Survival, growth and food conversion of cultured larvae of Pangasianodon hypophthalmus, depending on feeding level, prey density and fish density

Aquaculture 294 (2009) 52–59

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Survival, growth and food conversion of cultured larvae of Pangasianodon hypophthalmus, depending on feeding level, prey density and fish density J. Slembrouck a,⁎, E. Baras b, J. Subagja c, L.T. Hung d, M. Legendre b a

IRD, UR 175, c/o Loka Riset Budidaya Ikan Hias Air Tawar (LRBIHAT) Depok, Indonesia IRD, UR 175, BP 5095, Rue J.F. Breton 361, F-34196 Montpellier cedex 05, France Pusat Riset Perikanan Budidaya, 20 Jl. Raya Ragunan, Pasar Minggu, 12540 Jakarta, Indonesia d National University of Ho Chi Minh City, Faculty of Fisheries, Thu Duc, Ho Chi Minh City, Vietnam b c

a r t i c l e

i n f o

Article history: Received 2 December 2008 Received in revised form 23 April 2009 Accepted 24 April 2009 Keywords: Food density Catfish Aquaculture

a b s t r a c t In young fish larvae feeding efficiency is generally proportional to prey density, so feeding in excess is needed to maximise growth and survival. Increasing fish density might contribute to improve food conversion, but it can also impact negatively on fish growth or survival. Larvae of Pangasianodon hypophthalmus were raised until 192 h after hatching (hah) in 30-L tanks in a recirculating system (light regime: 12L:12D, 29.6 ± 1.2 °C) at three stocking densities (10, 30 and 90 fish L− 1), and fed every 3 h with Artemia nauplii at 1, 3 or 9 times a reference feeding level (RFL; 50% increase per day), thereby producing five different prey densities (from 10 to 810 RFL L− 1). Except for the highest prey density, survival (20–60%) was dependent on feeding level, whereas fish growth (12.5–17.6 mm TL at 192 h AH) was more influenced by prey density than by feeding level. Both variables were negatively affected by fish density, but to a much lesser extent than by food availability. At all fish densities, the gross conversion efficiency (GCE, 0.13–0.42) was highest at 1 RFL, and decreased for higher feeding levels, but not between 1 and 3 RFL at 90 fish L− 1, which provided the best compromise between survival, growth and GCE in this study. Temporal variations in the effects of food availability and fish density are interpreted in respect to the developmental pattern of P. hypophthalmus. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The definition of adequate feeding strategies in fish culture is proportionally more crucial during the early life stages, when suboptimal feeding can impact not only on growth but also on survival. The larvae of most fish species have a narrow resource spectrum and they generally require live feed which are expensive, manpower demanding or complicated to produce. On the other hand, live feed possess one major advantage in that they remain alive and accessible for a longer time (especially for species that do not feed on the bottom), in contrast to inert food particles. Hence, when a live prey has been found adequate for a particular life stage, the feeding strategy essentially consists of determining how much food should be delivered to fulfil the requirements of the larvae. This can be deduced from the relationships between fish size and food intake for meal size, and between fish size and gut transit rate for meal frequency. Such theoretical calculations rely on the assumption that fish are capable of finding and eating all food particles that are offered. This might be a false premise in fish, because the sensorial equipments and food searching behaviours are generally less elaborate in larvae than

⁎ Corresponding author. E-mail address: [email protected] (J. Slembrouck). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.04.038

in juveniles or adults. The relationships between food density and food intake vary among species (e.g. Perca fluviatilis, Wang and Eckmann, 1994; Gadus morhua, Gotceitas et al., 1996; Puvanendran and Brown, 1999; Paralichthys olivaceus, Dou et al., 2000; Rhombosolea tapirina, Shaw et al., 2006), and among developmental stages (e.g. Solea solea, Day et al., 1996; Sparus aurata, Parra and Yúfera, 2000; Sander lucioperca, Ljunggren, 2002). These relationships also depend on prey types (e.g. rotifers versus cladocerans in Chirostoma riojai, Morales-Ventura et al., 2004). However, with few exceptions (e.g. Sebastes spp., Laurel et al., 2001), the consumption of live prey by fish larvae does not become asymptotic unless food is given in excess (Houde and Schekter, 1980). Growth and survival follow similar asymptotic curves (Werner and Blaxter, 1980), except when excessively high prey densities alter the water quality (Puvanendran and Brown, 1999). Not only do low prey densities affect fish growth through their effect on food intake, but they can also impair growth by increasing the swimming costs during foraging (Ruzicka and Gallager, 2006). A possible way of maintaining high survival and fast growth without compromising the conversion efficiency would consist of increasing the fish density while keeping the same feeding level (i.e. number of prey per fish). This would increase the prey density (i.e. number of prey per volume unit) and thus the probability of prey encounter. However, a number of side effects are generally associated with higher fish densities, especially among larvae that exhibit cannibalistic behaviours

