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JOURNAL OF MORPHOLOGY 273:49–56 (2012)

Digestive Flexibility During Fasting in the Characid Fish Hyphessobrycon luetkenii Lucia Gaucher,1 Nicola´s Vidal,1 Alejandro D’Anatro,1 and Daniel E. Naya1,2* 1

Departamento de Ecologıá y Evolucioń, Facultad de Ciencias and Centro, Universitario de la Regional Este, Universidad de la Repu´blica, Uruguay 2 Departamento de Ecologıá, Center for Advanced Studies in Ecology & Biodiversity, Pontificia Universidad Cato´lica de Chile, Chile ABSTRACT Digestive flexibility is a widespread phenomenon among animals, and the congruence between empirical data and optimal digestion models strongly supports the idea that it has evolved by natural selection. However, current understanding of the evolution of this amazing flexibility is far from being comprehensive. Evidence from vertebrate tetrapods suggests that there are two major mechanisms for intestinal down-regulation during fasting periods: a decrease in the number of enterocytes in the mucosal epithelium in endothermic species, and a transitional epithelium in concert with a marked hypotrophy of enterocytes in ectothermic species. Here, we analyze the intestinal changes, at the morphological and histological levels, occurring after 9 and 16 days of fasting in a small characid fish species (Hyphessobrycon luetkenii). We found that short-term fasting was correlated with a marked down-regulation of gut size (i.e., caeca and intestine dry mass fall to a 42.3%, while intestinal length was reduced to a 73.9% of the feeding values) and that these changes were accompanied by a shift in intestinal epithelial organization from a simple columnar to pseudostratified one. This result, in conjunction with data on changes in enterocyte turnover rates during fasting in other fish species, suggests that gut regulation at both levels, cell renewal rate and epithelia configuration, is the basal condition to all tetrapods. More data, especially in some key taxonomic groups (e.g., fish that follow an endothermic strategy), will be needed in order to reach a clear understanding of digestive flexibility evolution. J. Morphol. 273:49–56, 2012. Ó 2011 Wiley Periodicals, Inc. KEY WORDS: fasting; fish; organ size, physiological flexibility, phenotypic plasticity

maximization of energy and nutrient return from the specific diet that is consumed. Second, it allows for the avoidance of the maintenance costs associated with one of the most expensive systems in terms of energy and protein metabolism. In line with these ideas, it is widely known that: (1) Gut size increase in parallel to the amount of recalcitrant material in the diet (e.g., Young Owl and Baltzi, 1998; Starck, 1999a; Liu and Wang, 2007); however, if diet quality is further reduced beyond some point, this relationship is reverted and downregulation of gut features occurs (e.g., Loeb et al., 1991; German et al., 2010); (2) Gut regulation parallels the energy demands imposed upon an organism (e.g., Toloza et al., 1991; Hammond et al., 1994; Naya et al., 2007), although at very high demand levels this relationship reach a plateau, which is probably related to organism limits in other processes (e.g., heat dissipation; Speakman and Krol, 2005; Krol et al., 2007); (3) Gut down-regulation occurs concomitant with a reduction in food consumption (Kristan and Hammond, 2001; Naya and Bozinovic, 2006), being particularly noticeable after fasting periods (e.g., Burrin et al., 1988; Secor and Diamond, 1998; Cramp et al., 2005). Two major strategies have been proposed for the downregulation of digestive functions in vertebrate tetrapods (Secor et al., 2000; Starck, 2005; Starck et al., 2007). For endothermic species that have high energetic demands and therefore the gut is rarely empty, gut down-regulation mainly involves adjustments in intestinal cell turnover rates, while the structure of the intestine

INTRODUCTION The intestinal tract represents the major interface, in terms of material and energy interchanges, between animals and their environment. Accordingly, flexibility in digestive attributes, from genes to intestinal gross morphology, has been repeatedly reported in representatives of numerous animal taxa (Piersma and Lindstrom, 1997; Penisi, 2005; Karasov et al., 2011). Digestive flexibility allows animals to cope with changes in the functional demand imposed upon this system, which is important for at least two reasons. First, it permits the Ó 2011 WILEY PERIODICALS, INC.

