Diet-induced developmental plasticity in life histories and energy metabolism in a beetle

Adaptive phenotypic plasticity, has been recognized as an important strategy by which organisms maximize fi tness in variable environments, which vary through development. A disassociation among stages should represent a null effect of the environment experienced during early ontogeny in the expression of adult traits. Food quality greatly infl uences survival, development and reproduction in many arthropod herbivores. We examined the effects of diet protein in physiological and life-history traits in the yellow mealworm beetle Tenebrio molitor through ontogeny. We established four experimental treatments: Low Protein (LP), Low Protein Control (LPC), High Protein (HP), and High Protein Control (HPC) with recently eclosioned larvae each. Individuals were maintained on the same diet or transferred to the opposite diet for all pupae period and almost all adult period. Contrary to the expected, the duration of life-cycle, larval growth rate and body mass in T. molitor were similar in diet treatments. We found intra-individual trade-offs between environmental diet (rich or poor in protein content) during larval phase and egg number. Larvae fed on a protein-defi cient diet exhibited signifi cantly higher respiratory rates than larvae fed on a rich protein diet. Compensatory feeding could act in T. molitor larvae indicating differences in metabolism but not in growth rate, body mass and life-cycle characteristics. Our results demonstrate the plasticity of reproductive and metabolic traits and lifecycle characteristics of T. molitor and how changes that occur in relation to diet can have profound effects on progeny and female fi tness.


INTRODUCTION
A d a p t i v e p h e n o t y p i c p l a s t i c i t y, t h e environmentally induced phenotypic change, has been recognized as an important strategy by which organisms maximize fitness in variable environments (Bouvet et al. 2005, Pigliucci 2005).Phenotypic plasticity generally refers to dif ferent traits, that could be connected with an allometric relationship, which vary along the individual's development (Schlichting & Pigliucci 1998).
Virtually, hundreds of studies have been published characterizing adaptive phenotypic plasticity in plants and animals, changing the way of thinking about how evolution works at the population and organismal level (see Via et al. 1995, De Witt et al. 1998, Pigliucci 2007).However, the role that early environment plays in determining the adult phenotype and per formance, also known as developmental plasticity, have been less explored (Hoffmann 1999, Diggle 2002, Castañeda et al. 2010a).
Insects are animals with a wealth variety in complex life cycles, especially among the coleoptera order.One of the explanations of abundance and persistence of complex life cycles in animals, is that they present adaptive mechanisms to face variable and unpredictable environments depending on the developmental stage (Moran 1994, Boggs 2009).According to the "Benefi cial Acclimation Hypothesis" (BAH), individuals acclimatized to a given environment might per form better in that environment compared with individuals acclimatized to a dif ferent environment (Leroi et al. 1994, Wilson 2002).However, tests of BAH have been attempted only in bacterial species or invertebrates with simple life cycles or within the same ontogenetic stage (Woods 1999, Woods & Chamberlin 1999, Woods & Harrison 2001, Lardies et al. 2004).In general, these studies have not supported the BAH but what these rejections imply about the adaptive value of physiological change remains unclear.
In addition, the allocation principle (Cody 1966, De Jong 1992, Kozlowski 1992) indicates that in a changing world where energy is limited, organisms will allocate resources in an optimal way, hence diver ting energy and nutrients in paralell to vital functions such as growth, reproduction, movement and maintenance.For example, a number of authros have found that resource constraints during ontogeny play pivotal roles in the response to temporal variation in several selective factor in adults, such as predation risk, physiological performance and dispersion capacity (Greene 1989, Castañeda et al. 2006, Hoverman & Relyea 2007).
Food quality greatly influences sur vival, development and reproduction in animals (Karowe & Martin 1989, Yang & Joern 1994, Rotem et al. 2003, Castañeda et al. 2010b).Also, different species of plants have been shown to vary greatly in their suitability and availability as a herbivore food source, as there are different populations of plants from the same species (Stockhof f 1993, Awmack & Leather 2002, Castañeda et al. 2010a).This variation depends on geographical and/or seasonal factors, and may also depend on the stage of the plant's development or its physiological state, which may also fl uctuate in space and time (Schultz et al. 1982, Stockhoff 1993, Alonso & Herrera 2000).Phytophagous insects, such as coleopterans of the Tenebrionidae family, feed primarily on live vegetal material and fungi, some of them feed on stored grains (Cotton 1927), hence being a postharvest pest (FAO 1992).
In this study we examined the effects of diet protein in physiological and life-history traits in the yellow mealworm beetle Tenebrio molitor Linnaeus (Polyphaga, Tenebrionidae), which is an ectotherm with a complex life cycle and a cosmopolitan pest of stored grains (responsible of 10-50 % losses of world grain production, FAO 1992) that can be easily reared in the laboratory (Worden & Parker 2001).