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at an early developmental stage. Indeed, early cannibalism does not require a size differential and is generally proportional to the probability of encountering siblings, and thus to fish density (for catfishes, see Hecht and Appelbaum,1988; Baras,1999; Baras and d'Almeida, 2001). Not only does early cannibalism impact directly on survival, but it also increases size heterogeneity, which favours the transition to complete cannibalism, and further impacts on survival in a cascading pattern that can hardly be mitigated among fish that are too small and delicate to be sorted mechanically (syntheses in Hecht and Pienaar, 1993; Baras and Jobling, 2002). In brief, these considerations can be summarised by one practical questioning: “should we feed the fish or the tank?” which in turn, requires examining the interactions between fish density, feeding level and prey density (per volume unit), and their consequences on fish survival, growth, size heterogeneity and food conversion. The present study was designed to investigate this issue in larvae of the catfish Pangasianodon hypophthalmus (Sauvage, 1878) (Siluriformes, Pangasiidae; formerly Pangasius sutchi or Pangasius hypophthalmus). Pangasiid production in aquaculture in 2007 exceeded 1 million tons per year (Lazard et al., 2009). It relies essentially on the culture of P. hypophthalmus, which originates from the Mekong and Tchao Praya river basins, but has been introduced throughout South-East Asia. Larvae of this species reputedly exhibit an intense cannibalistic behaviour at an early age (about 36 h after hatching; Subagja et al., 1999), so the issues of fish density and feeding strategy are crucial to define appropriate rearing strategies in respect to local production constraints in terms of fish survival, growth and food conversion. 2. Methods 2.1. Larval rearing The fish used in this experiment were obtained from the artificial reproduction of 3–5 year old broodfish raised in ponds at the RCA (Research Centre for Aquaculture) research station in Sukamandi, Indonesia (for detailed information on hormonally induced reproduction, egg fertilisation and incubation, see Legendre et al., 2000). Twelve hours after hatching (hereafter hah), fish were counted and transferred in an indoor recirculating system. Fish were stocked in 30-L tanks with little aeration after preliminary experiments demonstrated that strong aeration resulted in greater mortality (Hung, 1999). The water flow was 0.25 L min− 1 until fish were 72 hah, and 0.5 L min− 1 thereafter. The light regime was 12L:12D, with abrupt transitions between light and dark phases, and light intensities of 50–100 Lx, and b0.01 Lx, respectively. Water temperature was monitored continuously throughout the experiment. Water quality (pH, nitrite and ammonia concentrations) was examined at daily intervals in the system. Oxygen concentrations were measured daily in each tank. During the experiment, a 10 mg L− 1 oxytetracycline concentration was maintained in the recirculating system, following the conclusions of Subagja et al. (1999) that an early application of disinfectant or antibiotic strongly reduced the mortality among young larvae of P. hypophthalmus. Food delivery started at 36 hah. From this moment on, newly hatched brine shrimp (Artemia) nauplii were delivered every 3 h (eight times per day). Brine shrimp nauplii were produced twice a day (20-h incubation of cysts at 29 °C in brackish water [25 g NaCl L− 1]) and maintained in brackish water in a refrigerator (4–6 °C) during the 12 h following hatching. The concentration of brine shrimp nauplii was determined by volumetric counts (mean of three replicates) in 1mL samples that were collected with a pipette. At each meal, an identical volume of brackish water was collected, brine shrimp nauplii were rinsed and placed in fresh water from the recirculating system a few minutes before distribution to the experimental tanks, in order to minimise the amount of salt that was transferred to the rearing environment. At the time of food delivery, water flow in the tanks was stopped for 30 min in order to maintain the prey density as

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homogeneous as possible throughout the tank (i.e. otherwise brine shrimp nauplii tend to drift and concentrate near the exit of the tank). This 30-min period was adequate for the fish to feed maximally, by reference to observations of food intake in larvae allowed to feed over 15 and 30 min (E. Baras, unpublished data).