Contract grant sponsors: CSIC and ANII (Uruguay) and FONDAP 1501-0001 (Chile). *Correspondence to: Daniel E. Naya, Departamento de Ecologıá y Evolucioń, Facultad de Ciencias, Universidad de la Repu´blica, Igua 4225, Montevideo 11400, Uruguay. E-mail: [email protected] Received 23 February 2011; Revised 5 May 2011; Accepted 30 May 2011 Published online 10 August 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jmor.11005

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epithelial layer remains unaltered, i.e., a simple columnar epithelium (Burrin et al., 1988; DunelErb et al., 2001; Hume et al., 2002). Changes in enterocyte size and microvilli length are modest, and consequently the modulation of gut mass-specific function occur as changes in enzymes and transporters specific activity and (or) density (Secor and Diamond, 1998; Dunel-Erb et al., 2001). Accordingly, gut mass-specific rates of absorption do not change, or are even increased, during longterm fasting periods (Karasov and Diamond, 1983; Carey, 1990; Carey, 2005; Habold et al., 2007). In contrast, in ectothermic species the number of enterocytes appears to stay fairly constant during fasting periods, but the epithelial configuration of the intestinal mucosa is tranformed from a single layered columnar epithelium to a pseudostratified epithelium (Starck and Beese, 2001; Starck and Beese, 2002; Cramp and Franklin, 2005; Christel et al., 2007; Secor, 2008; Naya et al., 2009). This transformation is related to two other adjustments, a fall in blood flow to the small intestine and a noticeable reduction in the size of the enterocytes (Starck and Wimmer, 2005). The change in enterocyte size is mainly due to the disappearance of supranuclear lipid droplets, which, in turn, result in the folding of cellular membranes (Starck and Beese, 2001; Starck and Wimmer, 2005; Lignot et al., 2005; Starck et al., 2007). Finally, a noticeable decrease in microvilli length has been reported for ectothermic species that naturally experience long periods of fasting, but not for ectothermic species that feed relatively frequently in nature (Secor et al., 2000; Secor, 2005a,b; Christel et al., 2007). These differences between species with different feeding habits are correlated with variations in the ability to down-regulate mass-specific digestive function (Secor, 2005b; Cox and Secor, 2008). These two strategies probably represent end points of a continuous variation, and the relative contribution of each specific process to the overall digestive response may change from one taxonomic group to another. In line with this, it is known that (1) enterocyte turnover rates in some ectothermic tetrapods are similar to those observed in some bird species (McAvoy and Dixon, 1977; Kiss et al., 1986; Starck, 1996); (2) Retraction of lacteals and contraction of myofibrils in the lamina propia may be an important mechanism underlying villus atrophy during fasting in endothermic species (Habold et al., 2007). The model of gut regulation stated above for endothermic animals is based on evidence from different species of mammals and birds, while the model proposed for ectothermic animals is based on evidence from different species of amphibians and reptilian sauropsids (Starck, 2005). However, information about intestinal adjustments during fasting in fish has not been considered within this theoretical framework (Starck, 1999b; Starck, 2005; but see Krogdahl and Bakke-McKellep, Journal of Morphology

2005). Consequently, the aim of the present study are: (1) To analyze the intestinal changes, at the morphological and histological levels, occurring during fasting in a characid fish species (Hyphessobrycon luetkenii); and (2) To review previously published evidence of gut down-regulation during fasting in fishes. If ray-finned fishes also follow the suggested ectothermic tetrapod model, the phylogenetically most parsimonious explanation will be that the common ancestor of ray-finned fishes and tetrapods also followed this model. Hyphessobrycon luetkenii belongs to the family Characidae, one of the most important components of the Neotropical ichtyofauna. Within this family, the genus Hyphessobrycon includes more than 100 valid species, occurring from Mexico to Buenos Aires province in Argentina (Miquelarena and Lo´pez, 2006). Several species of this genus have been recorded for the Rıó de La Plata basin, with H. luetkenii being one of the most abundant representatives for this region. H. luetkenii is a smallsized omnivorous species, with a diet composed by algae, detritus and small invertebrates (Soneira et al., 2006). MATERIALS AND METHODS Experimental Design Fifteen adult animals were collected, using a trawl net, on June 19, 2010 (austral winter) in Punta Negra creek, Maldonado, Uruguay (348530 s - 558130 W). Animals were transported to the laboratory the same day of their capture, and then randomly assigned to one of three groups: fish in the first group were euthanized after 2 days of fasting (Control group, n 5 5), fish in the second group were euthanized after seven additional days of fasting (9-day fasting group, n 5 5), and finally, fish in the third group were euthanized after fourteen additional days of fasting (16-day fasting group, n 5 5). Individuals in the control group were fasted for 2 days in order to: (i) obtain empty guts without a severe manipulation of the digestive tissue, (ii) reduce differences in nutritional status between fish. In the laboratory, animals were placed in individual plastic aquariums (90 3 110 3 230 mm), and kept at an ambient temperature of 17 6 18C under natural photoperiod (11L:13D), without food. For each individual initial body mass (Imb) was measured with an electronic balance (60.0001 g; AND HR-200; Tokyo, Japan), and total and standard length (TL and SL, respectively) were measured with a plastic ruler (61 mm).