Animals, maintenance and culture
The common yellow mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera, Tenebrionidae), is a widespread distributed species that is found throughout Chile (Vidal & Guerrero 2007).We obtained T. molitor from a laboratory colony managed at the P. Universidad Catolica de Chile, Santiago.Beetles were housed in plastic cages containing a paper shelter and ad libitum water and food (bram and wheat middlings).The cages were maintained at room temperature (20-22 ºC).
We obtained about 300 adults from a colony that was transported and maintained in our laboratory.Adults were allowed to mate and then females were separated into petri dishes.Individuals (eggs) were separated and after hatching, larvae were placed individually on 6-well plates, diets were provided ad libitum.Individuals were maintained in an experimental chamber and rearing temperature and photoperiod were 18 ± 1 ºC and 12L:12D respectively.During larval period, body masses of individuals (mb) were weighed every 14 days (starting on the 21 st day of life), during pupal period and 10 days after, and during adult period, every 10 days using an analytical balance with a precision of ± 0.01 mg (ADAM AFA-180 LC).Larvae were reared from hatching to pupae on either a high-or low-protein diet (the "rearing diet", see Table 1).Pupae of one or two days were maintained on the same diet (controls) or transferred to the opposite diet (the "test diet") for all pupae period and almost all adult period prior to the measurements of the physiological and life-history traits.Thus we established four experimental treatments: A) Low Protein (LP), B) Low Protein Control (LPC), C) High Protein (HP), and D) High Protein Control (HPC) with 40 recently eclosioned larvae each.
The composition of the diets included ingredients listed in Martin & Hare (1942) who described the nutritive requirements for Tenebrio molitor larvae to obtain the optimum growth rates (see Table 1).Total protein (%) in the two different diets were 25.25 % for the high protein diet (from here HP) and 10.25 % for the low protein diet (from here LP).Diets were composed of the same ingredients but at different percentages, and they were also isocalorifi c (see Table 1).

Life-history traits
Relative Growth Rate (RGR) (van Emden 1969) was calculated using the periodical weights of larvae using the equation: RGR = [ln(mb t1 )-ln(mb t0 ) T -1 ]; where mbt 0 is the body mass (mg) at the start of time interval, mbt 1 is the body mass at the end of the time interval, and T is the number of days between the start (t 0 ) and fi nal (t 1 ).We determined larval growth rate (LGR) in intervals during life cycle and adult growth rate (AGR).
After sexing experimental individuals following Bhattacharya et al. (1970), females were bred for fi ve days to external males randomly chosen (obtained from our adult colony), then eggs were recollected after 15 days, and preserved in 70 % ethanol.All eggs were counted and measured under a stereomicroscope with ocular micrometric calibration (in mm, Olympus SZX1 stereomicroscope).

Metabolism
To estimate the rate of metabolism of experimental individuals, we measured CO 2 production through respirometry.The respirometry system used was similar to that described by Nespolo et al. (2007).CO 2 production was measured continuously using an infrared CO 2 analyzer (LI-COR LI6262, Lincon, NV, USA) capable of resolving differences of 1 ppm of CO 2 in air.The analyzer was calibrated periodically against a precision gas mixture.Baseline measurements taken before and after each recording we performed.Flow rates of dry CO 2 -free air (ambient air scrubbed free of water vapour and CO 2 using a Drierite/soda lime column) were maintained at 50 ml min -1 ± 1 % by a Sierra mass flow controller (Henderson, NV, USA).Metabolic chamber used was of 25 ml, each record was automatically transformed by a macro program recorded in the Datacan software (Sable Systems), in order to (1) correct the six-second lag introduced by the distance between the analyzer and the chamber and then to match the activity record with the CO 2 record, and (2) to transform the measure from parts per-million to ml-CO 2 per hour, taking into account the fl ow rate.For this, we used the standard equation when fl owmeter is downstream and CO 2 is scrubbed prior to flow measurement: CO 2 = STP(FeCO 2 -FiCO 2 )FR (1-FeCO 2 + FiCO 2 /RQ) -1 , where STP = standard temperature and pressure correction (equal to one with mass-fl ow controllers), FeCO 2 = excurrent fractional concentration of CO 2 , FiCO 2 = incurrent fractional concentration of CO 2 , FR = fl ow rate, RQ = respiratory quotient for herbivorous animals.Each individual was measured over a 25 min period (excluding the 5 min baseline mentioned above) and the chambers were not uncovered for visual monitoring, therefore we determined resting metabolism.Temperature during recording times was 20 ºC ± 2.

