 or 31 dph. 3.4. Experiment 3: the effect of prior culture history on feeding performance of larvae fed B. plicatilis at 1 ml− 1 in a range of algal cell induced turbidities Nine dph there was no effect of turbidity treatment, prior culture history or an interaction between turbidities and prior culture history on the consumption of B.

plicatilis (Fig. 4A, Table 4). The percentage of larvae feeding from clear water, 96.8 ± 1.5% (n = 12) was significantly higher (two factor ANOVA, F = 147.509; df 1,16; P b 0.001) than those from green water, 91.0 ± 2.4% (n = 12). Sixteen dph there was no significant interaction between prior culture history and turbidity on the consumption of B. plicatilis; however, both prior culture history and turbidity treatment had a significant effect on consumption (Fig. 4B, Table 4). Larvae coming from a green water environment consumed significantly more B. plicatilis (mean consumption of 14.30 ± 0.47 rotifers 0.5 h− 1 across all turbidity levels; n = 223) than did larvae from a clear water environment (mean consumption of 6.24 ± 0.46 rotifers 0.5 h− 1 across all turbidity levels, n = 218; Fig. 4B). Increased consumption of B. plicatilis occurred at moderate turbidities irrespective of prior culture environment (Fig. 4B). Mean consumption at 10 NTU of green water and clear water reared larvae combined was 3 times higher than at 0 NTU, 13.43 ± 0.91 rotifers 0.5 h− 1 (n = 105) and 4.12 ± 0.54 rotifers 0.5 h− 1 (n = 98), respectively. There was a significant interaction between prior culture history and turbidity treatment on the percentage of larvae feeding (two factor ANOVA, F = 3.923; df 3, 15; P = 0.030), with significantly less larvae from the clear water environment feeding in 0 NTU (38.5 ± 10.3%) and 10 NTU (76.3 ± 13.2%) treatments compared with any treatment containing larvae from green water (100% feeding). Neither prior culture history nor turbidity level had a significant effect on rotifer consumption 20 dph and

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12

A

25 dph

3.5. Experiment 4: the effect of prior culture history on feeding performance of larvae fed Artemia at 1 ml− 1 in a range of algal cell induced turbidities

High densities

10 8

Thirty-one dph larval consumption of Artemia was not significantly different between prior culture history or turbidity treatments (Fig. 5A, Table 4) with greater than 89% of larvae in each treatment feeding. In contrast, metamorphosed fish 38 dph from a clear water environment consumed significantly

Low densities

6

a

ab b

4

*

0 12

B

31 dph

25

A

9 dph

B

16 dph

10 High densities

20

8 a

a

(larva-1 h-1)

Low densities

6

b

4

15 10

2 5 0

C

39 dph x

High densities y y

20 Low densities

15

a

a

b

10 5 0 0.001

0.01

0.1

1

10

0 25 20 (larva-1 0.5 h-1)

25

Mean number of B. plicatilis consumed

Mean number of Artemia consumed (larvae-1 10min-1)

2

there was no interaction between prior culture history and turbidity (Fig. 4C, Table 4). Furthermore, turbidity level (two factor ANOVA, F = 0.914; df 3, 16; P = 0.456) and prior culture history (two factor ANOVA, F = 1.613; df 1, 16; P = 0.222) had no effect on the percentage of larvae feeding with greater than 95% of larvae feeding in each treatment.

∗

10 5 0 a

Artemia density (ml-1)

25

b

bc

c

20 dph

C

20 (larva-1 20 min-1)

Fig. 3. Mean number of Artemia consumed (±se) by greenback flounder larvae (n = 15) of cohort 3 reared and fed in either a green water (●) or clear water (○) environment A) 25 dph, B) 31 dph and C) 39 dph. Larvae were fed at low (0.01, 0.025, 0.05 ml− 1) or high (0.1, 0.5, and 2 ml− 1) prey density. There was a significant interaction between high prey densities and environment in B and between low prey densities and environment in C. Subscripts within low or high prey densities indicate significant differences in feed intake. The large asterisk indicates a significant difference between green and clear water reared larvae across the low densities. Note the change in scale on C and log scale of density axis.

15

15 10 5 0 0

10

20

30

40

Nominal Turbidity (NTU)

Fig. 4. Mean consumption of B. plicatilis by greenback flounder larvae 9, 16 and 20 dph of cohort 1, reared in either a green water (●) or clear water (○) environment and fed at a range of algalinduced turbidities. Larvae from each prior culture environment were fed B. plicatilis at a density of 1 ml− 1 for; A) 1 h, 9 dph, B) 0.5 h, 16 dph and C) 20 min, 20 dph. Subscripts indicate significant differences in feed intake between turbidity treatments. The large asterisk indicates significant difference between green and clear water reared larvae.