Morphological Determinations Fish were anesthetized with 2-phenoxyethanol and final body mass (Fmb) was measured with an electronic balance (60.0001 g). Then, animals were sacrificed by spinal transection, and their stomach, pyloric caeca, intestine, liver, and visceral fat were removed. Stomach and intestine were carefully dissected to avoid tissue stretching and then their lengths were measured with a digital caliper (60.01 mm; Litz Professional; Germany). Pyloric caeca were counted under a binocular microscope (Nikon SMZ-445; Tokyo Japan). Stomach, pyloric caeca and intestine were washed and rinsed with a 0.9% NaCl solution, carefully dried with paper towels, and weighed (60.0001 g). We did not attempt to separate the part of the intestine attached to the pyloric caeca from the remaining intestine (see Krogdahl and Bakke-McKellep, 2005). A ring of 1 cm in length from the proximal intestine (beginning immediately after the distal most

DIGESTIVE FLEXIBILITY IN FISH

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TABLE 1. Initial body mass (in g), final body mass (in g), total length (in cm), standard length (in cm), body condition index (BCI, in g cm23), weight loss (in %), liver and visceral fat dry mass (in g), for each experimental group Control (n 5 5) Initial mb Final mb Total length Standard length BCI Weight loss Liver Visceral fat

2.54 (60.14) 2.54 (60.14)a 6.2 (60.1) 4.9 (60.1) 12.5 (61.0)a — 0.0079 (60.0014)a 0.0030 (60.0009)

Fasting 9-days (n 5 5)

Fasting 16-days (n 5 5)

2.10 (60.13) 1.89 (60.11)b 5.7 (60.1) 4.6 (60.1) 8.7 (6 0.6)b 10.3 (60.9) 0.0073 (60.0012)a,b 0.0013 (60.0008)

2.33 (60.15) 2.02 (60.14)b 6.0 (60.1) 4.8 (60.1) 9.7 (60.9)b 13.4 (61.5) 0.0050 (60.0012)b 0.0006 (60.0007)

F and P values F2,12 F2,12 F2,12 F2,12 F2,12 F2,11 F2,11

5 5 5 5 5

2.38; P 5 0.14 7.12; P 5 0.01 2.72; P 5 0.11 2.00; P 5 0.18 5.50; P 5 0.02 — 5 4.09; P 5 0.05 5 1.75; P 5 0.22

Values are least square adjusted means 6 1 s.e.m. (for liver and peri-visceral dry mass) or absolute means 6 1 s.e.m (for the remaining variables). Different letters indicate significant differences between means (after a Tukey HSD test).

pyloric caecum) was obtained from each individual and immediately fixed in 10% formalin buffered saline solution for histological analysis (see below). Finally, all the organs, together with the animal’s carcass, were dried in an oven at 608C until constant weight (4 days), and then weighed individually (60.0001 g). We estimated intestine dry mass as the total intestinal wet mass multiplied by the dry-mass to wet-mass ratio calculated from the intestine section that was not used for histological investigation.

Histological Methodology

level. All the analyses were performed using the statistical package Statistica version 6.0.

RESULTS Intial body mass, total length and standard length did not differ between groups (Table 1). However, final body mass was greater in control animals, which corresponded with a reduction in body mass of 10% and 13% after 9- and 16-days of

The previously fixed intestinal tissue was washed in running water, dehydrated through an increasing ethanol series, and embedded in paraffin wax, according to standard protocols (Bancroft and Stevens, 1996). Tissues were transversely sectioned at 6 lm and mounted onto glass slides. Eight to twelve serial sections per animal were then stained with hematoxylin and eosin and photographed using a light microscope (Nikon Eclipse 80i; Tokyo, Japan) with a mounted digital camera (Nikon Digital Sight DS–5MC; Tokyo, Japan). Images were captured digitally at different magnifications (40, 100, 200, and 4003) and subsequently downloaded to the software NIS Elements AR version 3.1, with which measurements were taken using line morphometry. For one tissue section of each specimen we measured: (1) the intestinal diameter, (2) the width of the muscularis externa, which includes the circular and longitudinal muscle layers, (3) the total number of folds, (4) the height of five randomly selected folds, taken from the innermost edge of the circular smooth muscle layer to the outermost edge of the mucosal layer, (5) the width of five randomly selected folds, taken at the middle point of each one, and (6) the height and diameter of five randomly selected enterocytes at the tip of the folds.