Statistical analysis
To compare the duration of entire life-cycle, number of days of the different ontogenetic stages and the growth rate of different stages an one-way ANOVA was utilised with protein treatment as the factor.The same analysis also was utilized in body mass and egg volume comparison among treatments.One-way ANCOVA was used to compare the egg number among dietary treatments using female body mass as covariable.The metabolism data were analyzed by a two-way ANCOVA, with diet and ontogenetic stage as main factors and body mass as covariate.All data were analyzed for normality and homoscedasticity.When differences in the means were signifi cant at the P < 0.05 level they were also tested with a posteriori Tukey test (HSD) (Sokal & Rohlf 1997).All statistical analyses were conducted using SPSS 12.0 for windows software.Data are presented as mean ± standard error (SE).

Life-history traits
The duration of the entire life cycle was very similar in the different diet treatments with an average of 206.5 ± 25.5 days in HP and 216.0 ± 19.6 days in LP diet, and a similar pattern was obser ved in the control treatments (F = 1.714, df = 3.160, P = 0.1542) (see Fig. 1).The same results were obtained when the duration of pupae stage was considered (F = 0.926, df = 3.160, P = 0.2349) (Fig. 1).Lar val growth rate from all four diets were similar from the beginning to the 105 th day of the experiment (F = 0.03, df = 3.47, P = 0.993) (Fig. 2A).From the 105 th to 175 th day of the experiment LGR increased signifi cantly in all treatments compared with rates of previous periods (Repeated measures ANOVA; F = 15.27,df = 2.92, P = 0.0021).However, differences among diet treatments were neither obser ved in the last period (F = 0.45, df = 3.47, P = 0.4521) (Fig. 2A).In general, for all four treatments AGR was negative, with adults from low diets protein with higher rates of mass loss (Fig. 2B).We did not obser ve statistical differences in AGR in the two time periods of adult life of the experimental individuals (F = 1.77, df = 3.169, P = 0.155 and F = 1.28, df = 3.69, P = 0.086 for 205-215 and 215-225 life days range, respectively).
Egg size and egg number of female T. molitor developed in markedly different ways with respect to food quality.Mean egg number varied from 12 ± 4.17 for females on HPC diet, to 5.42 ± 3.81 for females on the LPC diet (Table 2).Female body mass was not significantly dif ferent between treatments (F = 1.816, df = 3.36; P = 0.215) (see Fig. 1).Signifi cant differences were obser ved in egg number per female between the HP and HPC diet comparing with the other two diets (F = 21.715,df = 3.36, P = 0.0001).The effect of rearing diet was signifi cant in egg number and individuals maintained during lar val stage in LP diet and switched to HP in pupal stage did not recuperate the egg production capacity as adults.The size of recently released eggs were similar between all treatments (F = 0.826, df = 3.150, P = 0.358) (Table 2).

Energy metabolism
A significant interaction between protein treatments and ontogenetic stage on CO 2 production was found in T. molitor (twoway ANCOVA; F = 8.335, df = 6.69,P = 0.0001 for interaction term).The ef fect of protein was significant in the lar val stage (two-way ANCOVA; F = 3.249, df = 3.69, P = 0.0269), where the low protein diet had higher CO 2 production comparing with HP-HPC treatments (see Fig. 3 and Table 3).As expected, pupae stage show significant lower values of metabolic rate in all diet treatments with a mean of 0.160 ± 0.063 µl min -1 (two-way ANCOVA; F = 44.298,df = 2.69, P = 0.0001), comparing with values of 0.625 ± 0.137 and 0.539 ± 0.050 µl min -1 for lar vae and adult stage, respectively (Fig. 3).Adult and lar vae stage from different dietar y protein treatments did not show differences in mass-specific CO 2 production (Tukey's HSD, P > 0.05).Insects that were switched from HP to LP as pupae had adult metabolic rates comparable to those insects continuously reared in HP (high protein control) and had the same pattern found in insects switched from LP to HP diet (see Fig. 3).Lar val rearing diet did not have effects on adult metabolism (Tukey's HSD, P > 0.05).