G.W. Shaw et al. / Aquaculture 253 (2006) 447–460

455

Table 4 Results of ANOVAs of feed intake for Experiments 3, 4 and 5; effect of culture environment and turbidity on feed intake of larvae fed prey at a density of 1 or 0.1 prey ml− 1 Experiment

Age (dph) 9

16

20

Factor

F

d.f.

P value

F

d.f.

P value

Environment Turbidity Interaction

1.807 2.964 3.866 31

1, 16 3, 16 3, 16

0.198 0.063 0.890

41.856 9.412 1.359 38

1, 15 3, 15 3, 15

0.000 * 0.001 * 0.293

Environment Turbidity Interaction

0.012 0.165 0.042 10

1, 16 3, 16 3, 16

0.915 0.918 0.988

59.460 1.688 0.185 14

1, 12 2, 12 2, 12

0.001 * 0.226 0.833

0.185 3.894 1.676

1, 20 4, 20 4, 20

50.843 8.165 1.369

1, 20 4, 20 4, 20

F

d.f.

P value

1, 16 3, 16 3, 16

0.911 0.097 0.301

1, 20 4, 20 4, 20

0.002 * 0.007 * 0.590

3.

4.

5. Environment Turbidity Interaction

0.673 0.017 * 0.195

0.013 2.493 1.323

18 0.001 * 0.001 * 0.280

13.362 4.775 0.717

Turbidity and culture environment were fixed factors with aquarium nested as a random factor. * Significant effect.

Feed intake by larvae 10 dph was significantly affected by turbidity; however, there was no effect of prior culture history and no interaction between prior culture history and turbidity on the consumption of B. plicatilis (Fig. 6A, Table 4). Mean consumption of B. plicatilis by larvae from both environments was significantly reduced at 0 NTU (2.43 ± 0.60 rotifers h− 1; n = 30) compared to feeding at 10 NTU (6.30 ± 0.67 rotifers h− 1; n = 30) and 15 NTU (5.34 ± 0.83 rotifers h− 1; n = 29). The percentage of larvae feeding was not affected by turbidity (two factor ANOVA, F = 1.931; df 4, 40; P = 0.124) or prior culture history (two factor ANOVA, F = 1.399; df 1,40; P = 0.244). Fourteen dph there was no interaction between turbidity and prior culture history on the consumption of B. plicatilis; however, the effects of turbidity and prior culture history were significant (Fig. 6B, Table 4). Mean consumption of all larvae from a clear water

14

A

31 dph

B

38 dph

(larva-1 10 min-1)

12

Mean number of Artemia consumed

3.6. Experiment 5: the effect of prior culture experience on feeding performance of larvae fed rotifers at 0.1 ml− 1 in a range of algal cell induced turbidities

environment was 5.27 ± 0.46 rotifers h− 1 (n = 74), whereas mean consumption of all larvae from green

10 8 6 4 2 0 14 12

(larva-1 3 min-1)

more Artemia compared to larvae from a green water environment but Artemia intake was not affected by turbidity level (Fig. 5B, Table 4). Mean consumption for larvae from clear water was 9.74 ± 0.56 Artemia 3 min− 1 (n = 90), significantly higher than the mean consumption for larvae from green water 4.72 ± 0.45 Artemia 3 min− 1 (n = 60). Greater than 80% of larvae in each treatment were feeding.

10 8

∗

6 4 2 0 0

10

20

30

40

Nominal Turbidity (NTU)

Fig. 5. Mean consumption of Artemia by greenback flounder 31 and 38 dph of cohort 1 reared in either a green water (●) or clear water (○) environment and fed at a range of algal-induced turbidities. Larvae from each prior culture environment were fed Artemia at a density of 1 ml− 1. Large asterisks indicates significant difference between green and clear water reared larvae.