2.2. Experimental design Larvae of P. hypophthalmus aged 24 hah were stocked at three densities (10, 30 and 90 fish L− 1). Three feeding levels (1, 3 and 9 times a “reference feeding level”, hereafter RFL) were tested for each fish density, with two replications per treatment (i.e. fish density × feeding level). In the rest of the manuscript, each treatment is designated as x: y, where x is the fish density (fish L− 1) and y is the number of RFL per meal (Fig. 1). In addition to the nine aforementioned treatments, a tenth treatment was evaluated, with larvae raised at 10 fish L− 1 and using a very high feeding level (81 RFL, thereby producing the same prey density as in treatment 90:9). This aimed to test whether growth was maximal with 9 RFL. No counterpart with very high feeding level was evaluated for higher fish densities, essentially to minimise the risk of degradation of water quality. Groups were randomly assigned to the 20 tanks of the water recirculation system. As a result of the experimental design, the prey density (number of brine shrimp nauplii per mL) was identical among treatments 10:3 and 30:1, among treatments 10:9, 30:3 and 90:1, among treatments 30:9 and 90:3, and among treatments 90:9 and 10:81 (see Fig. 1). The design thus enabled testing within a single experiment the effects of fish density, feeding level (nauplii per fish) and their interactions, as well as those of prey density (nauplii per volume unit). The RFL was based on feeding schedules that were determined after preliminary culture trials in Viet Nam (Slembrouck, 1997), and stood as Log (RFL) = 0.377 + 0.176 A, where RFL is the number of brine shrimp nauplii delivered per meal per fish stocked, and A is the age of the fish (days after hatching, hereafter dah). In practice, the RFLs on 2– 8 dah were 4, 6, 8, 12, 18, 27 and 41 nauplii per fish per meal, respectively (i.e. a 50% increase per day).

Fig. 1. Experimental design for testing the combined effects of fish density, feeding level (prey per fish) and prey density (prey per volume) on the survival, food intake, growth and food conversion in larvae of P. hypophthalmus. Identical symbols connected by plain lines highlight identical food densities (10–810 RFL L− 1), obtained with different combinations of fish densities and feeding levels (i.e. N times the reference feeding level, RFL). An additional treatment (not shown on the figure) was also evaluated with fish raised at 10 fish L− 1 offered 81 RFL.

2.3. Measurements and calculations At 48, 96, 144 and 192 h after hatching, 10 fish were sampled in each tank 30 min after the distribution of brine shrimp nauplii. They

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were anaesthetised with 2-phenoxy-ethanol (0.4 mL L− 1) and their total body length (TL, mm) was measured under the stereomicroscope with a micrometer (accuracy ranging from 0.05 to 0.1 mm, depending on fish size and magnification). The anaesthetised fish were killed with a lethal solution of 2-phenoxy-ethanol (over 2 mL L− 1) and preserved in 5% formalin. Thereafter their guts were dissected under the stereomicroscope and food intake was determined by counting the number of brine shrimp nauplii in the foregut (then stomach when differentiated). To evaluate growth, body length was examined preferentially to body mass, since the fish were fed every 3 h and rarely had an empty gut, so growth estimates from body mass measurements would have been biased to variable extents depending on food intake and thus presumably on treatment. Fish wet body mass (WM) was backcalculated from the fish's total length (TL) with a mass-to-size model for larvae of P. hypophthalmus with empty guts (E. Baras, unpublished data), i.e. Log WM = 176.0 − [882.8 Log(TL)] + [1737.3 log(TL)2] − [1684.4 Log(TL)3] + [808.9 Log(TL)4]) − [154.1 Log(TL)5]. The model is unusually complex for a mass-to-size relationship in fish, but it accurately depicts the various stages of allometric growth that occur throughout the larval stages of P. hypophthalmus (here from 0.9 to 80 mg WM, R2 N 0.99, df = 350). At 192 hah, all tanks were emptied and survivors were counted. During the experiment, the tanks were siphoned twice a day to remove uneaten food and dead fish. However, larvae of P. hypophthalmus are small (about 0.9 mg at the start of exogenous feeding, at 36 hah), and they undergo a rapid decay in warm water. Hence, many dead fish probably passed unnoticed and the mortality dynamics could not be traced accurately during this experiment. The knowledge of the mortality dynamics is unimportant in respect to prey density (nauplii per volume unit), whereas it matters for determining the actual number of prey available per survivor, and its consequences on food intake, growth and size heterogeneity. In absence of additional information, daily mortality (M, % day− 1) was deemed almost constant throughout the experiment, and was estimated as M = [Ln (Ni) − Ln (Nf)] / 6.5, where Ni and Nf are the initial and final numbers of fish in a tank, and 6.5 is the duration of the experiment (days). The number of survivors at a particular age in each tank was estimated from these. Using the information on growth and survival, fish production was calculated and compared to the food offered in order to estimate the gross conversion efficiency (GCE = production/food offered). Dry mass (DM) rather than wet mass (WM) ratios were used for calculating the GCE, because the water content varies substantially during the ontogeny of P. hypophthalmus, so comparisons between fish of different WMs would have been biased. A standard DM:WM ratio of 16.5% was used for brine shrimp nauplii (Sorgeloos et al., 1986). The DM:WM ratios for fish larvae were calculated from independent data sets produced during experiments where larvae were fed maximally with brine shrimp nauplii (E. Baras, unpublished data). At the start of exogenous feeding (fish of 0.9 mg WM), the DM:WM ratio is about 11%. Thereafter, the ontogenetic changes in the proportion of dry matter during the larval stages of well-fed P. hypophthalmus can be described by the following model: DM:WM (%) = 9.048 [0.187] + 3.689 [0.269] Log WM (R2 = 0.969, F = 187.70, P b 0.0001 both for the intercept and slope) (where DM and WM are the dry and wet body mass of fish, and the values between brackets are the standard errors of coefficients).