DISCUSSION
Food quality describes the chemicals components required by an organism for its optimal growth, tissue maintenance, reproduction and energy expenditure.Food quality affected the life histor y traits in our study model in contrasting ways.The duration of the complete life-cycle was comparable with what is reported for T. molitor standarizing by the different rearing temperatures (see Gerber 1975, Damborsky et al. 1999).Contrary to what it could be expected, the duration of the lifecycle, lar val growth rates and body mass in T. molitor were similar among diet treatments despite their contrasting differences in protein concentration.In general, low protein lead to reduced rates of growth (Scriber 1984, Woods 1999).However, our data shows that experimental diets did not delay the lar val and pupae development nor body mass in the different ontogenetic stages.The little variation among treatments in the number of days of the life cycle, body mass, as well as growth rate could be the result of three non exclusive processes.First, growth depends on nutrient ratios, and insects may use behavioral and postingestive mechanisms to compensate for nutrient imbalance (Raubenheimer & Simpson 1993, 1997).Beetles appear to perform best on diets containing protein and carbohydrate in roughly equal amounts, and beetles may have consumed low-protein food at higher rates to provided nutrients needed (see Woods 1999).However, whether ingestion rate was different between treatments is unknown.Second, the experimental diets were isocaloric therefore T. molitor could respond to total energy ingested and not to par ticular changes in essentials elements like dietar y nitrogen, as occurs in several ar thropod (see Joern & Behmer 1997, Blanco et al. 2009).That is, diet quality (especially variation in diet protein as total nitrogen content), does not affect the duration of the life-cycle, and at the same time, neither body mass nor larval growth rate.Third, plasticity is common in early post-embr yonic or adult development, but many organisms also exhibit development inflexibility during ontogeny, which is the lack of the ability to change reproductive tactics during phases of the lifecycle (Hatle et al. 2004, Hatle et al. 2005).Our results showed that traits such as duration in  Offspring number/day and size in females of Tenebrio molitor maintained through the entire life cycle on the different protein diets.Values show are mean ± (SE).N indicates number of females analyzed.A different letter indicates signifi cant differences at the 5 % level.

High protein High protein control Low protein Low protein control
Egg number/day 11 (± 6.28) a 12 (± 4.17) a 6.13 (± 2,01) b 5.42 (± 3.81) b Egg size (mm) 1.76 (± 0.13) 1.70 (± 0.05) 1.73 (± 0.12) 1.68 (± 0.21) N 1 1 6 1 4 7 days of ontogenetic stages, growth rate in larval and adult stage and egg size and body mass in response to different diet would be canalized in T. molitor.This last affi rmation is supported by our results since the diet switches realized in pupal phase did not affect the timing of moulting and metamorphosis nor the adult growth rate.
Number and size of eggs are life histor y traits of key relevance for fitness in nature (Stearns 1992), determining the persistence of populations.Our results showed that egg size did not change among diets, but the clutch size was larger in HP diet compared with the LP diet.A classical trade-of f in life-histor y theor y predicts that if parental resources are limited, increasing resource allocation to one offspring means that the other offspring must either be smaller or fewer in number.These trade-of fs between of fspring size and number have received a great deal of theoretical and empirical support (Holloway et al. 1987, Bernardo 1996, Fox & Czesak 2000, Vijendravarma et al. 2009).In our study, egg number was af fected with no evidence of a trade-of f between number of eggs and their size, resulting in the obser vation that the quality of the diet ingested during early ontogeny can be the main cause of changes in some trait values.Several authors are agreed that, especially for herbivores ar thropod, mothers in environments of rich or poorresource produce eggs of similar size (Geister et al. 2008), being a non-plastic (fi xed) trait probably due to the fact that egg size is directly related to the fi tness of individual offspring in many arthropods (Capinera 1979).Similar TABLE 3 Rate of CO 2 emission and mass-specifi c metabolic rate of Tenebrio molitor in different ontogenetic stages at 20 ºC.Data for the treatments (HP = High Protein and LP = Low Protein) and their respective controls (CHP and CLP) were combined.Values show are mean ± (SE).
Tasa de producción de CO 2 y metabolismo masa-específi co de Tenebrio molitor en diferentes estadíos ontogenéticos a 20 ºC.Los datos para los tratamientos (HP = Alta Proteína y LP = Baja Proteína) y sus respectivos controles (CHP y CLP) fueron combinados.Los valores mostrados corresponden al promedio ± (EE).(Joern & Behmer 1997, Aron et al. 2001, Romeis & Wäckers 2002, Wall et al. 2002, Lardies et al. 2004, Kyneb & Toft 2006).Dietar y restriction in Drosophila results in a situation that is characterized by reduced fecundity and increased star vation resistance (Piper et al. 2005).Interestingly, the switch of individuals in pupal stage from LP diet to HP diets showed the same result as the control (LPC), that is to say, the egg number of females is determined by diet ingested in larval stage no matter if adult are fed with HP or LP diet.A more complete explanation for this change in fecundity is that this trait can be switched on or off as a consequence, or as a response to, environmental challenge (Spicer & Gaston 1999).In other words, an early stimulus or environmental challenge, that operates at a critical or sensitive period (lar val phase) can result in a long-term change in the structure or traits of an organism, in this case female egg number.This phase may exist because there are physiological limitations on how rapidly an organism can respond developmentally to environmental challenges (Schlichting & Pigliucci 1998).The last assertion is supported in T. molitor because the duration of the adult period (about 25 days) is shorter than the larval stage (about 165 days).A trade-off between female fecundity and subsequent of fspring fitness can be an impor tant selective factor in the evolution of fecundity (McGinley & Charnov 1988, Lardies et al. 2004).