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G.W. Shaw et al. / Aquaculture 253 (2006) 447–460 14

A

Similarly, 18 dph, when feeding duration was reduced to 30 min, both turbidity and prior culture history had a significant effect on the consumption of B. plicatilis, while there was no significant interaction (Fig. 6C, Table 4). Mean consumption of all larvae from a clear water environment was 7.72 ± 0.59 rotifers 0.5 h− 1 (n = 72), whereas mean consumption of all larvae from green water was 10.27 ± 0.60 rotifers 0.5 h− 1 (n = 74; Fig. 6C). The mean consumption of B. plicatilis was significantly lower at 0 NTU (6.17 ± 0.93 rotifers 0.5 h− 1; n = 29) compared to 5 NTU (10.55 ± 0.96 rotifers 0.5 h− 1; n = 29) and 10 NTU (10.17 ± 1.00 rotifers 0.5 h− 1; n = 29). Higher turbidity levels reduced consumption to levels not significantly different to consumption at 0 NTU (Fig. 6C). Prior culture environment had a significant effect on the percentage of larvae feeding (two factor ANOVA, F = 5.439; df 1 40; P = 0.025) with 100% of green water reared larvae and 87.3 ± 5.2% of clear water reared larvae feeding.

10 dph

12 (larva-1 h-1)

10 8 6 4 2 a

14

ab

B

b

b

ab

b

b

14 dph

12 10 (larva-1 h-1)

Mean number of prey consumed

0

∗

8 6 4 2 a

0 14

b

ab

4. Discussion

C

18 dph

4.1. Ontonogentic changes in prey intake

(larva-1 0.5 h-1)

12

∗

10 8 6 4 2

a

0

b

b

ab

ab

5

10

15

20

25

Nominal Turbidity (NTU)

Fig. 6. Mean consumption of rotifers by greenback flounder larvae 10, 14 and 18 dph of cohort 2 reared in either a green water (●) or clear water (○) environment and fed at a range of algal-induced turbidities. Larvae from each prior culture environment were fed rotifers at a density of 0.1 ml− 1 for 1 h A) 10 dph and B) 14 dph or 0.5 h C) 18 dph. Subscripts indicate significant differences in feed intake between turbidity treatments. Large asterisks indicates significant difference between green and clear water reared larvae.

water was 8.53 ± 0.46 rotifers h− 1 (n = 75; Fig. 6B). Again, the mean consumption of B. plicatilis was significantly lower at 0 NTU (4.34 ± 0.53 rotifers h− 1; n = 29) compared to 5, 15 and 20 NTU where mean consumption ranged from 7.2 ± 0.88 rotifers h − 1 (n = 30) to 8.07 ± 0.83 rotifers h− 1 (n = 30; Fig. 6B). There was a significant effect of prior culture history on the percentage of larvae feeding (two factor ANOVA, F = 2.599; df 1, 40, P b 0.001) with 98.7 ± 1.3% of green water reared larvae and 83.3 ± 4.1% of clear water reared larvae feeding.

Intake of both rotifers and Artemia increased with larval age for larvae reared in clear and green water environments, concomitant with improved visual acuity and visual range (Pankhurst and Butler, 1996) and in agreement with a previous study on flounder (Shaw et al., 2003). All three factors tested, prey density, turbidity and prior culture experience, had significant effects on prey intake; however, prey intake enhancement was only strongly seen during the rotifer feeding period. Despite green water enhancing rotifer intake, the growth in length or width (data not shown) of larvae reared in clear or green water was significantly different in only 1 of the 3 larval cohorts. The lack of a strong growth differential suggests that differences in prey consumption rates of larvae from green and clear water environments were not a result of differential size and therefore competence of larvae. However, we cannot discount a differential growth effect because body weight rather than length is a more reliable predictor of larval fish growth (Fielder et al., 2002; Moksness et al., 2004) and we did not measure body weight. Furthermore, species that benefit from a green water environment typically have improved survival (Naas et al., 1992; Reitan et al., 1993; Tamaru et al., 1994), as was evident in the current study in the single cohort of fish in which survival was assessed. As larvae age and their predatory ability improves, the increased survival by larvae reared in green water would have had the

G.W. Shaw et al. / Aquaculture 253 (2006) 447–460

effect of decreasing the ration size available per larva. A reduction in available ration may have masked any potential growth improvement in a green water environment. Alternatively, improved feeding in greenwater during the short feeding period tested may not translate to improved feeding over a full light period i.e., 16 h.

457

prey. Presumably, under a given set of environmental conditions, the only limit to the rate of prey consumption at prey densities above the density-dependent threshold, assuming an empty stomach, is the time taken to orient towards, capture, and ingest the prey (i.e., handling time). 4.3. Effect of turbidity level on prey consumption