Statistical Analysis Differences between groups in body size (Imb, Fmb, TL, and SL), body condition index (BCI 5 Fmb/SL3), number of pyloric caeca, width of the muscularis externa, and number of folds, were evaluated separately by one-way ANOVAs. Differences in intestinal diameter, organ length and organ dry mass were evaluated by one-way ANCOVAs, using Fmb, SL and carcass dry mass as covariates, respectively. Differences in the height and width of folds and in the height and diameter of enterocytes were analyzed through repeated measures one-way ANOVAs. In all the cases post hoc comparisons among groups were evaluated by Tukey HSD tests. Prior to all statistical analyses, data were examined for assumptions of normality and homogeneity of variance, using Kolmogorov - Smirnov and Levene tests, respectively. In some cases, data were log-transformed (e.g., intestine diameter, organ dry mass) to meet the assumptions of the analyses. Values presented are means 6 1 s.e.m., and statistical significance was established at the 0.05

Fig. 1. Stomach (A) and intestine (B) length for each experimental group. Values are least square adjusted means 6 1 s.e.m. (covariate: SL 5 4.8 cm). Different letters indicate significant differences between means (after Tukey HSD test).

Journal of Morphology

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unaffected after 9 days of fasting; however, after 16 days of fasting liver mass had declined by 36.7% (Table 1). Finally, although visceral fat dry mass showed a tendency towards a gradual decline as fasting progressed, differences among groups were not statistically significant (Table 1). At the microscopic level, we found that intestinal epithelium of control animals was a single-layered columnar epithelium, with the nuclei of epithelial cells positioned basally or medially (Fig. 3A). The cytoplasm of the enterocytes was filled with supranuclear translucent lipid droplets, enterocytes membranes were well-defined, and there was a clear brush border (Fig. 3B). During fasting, the diameter of the proximal intestine was significantly reduced (Table 2), and intestinal mucosa acquired the appearance of a pseudostratified epithelium (Fig. 3C). In addition, fewer folds were observed and their length was markedly reduced (Table 2). Enterocytes showed a noticeable decrease in their size, and there was little evidence of the apical enterocyte brush border or supranuclear lipid droplets (Table 2; Fig. 3C). The width of the muscularis externa was greater in 16-days fasted animals than in fed specimens (Table 2). DISCUSSION

Fig. 2. Stomach (A), pyloric caeca (B), and intestine (C) dry mass for each experimental group. Values are least square adjusted means 6 1 s.e.m. (covariate: carcass dry mass 5 0.48 g). Different letters indicate significant differences between means (after Tukey HSD test). Sample size for pyloric caeca was 13 (instead of 15).

fasting, respectively (Table 1). Similarly, body condition was greater in the control group than either fasted groups (Table 1). Stomach and intestine were longer in control animals (Fig. 1), while total number of pyloric caeca did not differ between groups (Table 2). Stomach, pyloric caeca and intestine were heavier in control animals than in the two fasted groups (Fig. 2). Liver dry mass was Journal of Morphology

Current evidence for tetrapods suggest that there are two major mechanisms by which intestinal morphology may be modulated during fasting: a reduction in the number of enterocytes in the mucosal epithelium in endothermic species, and the use of a transitional epithelium, in concert with a marked reduction in the size of the enterocytes, in ectothermic tetrapods. In what follows, we discuss the results obtained herein for Hyphessobrycon luetkenii, together with similar information for other fish species, within the framework of digestive function regulation. We found that the characid fish H. luetkenii responds to fasting by a noticeable reduction in the size of their digestive organs. Compared with data for other fish species (Table 3), gut size downregulation in H. luetkenii appears to be greater, which is noteworthy for at least two reasons. First, we use a short fasting period in comparison with most of the other studies (Table 3). Second, our estimation of gut down-regulation is conservative, because our control group (i.e., the baseline for fasting comparisons) underwent 48 hours of fasting before the beginning of the experiment. Although this procedure has been previously done to standardized feeding conditions of collected animals (e.g., Be´langer et al., 2002; Blier et al., 2007), some reduction of gut size already occur during the first 2 days of fasting (Krogdahl and BakkeMcKellep, 2005). Histological features of the proximal portion of the intestine of H. luetkenii are similar to those