The 10:81 treatment could not be included into these analyses (since the 81 RFL feeding level was not tested for all densities), so additional post hoc tests (Scheffe F-tests) were used for pairwise comparisons between all 10 treatments. These analyses also permitted to test for differences between treatments with identical prey densities but different combinations of fish density and feeding level (e.g. 10:9 versus 30:3 and 90:1). Stepwise multiple-regression analyses were used to test for the preponderant influence of variables (feeding level, prey density, fish density) on the variables under study (survival, growth, food conversion). Null hypotheses were rejected at P b 0.05. 3. Results Temperature varied between 28.4 and 30.9 °C during the experiment. Water pH was 8.4–8.5 throughout, whereas nitrite and ammonium increased slightly in the course of the experiment (from −1 −1 0.005 to 0.012 mg NO− , and from 0.01 to 0.02 mg NH+ ). At 2 L 4 L the very end of the experiment, however, the ammonium concentra−1 tion in the system soared to 0.22 mg NH+ , but no mass mortality 4 L was observed by then. The oxygen level remained stable (around 7.6 mg O2 L− 1) throughout the experiment in the tanks with the lowest prey density (10 RFL L− 1, treatment 10:1). In all other treatments, the oxygen level declined progressively, and proportionally to the prey density (mean levels of 7.5, 7.0, 6.3 and 5.5 mg L− 1, at prey densities of 30, 90, 270 and 810 RFL L− 1). The lowest oxygen level (4.4 mg O2 L− 1) was observed at 192 hah in a tank of the 90:9 treatment but no mass mortality was observed on this day. 3.1. Survival Survival at 192 h PH ranged from 20 to 60.5% (Fig. 2). It was significantly dependent on feeding level (P = 0.0003) and independent from fish density (P = 0.1080). At 10 and 30 fish L− 1, survival of fish fed 1 to 9 RFL was proportional to the feeding level. By contrast, at 90 fish L− 1, survival was slightly lower among fish that were offered 9 RFL than among those receiving 3 RFL (33.5 versus 39%). A stepwise multiple regression analysis revealed that survival at 192 hah (S192h, % initial; Log form) was more closely dependent on the feeding level (FL, nauplii fish − 1 meal− 1, Log form, partial R2 = 0.718) than on prey density (PD, nauplii mL− 1, Log form, partial R2 = 0.190),

2.4. Statistics The heterogeneity of food intake or fish size was expressed as the coefficient of variation, CV = SD mean− 1, where SD is the standard deviation of the sample. GCE, food intake, survival and growth rates were compared between treatments by 2-way analysis of variance (ANOVA), using fish density and feeding level as independent factors.

Fig. 2. Survival of larvae of P. hypophthalmus at 192 h after hatching (hah), as a function of fish density and feeding level (see Fig. 1). Bars and whiskers are the means and differences between two replicates. Bars with the same shade correspond to treatments with identical prey density (nauplii per volume). Statistics (2-way ANOVA, excluding the 10:81 groups): fish density: P = 0.1080, feeding level: P = 0.0003, interaction: P = 0.0663. Bars that share at least one superscript in common do not differ at P b 0.05 (Scheffe F-tests between all 10 treatments).