4.2. Effect of prey density on consumption The ability of larvae to feed on rotifers did not differ at high prey densities between 0.5 and 5 rotifers ml− 1 (Fig. 2), suggesting that prey consumption was independent of prey density across this prey density range and under the particular suite of environmental parameters tested. However, at lower prey densities (0.025–0.1 rotifer ml− 1), prey consumption was clearly density-dependent, increasing significantly with increasing prey density, irrespective of a larva's culture environment. Other larval fish studies have shown that increasing prey density generally results in a hyperbolic increase in prey consumption until an asymptote is reached (Morales-Ventura et al., 2004). However, response curves vary between fish species (Houde and Schekter, 1980) and for different prey types fed under varying conditions (Dou et al., 2000; Morales-Ventura et al., 2004). Japanese flounder, Paralichthys olivaceus (Temminck and Schlegel), show density-dependent feeding on rotifers but shift to density-independent feeding on Artemia at densities above 1 ml− 1 (Dou et al., 2000). With one exception (fish 39 dph), feeding on Artemia in the current study was also density-independent at densities above 1 ml − 1 ; however, prey consumption by green back flounder was still dependent on prey density at Artemia densities less than 1 ml− 1. The consumption of Artemia and the percentage of greenback flounder feeding 39 dph increased even at high prey densities. Increased Artemia consumption 39 dph may be a result of the significant changes associated with flatfish metamorphosis which, depending on temperature, occurs during the period 30 to 40 dph in greenback flounder (Pankhurst and Butler, 1996). Metamorphosis of flatfishes coincides with a shift from a pelagic to benthic habitat, marked alterations in morphology and body orientation and alterations to the visual field in conjunction with eye migration (Evans and Fernald, 1993; Pankhurst and Butler, 1996). Accordingly, the point at which a larva's reactive field is ‘saturated’ with prey; i.e., the threshold between density-dependent and density-independent feeding, is thus dependent on the larva's stage of sensory development and resultant ability to detect and capture

The effect of turbidity on prey intake, growth and survival of larvae is often species specific and dependant on larval size and age. Young larvae that have small reactive fields often show improvements in feeding at moderate turbidities typically less than 30 NTU [e.g., Pacific herring, C.harengus pallasi (Boehlert and Morgan, 1985), Atlantic halibut, H. hippoglossus (Naas et al., 1992), turbot, Scophthalmus maximus L. (Reitan et al., 1993), striped mullet, Mugil cephalus L. (Tamaru et al., 1994)], whereas older larvae and juveniles are often negatively affected by the same turbidity levels [e.g., bluegill, L. macrochirus (Vinyard and O'Brien, 1976), rainbow trout, Oncorhynchus mykiss (Walbaum) (Barrett et al., 1992) Goby, G. flavescens (Utne, 1997)]. Higher turbidities during the larval phase can also cause decreased feeding rates; for example, sediment or volcanic ash suspensions at levels of 1000 or 500 mg l− 1 significantly increased feeding incidence of Pacific herring larvae, but concentrations above these dramatically reduced feeding rates (Boehlert and Morgan, 1985). Excluding the effects of prior culture environment, larvae in the current study of a given age and fed at a low prey density, consumed more rotifers, and a higher percentage of larvae fed, across the full range of turbidities tested (5–20 NTU) compared to larvae feeding in clear water. A significant decline in feeding in the highest turbidity treatments was not seen; hence it is possible that turbidity levels higher than those tested may be beneficial to this species. Turbidity also enhanced the rate of prey consumption and percentage of larvae feeding on rotifers 16 dph at high prey density (1 ml− 1; Fig. 4). However, rotifer intake of larvae 9 and 20 dph fed at high prey density (1 ml− 1; Fig. 4) showed no feeding enhancement across the range of turbidities tested, suggesting that high prey density masked any potential benefit of green water. The reason why prey consumption was enhanced in larvae 16 dph under conditions of high prey availability is unknown, but may reflect an age-specific developmental event. Turbidity had no effect on consumption rates or the percentage of older greenback flounder feeding on Artemia. Similarly, growth of lake