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Fig. 3. Transverse sections of the proximal intestine of a control (A,B) and a fasting (C) specimen of Hyphessobrycon lutheri. Abbreviations: f 5 intestinal fold; g 5 globet cell; lp 5 lamina propria; me 5 muscularis externa; n 5 enterocyte nucleus; bm 5 basolateral membrane; sm 5 submucosa layer; sl 5 supranuclear lipid droplet. Scale is given inside each panel.

reported for other fish (e.g., Hall and Bellwood, 1995; Baeverfjord and Krogdahl, 1996). We did not observe intricate branched folds (rugae), nor cryptlike structures, as was observed in other fish species (e.g., Hellberg and Bjerkas, 2005). During fasting a noticeable reduction in the diameter of the proximal intestine occurs, and mucosa epithelium changed its appearance from a simple colum-

nar epithelium to a pseudostratified one. In addition, folds heights were reduced to one half of control values, and enterocytes showed a noticeable reduction in their size together with the disappearance of supranuclear lipid droplets. All of these major changes have been documented for other fish species (e.g., Gas and Noailliac-Depeyre, 1976; McLeese and Moon, 1989; Hall and Bellwood,

TABLE 2. Number of pyloric caeca, proximal intestine diameter (in mm), muscularis externa width (in lm), number of folds, fold height (in lm), fold width (in lm), enterocyte height (in lm), and enterocyte diameter (in lm) for each experimental group

No. pyloric caeca Proximal intestine diameter Muscularis e. width Number of folds Fold height Fold width Enterocyte height Enterocyte diameter

Control

Fasting 9-days

Fasting 16-days

9.6 (60.5) 1.35 (60.09)a 16.97 (61.76)a 17.2 (61.4)a 509.6 (652.5)a 114.9 (611.0) 47.6 (63.7)a 8.3 (60.7)a

9.2 (60.4) 0.87 (60.08)b 21.94 (61.55)a,b 10.2 (60.7)b 275.3 (626.0)b 94.0 (69.3) 30.7 (62.3)b 5.3 (60.7)b

9.0 (60.3) 0.83 (60.07)b 27.50 (63.80)b 9.2 (61.5)b 243.5 (626.4)b 88.7 (612.0) 30.4 (62.2)b 3.8 (60.8)b

F and P values F2,12 5 0.56; P 5 0.59 F2,11 5 13.1; P < 0.002 F2,12 5 4.18; P < 0.05 F2,12 5 12.3; P < 0.002 F10,16 5 3.25; P < 0.02 F10,16 5 1.96; P < 0.12 F10,16 5 3.59; P < 0.02 F10,16 5 2.10; P < 0.09

Values presented are least square adjusted means 6 1 s.e.m. (for intestinal diameter) or absolute means 6 1 s.e.m. (for the remaining variables). Different letters indicate significant differences between means (after a Tukey HSD test).


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TABLE 3. The effect of fasting on intestinal length and wet mass for different fish species (only maximum down-regulation values for each study are reported) Species Hyphessobrycon luetkenii Pomacentrus coelestis Salmo salar Hoplias malabaricus Cyprinus carpio Hyphessobrycon luetkenii Pleuronectes platessa Salmo salar Salmo gairdneri Salmo gairdneri Pterygoplichthys disjunctivus Gadus morhua Gadus morhua Oncorhynchus mykiss Cyprinus carpio

Variable

fp

Gd

Source

(I) L (I) L (PC1I) L (PC1I) L (I) L (PC1I) WM (I) WM (PC1I) WM (I) WM (I) WM (I) WM (PC1I) WM (PC1I) WM (PC1I) WM (I) L

16 (2) 13 (1) 40 (0) 240 (0) 390 (0) 16 (2) 32 (3) 40 (0) 91 (0) 56 (0) 150 (0) 68 (2) 70 (2) 126 (21) 390 (0)