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and that fish density (FD, fish L− 1) impacted negatively on the survival of fish that were offered the same feeding level, i.e. LogS192 h = 1:517½0:084 + 0:352½0:051LogðFLÞ − 0:116½0:053Log FD;

where variables are presented in order of entrance in the model, and values between brackets are the standard errors of coefficients (F = 26.186, R2 = 0.789, df = 17, P of b0.0001, b0.0001, and 0.0469 for the intercept, FL and FD, respectively).

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is most unusual in fish. By contrast, no such decrease was observed in the groups fed 9 or 81 RFL. It was strongly suspected that this situation originated from a lower quality and survival of brine shrimp on these two days, which affected the fish fed the lowest feeding levels more than those fed in large excess. Since the experimental conditions were seemingly not fully respected, the data from the last feeding days were not taken into account in the growth model, which stood as: Log G = 0:372½0:108 + 0:691½0:061Log WM + 0:221½0:050Log PD − 0:259½0:067Log FD;

3.2. Growth and size heterogeneity The average size of fish at 192 hah varied between 12.5 and 17.6 mm TL, in the 10:1 and 90:9 groups, respectively (Fig. 3). Fish from the 10:81 group did not attain significantly larger sizes (17.7 mm TL on average). The coefficients of variation of fish size at 192 hah remained low in all groups (from 4.52 to 7.93%), and were not influenced significantly by fish density (2-way ANOVA: P = 0.0972), by feeding level (P = 0.9612) or by the interaction between the two factors (P = 0.4786). By contrast, at all fish densities and on all sampling dates, the feeding level had a significant positive effect on fish size (P b 0.0001; Fig. 3). Fish density also had a significant impact on growth (P from b0.0001 to 0.0027), but it acted in contrasting ways, depending on fish age and size. During the early feeding stages (from 36 to 48 hah), fish density impacted negatively on fish growth. Thereafter, growth progressively became faster among the fish stocked at medium or high density, at least among those that were offered 3 or 9 RFL. A stepwise multiple regression analysis was conducted for analysing the variations of fish growth (G, mg WM day− 1) against the wet body mass of fish (WM, mg), fish density (FD, fish L− 1), feeding level (FL, nauplii fish − 1 meal− 1) and prey density (PD, nauplii mL− 1). The analysis was restricted to the data collected until 144 hah for the following reason. From 144 to 192 hah, the growth of fish fed 1 or 3 RFL was slower than on the previous days (Fig. 3), which

where variables are presented in order of entrance in the model, and values between brackets are the standard errors of coefficients (F = 139.13, R2 = 0.948, df = 53, P of 0.0022, b0.0001, 0.0002 and 0.0008 for the intercept, WM, PD and FD, respectively). The influence of the feeding level was insignificant after the effect of prey density was taken into account by the model. 3.3. Gut content The gut contents of larvae at 48, 96 and 144 hah were significantly dependent on the feeding level (P b 0.0001) but independent from fish density (P of 0.1491, 0.0634 and 0.1766) and from the interaction between the two variables (P of 0.1235, 0.3420 and 0.0680). At all sampling dates, the gut contents of larvae that received 1 RFL were close to those predicted by the model that was used for calculating the reference feeding level (Fig. 4). By contrast, larvae that were offered larger amounts of food ate more than predicted. At 48 hah, the gut contents of fish fed 3 to 81 RFL were about three times higher than predicted by the model. Differences between predicted and observed food intakes at 3, 9 and 81 RFL persisted in older fish, because fish grew faster in this experiment than in the feeding trials that were used to elaborate the feeding schedule. At almost all fish densities, the coefficient of variation of food intake was reduced when the feeding level was increased (Fig. 4).

Fig. 3. Size (total body length, TL) of larvae of P. hypophthalmus as a function of fish age (hours after hatching, hah), fish density and feeding level. Bars and whiskers are the mean and standard errors of 20 fish (two replications per treatment, 10 fish per sample). Bars and whiskers are the means and standard errors of 20 fish for each treatment (10 fish per sample two replicate groups per treatment). Bars with the same shade correspond to treatments with identical prey density (nauplii per volume). Statistics (2-way ANOVA, excluding the 10:81 group): 48 hah: fish density: P b 0.0001, feeding level: P b 0.0001, interaction: P = 0.4497; 96 hah: fish density: P = 0.0015, feeding level: P b 0.0001, interaction: P = 0.3826; 144 hah: fish density: P = 0.0027, feeding level: P b 0.0001, interaction: P = 0.0018; 192 hah: fish density: P = 0.0013, feeding level: P b 0.0001, interaction: P = 0.0012. In each graph, bars that share at least one superscript in common do not differ at P b 0.05 (Scheffe F-tests between all 10 treatments).