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herring, Coregonus artedii L., feeding on Artemia was not affected by turbidity levels ranging from 0 to 38.4 NTU (Swenson and Matson, 1976). Importantly, larvae both experienced and naive toward feeding in a turbid environment showed feed intake enhancement in turbid waters and the effect of turbidity was independent of the effect of a larva's prior culture environment. Thus there was an immediate improvement in feed intake when naïve larvae were fed in green water. The mechanism by which green water enhances prey intake is not yet clearly understood. Bristow et al. (1996) showed that larval walleye, Stizostedion vitreum (Mitchill), reared in clay-induced turbid waters were more evenly distributed and had better growth, survival and feeding performance than those reared in coloured water, despite similar light attenuation characteristics in the tanks. This suggested that the particulate scattering of light in turbid waters was more important for feeding enhancement than light attenuation properties per se. A possible mechanism of feeding enhancement under turbid conditions may be an increase in the contrast of prey produced by algal cells scattering light and providing a bright, diffuse background against which larvae can view prey (Boehlert and Morgan, 1985; Naas et al., 1992; Miner and Stein, 1993). Improved contrast may be responsible for the increased prey consumption rates in both green and clear water reared larvae across a range of turbidities, particularly at low prey densities when prey detection would be more highly constrained by early stage of development (Pankhurst and Butler, 1996). Indeed, low turbidity levels have been shown to increase reaction distance and decrease contrast thresholds, thus increasing the reactive field of larvae (Utne-Palm, 1999). An increased reactive field may explain the improvement in feeding by green water reared fish fed at sub ‘saturation’ prey densities; however, it is insufficient to explain the improved feeding evident above the threshold limit. Alternatively, it has also been proposed that algae may release chemicals stimulating foraging behaviour (Lazo et al., 2000), thus increasing the rate of prey encounter and consumption. Given that the feeding rates of green back flounder larvae of a given age showed improvement in turbid conditions, but did not significantly differ across the range of turbidities tested (5–20 NTU), chemical stimulation of feeding cannot be discounted. 4.4. Effect of prior culture environment (green water) on feeding Irrespective of the turbidity level tested, larvae from a green water environment when fed at low prey

density consumed more prey and a higher percentage of larvae fed in comparison to clear water reared larvae. Green water reared larvae feeding in green water also outperformed clear water reared larvae feeding in clear water and this was consistent between larval cohorts (Figs. 2 and 6). Thus, although both green and clear water reared larvae benefited from a turbid environment, larvae reared in a green water environment consumed significantly more rotifers. Improved feeding may have resulted from at least four factors: First, experience of feeding in a turbid environment (i.e. learned behaviour), just as prior experience of a given prey type affects subsequent prey intake and selection (Coughlin, 1991; Cox and Pankhurst, 2000); second an improved capacity of green water reared larvae to orient, capture, ingest and digest prey; third, an undetected but biologically significant size/weight differential between clear water and green water reared larvae as suggested earlier and fourth, from differential development of the neural retina in response to differences in the light environment in the culture tanks. (Pankhurst, 1984, 1992; Raymond et al., 1988; Judge, 1990). Another possibility is that there was differential intake of nutrients of importance for retinal development in larvae reared in the two environments. It is likely therefore that a single mechanism resulting in improved prey intake is not in operation. Rather, knowing that green water enhanced the rotifer intake of larvae both with and without experience of feeding in a green water environment, it follows that mechanisms that can operate over the short term such as contrast enhancement (Boehlert and Morgan, 1985; Naas et al., 1992; Miner and Stein, 1993), and chemical stimulation of feeding (Lazo et al., 2000), as well as mechanisms that operate or develop over the longer term, such as possible differences in retinal development, improvements in handling times and experience of feeding in a green water environment, are likely responsible for improved larval performance in green water when feeding on rotifers. Older larvae showed a different response to their prior culture environment. Prey consumption by flounder 38 dph was higher in fish cultured in clear water compared with green water and this was contrary to the effect of prior culture environment on green and clear water reared flounder fed Artemia in the density experiments, and throughout the rotifer feeding phase (Figs. 3 and 5). If there was an undetected growth differential between fish reared in a clear or green water environment 38 dph this likely placed the larvae at different stages of metamorphosis and thus could

G.W. Shaw et al. / Aquaculture 253 (2006) 447–460

explain the reduced feeding of green water reared larvae compared with clear water reared larvae, for reasons mentioned previously. As hypothesized, prey density, turbidity and prior culture environment all affected the prey intake of larvae. Hypothesis one proved correct with the benefits of green water only strongly evident during the rotiferfeeding period. Once larvae were able to consume Artemia green water no longer enhanced feed intake. Hypothesis, two also proved correct with green water enhancing prey intake at low prey densities but improved feeding also occurred at high prey densities. Hypothesis three predicted an increase in prey consumption at moderate turbidities in comparison to clear and higher turbidity water. Green water enhanced feeding in comparison to clear water, however, there was no strong evidence that prey intake decreased even at the highest turbidity level tested. Finally, hypothesis four predicted larvae would consume more prey in the environment of which they had experience. Larvae with experience of feeding in a green water environment fed better in green water than clear water reared larvae. However, clear water reared larvae did not feed better in clear water in comparison to green water reared larvae. Since green water enhanced feed intake of both larvae naive and experienced of a green water environment it is likely that mechanisms that act over both the short and long term are responsible for enhanced rotifer intake. The consequences of short term prey intake enhancement on long term growth and survival are unclear and should be the basis of further research.

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