64.8 81.8 80.7 61.2 82.2 37.7 57.8 55.0 47.9 43.2 56.0 45.0 58.8 59.8 55.9

This study Hall and Bellwood (1995) Krogdahl and Bakke-McKellep (2005) Rios et al. (2004) Gas and Noailliac-Depeyre (1976) This studya Jobling (1980) Krogdahl and Bakke-McKellep (2005) Weatherley and Gill (1981) Boge et al. (1981) German et al. (2010) Blier et al. (2007) Be´langer et al. (2002) Simpkins et al. (2003) Gas and Noailliac-Depeyre (1976)

fp, fasting period in days (the days elapsed between the last meal consumption and dissection of feeding animals is given between brackets); gd, gut decrease (amount of tissue remaining at the completion of the experiment, expressed as percentage of feeding values); (I)L, intestinal length; (PC1I)L, pyloric caeca plus intestine length; (I)WM, intestinal wet mass; (PC1I)WM, pylocric caeca plus intestine wet mass. In all cases, data were adjusted for differences in body size between feeding and fasting groups (i.e., adjusted mean values or gut size to body size ratios). a For comparative purposes, data reported for H. luetkenii in this table are in a wet basis and not in a dry basis as reported in the text. In any case, both sets of values are practically the same.

1995), and for other ectothermic vertebrates (see introduction). Indeed, fasted animals showed other histological features that also agree with previous descriptions for ectothermic vertebrates: (1) A fairly evident increase in globet cell number in all the histological slides (Gas and Noailliac-Depeyre, 1976; Baeverfjord and Krogdahl, 1996); (2) A slight rise in muscularis externa thickness (MacLeod, 1978; Cramp et al., 2005; Naya et al., 2009); (3) A moderate infiltration of mononuclear cells (e.g., lymphocytes, leucoytes and macrophages) in the submucosa and lamina propria (Gas and NoailliacDepeyre, 1976; Baeverfjord and Krogdahl, 1996; Domeneghini et al., 2002; Cramp et al., 2005). Regarding to this last parameter, the increase in the number of inflammatory cells during fasting has been related to an increase in defensive function (e.g., Baeverfjord and Krogdahl, 1996; Domeneghini et al., 2002). However, a recent study in the common wolffish (Anarhichas lupus) suggests that the increase in macrophages number during fasting periods might be related to a rise in programmed cell apoptosis (Hellberg and Bjerkas, 2005), as was previously stated for mammals and birds (Hall et al., 1994; Mayhew et al., 1999). This observation suggests, in turn, that enterocytes turnover rate in fish may be adjusted to changes in functional demands during feeding and fasting cycles. Reviewing intestinal cell renewal in fish, two observations are noteworthy. First, cell turnover times for some species are relatively short and, although longer than those observed for mammals (e.g., mice and rats: 2–3 days, man: 3–6 days, Williamson and Chir, 1978; lamb: 2–6 days, Attaix and Meslin, 1991), they are not so different from Journal of Morphology

those reported for some bird species (e.g., quails: 9–17 days, starlings: 8–10 days; Starck, 1996). Renewal time has been estimated to be 6–9 days in Carassius auratus (Vickers, 1962), 8 days in the Cyprinus carpio (Gas and Noaillac-Depeyre, 1974), 10–15 days in Ctenopharyngodon idella (Stroband and Debets, 1978), and 9–14 days in the lamprey Petromyzon marinus (Youson and Langille, 1981). Second, at least for one species, the winter flounder (Pseudopleuronectes americanus), enterocytes turnover rate is greater during feeding than after a 7-day fasting period (Trier and Moxey, 1980). Hence, current evidence does not preclude that gut size regulation in fish could occur, to some extent, by modulation of cell turnover rate according to the amount of nutrients in the intestinal lumen (i.e., the typical mechanism of regulation of endothermic species). In conclusion, digestive flexibility is a widespread phenomenon among animals, and the congruence between empirical data and optimal digestion models strongly supports the idea that gut size is adjusted to the actual demands (Starck, 1999b; McWilliams and Karasov, 2001; Karasov and McWilliams, 2005; Starck, 2005; Naya et al., 2008; Karasov et al., 2011). However, our understanding of the detailed mechanisms and evolutionary pathways which are responsible for this flexibility is far from being comprehensive.

ACKNOWLEDGMENTS The authors thank all the people of the Evolution Lab (Facultad de Ciencias, Udelar) for their patience during the course of the experiment, to


Carolina Abud and one anonymous referee for valuable comments on the draft, and to Francisco Panzera for the use of microscope facilities. William Karasov kindly provided a useful list of references. Research conducted as part of this study conformed to national and institutional guidelines for research on live animals.

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