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Fig. 4. Gut content of larvae of P. hypophthalmus, depending on age, fish density and feeding level. The lower right graph illustrates the coefficients of variation (CV, %) of gut content between treatments. Bars and whiskers are the means and standard errors (10 fish per sample two replicate groups per treatment). Bars with the same shade correspond to treatments with identical prey density (nauplii per volume). The dotted horizontal lines in the first three graphs indicate the reference feeding level on these days (nauplii per meal per fish stocked). Statistics for gut content (2-way ANOVA, excluding the 10:81 group): 48 hah: fish density: P = 0.1491, feeding level: P b 0.0001, interaction: P = 0.1235; 96 hah: fish density: P = 0.0634, feeding level: P b 0.0001, interaction: P = 0.3420; 144 hah: density: P = 0.1766, feeding level: P b 0.0001, interaction: P = 0.0680. Bars that share at least one superscript in common do not differ at P b 0.05 (Scheffe F-tests between all 10 treatments).

A model of maximal food intake against fish size was constructed from the data on gut contents (Fig. 5). Based on this model, it became obvious that fish offered 1 RFL were underfed at the start of the experiment. The question whether this feeding level remained insufficient during the subsequent days cannot be answered directly, because it requires knowledge on the dynamics of growth and mortality. For each treatment and each rearing day, the number of

survivors was calculated on the basis of a constant mortality rate (see methods), fish size was modelled from growth curves constructed from samples at 2-day intervals, then fish body mass was backcalculated, using the model described in the methods. Finally, fish number and body mass were equated to determine how many nauplii would have been needed to feed the survivors maximally on each day, and this value was compared to the number of nauplii that was actually distributed on this particular day. The results of this analysis (Fig. 6) clearly indicate that, at all fish densities, the fish that were offered 1 RFL were underfed throughout, except for the last feeding day, and even those given 3 RFL were slightly underfed from 60 to 108 hah.

Fig. 5. Ontogenetic variation of food intake in larvae of P. hypophthalmus, based on the gut contents of fish sampled in all treatments at 48, 96 and 144 hah. A model of food intake against fish size was constructed from the fish with the highest gut contents relative to their size (circled symbols), which were deemed to have replenished, and stands as: FImax = − 74:01½3:39 + 14:51½0:34TL where FImax is the maximum food intake (nauplii fish− 1 meal− 1), TL is the total body length of fish (mm), and values between brackets are the standard errors of coefficients (F = 1840.62, R2 = 0.986, df = 27, P b 0.0001 both for intercept and slope).

Fig. 6. Comparison between the amounts of food that were actually distributed and those that would have been needed to feed maximally the survivors (nauplii available: needed). Calculations are based on fish survival, size and maximum food intake (Figs. 2, 3 and 5, see text for details). All points below the bold horizontal line correspond to situations where fish were fed less than maximally. The 10:81 treatment is not illustrated here.

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4.2. Effects on fish growth

Fig. 7. Comparison between the gross food conversion efficiency (GCE, DM:DM) of larvae until 144 hah, depending on fish density and feeding level. Bars and whiskers are the means and differences between two replicates. Bars with the same shade correspond to treatments with identical prey density (nauplii per water volume). Statistics (2-way ANOVA, excluding the 10:81 group): fish density: P = 0.9301, feeding level: P = 0.0006, interaction: P = 0.2583 Bars that share at least one superscript in common do not differ at P b 0.05 (Scheffe F-tests between all 10 treatments).

3.4. Apparent food conversion efficiency The apparent gross conversion efficiency (GCE, DM:DM) until 144 hah was significantly dependent on feeding level (P = 0.0006), but was not influenced by the fish density (P = 0.9301) or by the interaction between the two factors (P = 0.2583; Fig. 7). At all fish densities, the GCE was highest (≥0.35) at 1 RFL, and decreased significantly at higher levels (except between 1 and 3 RFL at 90 fish L− 1). 4. Discussion 4.1. Main outcomes of the study This study is among the very first to analyse the interactions between feeding level, prey density and fish density in fish larvae. It provided evidence that fish density and prey density, rather than feeding level, govern the growth of P. hypophthalmus larvae, whereas their survival is influenced to a greater extent by the feeding level than by prey density, and little affected by fish density. The results also provide information on how the rearing and feeding strategies can be defined in respect to production constraints, in terms of survival, growth or food conversion. In particular, the study permitted us to model the relationships between food intake and fish size during the larval stages of P. hypophthalmus, thereby giving the opportunity of adjusting feeding charts to fish size, which can be rapidly obtained from fish samples. The maximal meal size is low during the early feeding stages (12% WM at 5.5 mm TL and 0.72 mg), but increases rapidly as it amounts to 22 and 26% WM, respectively at 6.0 and 6.5 mm TL (corresponding WM of 1.2 and 1.6 mg). From 7 mm TL onwards, meal size starts decreasing in a curvilinear way, and it amounts to about 10% WM at 15 mm TL (WM of 25 mg). Based on stomach residence time (b3–4 h at 29 °C in 6.5–7.0 mm TL fish; E. Baras and Y. Moreau, unpublished data), the daily food intake can be 6 to 8 times higher than meal size, thus 155–205% WM. These estimates concur with those obtained during independent experiments where the food intake of cannibals was estimated from the number of siblings consumed during 24 h (i.e. up to 203% WM in a cannibal of 5 mg WM; E. Baras, unpublished data). They also support the “empirical” recommendation of Hardjamulia et al. (1981) that larvae should be fed 250% WM per day during the first feeding days.

The model between food intake and fish size emphasised that fish offered 1 RFL were underfed. This statement remained unchanged after survival rates were taken into account in the analysis, except for the last feeding day (Fig. 6). Similarly, it turned out that from 60 to 108 hah fish offered 3 RFL were not fed in excess but just maximally. However, these findings do not compromise the relevance of the experimental design, because the highest feeding level that was evaluated at all fish densities (9 RFL) was enough to produce maximal or near-maximal growth in this species (comparison with the 10:81 treatment; Fig. 3). The growth model that was constructed with a stepwise multiple-regression analysis provided evidence that prey density (per volume unit) prevailed over prey availability (per fish), and that fish growth at a particular prey density was inversely proportional to the fish density. The effect of prey density on the growth of P. hypophthalmus larvae can be accounted as follows. Young larvae of this species swim throughout the water column with their mouth wide open (Hardjamulia et al., 1981; Subagja et al., 1999) and possess little ability for manoeuvring, since their pectoral fins are not developed at the start of exogenous feeding (E. Baras, J. Slembrouck and M. Legendre, unpublished data). With such behavioural and morphological traits, the probability of encountering and capturing food items is directly proportional to prey density, to a greater extent than in larvae of other fish species with greater manoeuvring abilities. Whatever the intrinsic foraging efficiency of a larva of P. hypophthalmus, it is presumably reduced in presence of conspecifics, which either remove prey from the trajectory of a particular forager or interact with its movements. Hence, it presumably takes longer for a particular fish to replenish in the presence of numerous conspecifics, This interpretation is supported by the observation that the food intake of 48-h old larvae at a particular prey density was negatively correlated with fish density, even when food was in excess (Fig. 3). The longer the foraging time, the higher the swimming costs (Ruzicka and Gallager, 2006), and the slower the growth. There was no adverse impact of high prey densities on food intake or fish growth, in contrast to the situation in redfish Sebastes spp. (Laurel et al., 2001). Redfish larvae offered high prey densities frequently abandoned a prey capture sequence and re-orientated towards other prey, thereby resulting in slower growth. The absence of such confusion effect in P. hypophthalmus is consistent with their very simple foraging behaviour, which merely relies on constant swimming and little orientation towards prey. 4.3. Effects on food conversion efficiency At all fish densities, the lowest gross conversion efficiency (GCE, DM:DM) was observed when fish were offered 9 RFL (or 81 RFL at 10 fish L− 1): these feeding levels largely exceeded the maximal needs of fish (cf. Figs. 6 and 7), so food was inevitably wasted. The highest GCE at all fish densities (about 0.35–0.40) was obtained with the lowest feeding level. However, this finding is not an encouragement to feeding submaximally the larvae of this species, especially in view of the negative effects of underfeeding on fish growth and survival. Furthermore, the GCE was almost equally high with other treatments that produced significantly faster growth and higher survival than among groups fed 1 RFL (e.g. 90:3 treatment). The best GCE values during this study were just below those (0.45 DM:DM) obtained in experiments where the growth of cannibalistic larvae of P. hypopthalmus was compared to their actual food intake (E. Baras, unpublished data). This comparison suggests that, in spite of the apparent simplicity of their foraging behaviour, larvae of P. hypophthalmus are capable of finding and capturing food quite efficiently, even when prey are scarce or just abundant enough to fulfil their needs (Fig. 6).

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4.4. Effects on survival The effects of feeding level and fish density on fish survival were less straightforward than those on fish growth or food conversion. Survival increased with increasing feeding levels, except for the highest prey densities (90:9 and 10:81 treatments). Brine shrimp nauplii release nitrogenous metabolites, the accumulation of which in recirculating systems can impact fish survival, especially at densities above 8 nauplii mL− 1 (Houde, 1975; Léger et al., 1986; Puvanendran and Brown, 1999). The prey density culminated at 33 nauplii mL− 1 on the last feeding day of this study. Freshly hatched brine shrimp nauplii survive no more than a few hours in fresh water at 29 °C, and their decay alters the water quality. However, it is unlikely that in this study P. hypophthalmus larvae died from a decline in the chemical quality of the water. First, the total ammonia nitrogen (TAN) in the recirculation system remained low throughout the experiment, except for the very last day. However no high mortality ensued. Second, recent experiments indicated that larvae and juveniles of this species had a rather high tolerance to TAN (DL50 in 5-g fish is about 35 mg TAN L− 1 at 28 °C and pH 8.2; D. Caruso and E. Baras, unpublished data 2009). Finally, survival rates of 85% have been obtained for larvae of P. hypophthalmus raised from 8 to 28 dah in a recirculating system where nitrite and ammonia concentrations varied between 0.2 to 7.5 mg L− 1, and 0.5 to 1.0 mg L− 1, respectively (J. Slembrouck, unpublished data). Alternatively, it is likely that the large amounts of uneaten food at high prey density facilitate the proliferation of bacteria. Bacterial growth puts larvae at risk, especially those that grow long oral teeth and are prone to injure each other, as is the case of P. hypophthalmus. Yet, antibiotics were applied throughout the experiment (10 mg L− 1 oxytetracyclin). This concentration gives satisfactory results with standard feeding schedules (Subagja et al., 1999), but it might be insufficient for a rapid bacterial growth with high loads of wasted food. The low survival of P. hypophthalmus at low feeding levels cannot be blamed on fish dying out of hunger, since the P50 mortality of larvae deprived of exogenous food lies at 8–9 dah at 28 °C (E. Baras, unpublished data). Cannibalism is generally higher when food is rationed (syntheses in Hecht and Pienaar, 1993; Baras and Jobling, 2002). However, cannibalism generally causes the elimination of the smallest fish, while the largest fish gain a rapid growth advantage and become “jumpers”. If cannibalism had been the main source of mortality in this study, the survivors in underfed groups would have been expected to grow as fast as, or faster than in other groups, while the opposite trend was observed. Other factors can be invoked, in relation to the morphology of P. hypophthalmus larvae. At the onset of exogenous feeding, these larvae exhibit a large gape (18% TL), and long oral teeth (0.10 mm). They swim continuously throughout the water column but in absence of pectoral fins, they are probably incapable of fine swimming manoeuvres. Encounters result in deadly clashes, because the larvae cannot get rid of the siblings they grasped and rapidly suffocate. This situation is most transient, as the mouth and teeth exhibit a strongly negative allometric growth. The faster the fish grow through this interval, the shorter the risk period and the lower the mortality. Furthermore, fish probably need to forage over longer periods if food is scarce, thereby increasing the risks of deadly encounters with siblings during each meal. Altogether, these interpretations might account for why survival was about three times lower among the groups that were underfed than when food availability was higher. 4.5. Conclusions The study highlights how the production settings in nurseries of P. hypophthalmus could be adjusted if the premium is growth, survival or food conversion. These priorities can vary substantially between producers, depending on local constraints in terms of food supply, size and type of rearing system (ponds versus recirculating water

systems), number of broodfish relative to the production of weaned larvae or juveniles, water renewal rate or water quality. If growth, survival and food conversion are equally important, the best compromise until 192 hah is a density of 90 fish per litre, with slight overfeeding (3 RFL). It is possible that higher performance be obtained with intermediate combinations of feeding levels and fish densities, or with feeding schedules that better track the ontogenetic variations in fish behaviour and morphology. For example, it might be worth testing very high feeding levels during the first 24 or 36 h of exogenous feeding, in order to maximise early survival and growth, then decreasing the feeding level. This would have little negative impact on the food conversion, because young larvae of P. hypophthalmus consume few nauplii per capita.

Acknowledgements The authors acknowledge Kamlawi and Wawan (Sukamandi research station) for their technical assistance, and Rémi Dugué (IRD, UR 175, Montpellier) for the constructive criticisms on a draft of this manuscript. This work was part of a project on the biodiversity and culture of Southeast Asian catfishes supported by the European Union (contract IC18-CT96-0043). Mrs. Dominique Caseau contributed to improve the English style of the manuscript.

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