Body mass
It could be argued that nutrient limitation (i.e.protein) was not enough to af fect the life-cycle and life histor y traits of T. molitor.However, the experimental high protein diet used by us were the ones described by Martin & Hare (1942) which includes the optimum growth rates for lar vae and feundity for females.Life-cycle characteristics (i.e.duration of stages, body mass and growth rate) support the idea that these traits are unaf fected by diet quality.On the other hand, female egg numbers suppor t the hypothesis "ef fect of rearing diet" (see Woods 1999).This outcome may arise if long-term exposure to a particular concentration of protein affects the capacity for egg production but not for shor t-term compensation in adult stage (for an example see Simpson et al. 1990).This fact would refl ect that dietar y protein has long-term postive effects in fecundity.Despite that quantification of hexameric storage of proteins was not realized in this study our resuls implies that the amount of storage of proteins during larval stage could be the threshold for egg number in T. molitor.
In terms of energy metabolism, we found that this variable is affected by diet in larval stage showing that the lar vae reared in low protein environment have higher metabolic rate and this effect is not transferred to adult phase.Lar vae fed on a protein-defi cient diet exhibited signifi cantly higher respiratory rates on a µl min -1 basis than larvae fed on a rich protein diet.A similar observation have been reported in blowfl ies reared in different protein environments and where respiratory rate was directly correlated with the occurrence of a protein hunger drive (Calabrese & Stoffolano 1974).
In general our data are consistent with the view that herbivorous insects balance their nutrient budget.One means to stabilize nutrient intake, and thus growth and developmental performance, is through compensatory feeding (see Berner et al. 2005).Results showed that beetles differentially utilized ingested protein and carbohydrate and consequently achieved similar growth on all four diets despite having ingested very different amounts of protein and carbohydrate.Compensatory feeding could act in T. molitor lar vae indicating differences in metabolism but not in growth rate, body mass and life-cycle characteristics.It is unknown if the ingestion rate, as mentioned ealier, is higher in larvae reared on a low protein diet, however the previous implies a higher ingestion of carbohydrate.A possible explanation accounting for differences in metabolism lies in the fact that compensatory feeding implies a trade off in that one nutrient is always ingested in excess of the physiological requirements unless the nutrients are per fectly balanced (Zanotto et al. 1993, 1997, Simpson et al. 2004).The previous implies a mechanism where the diet-induced thermogenesis, that occur in most if not all organisms (including insects) and functions to maintain nutritional homeostasis by metabolically oxidizing excess of energy intake, to maintain a metabolic pool of nutrients that is balanced in both energy and nutrients (Trier & Mattson 2003).
It has been suggested that physiological traits are set, often refer red to as being 'programmed', by the nutritional environment experienced during early growth (Lardies et al. 2004).Such changes in physiological processes may serve to 'match' individual phenotypes to the environment that they are most likely to encounter as adults (Monaghan 2008).Contrary to expectation no differences in metabolic rates were detected in adult beetles, nevertheless, differences were detected in other traits such as fecundity.The female cost in fecundity would be triggered in early ontogeny (i.e.high metabolic rate) which is not reversible as demonstrated by beetles that were switched from low protein to high protein diet as pupae.There is no effect in metabolism of the rearing diet and neither in the test diet in adult stage.Beetles that ate a high-protein diet had lower metabolic rates than those that ate a low-protein diet as larvae, but the rearing diet had not effect in the posterior effects.This outcome suggests that either the physiology of late instars is not affected by dietary conditions in early instars or that later stages are less able to compensate for low-quality diets (see Woods 1999).An explanation could be the large time required for this acclimation is suffi cient in the larvae stage compared with the small period of life as an adult.Metabolic rate affects developmental time in insects which is dependent on moult number and time inter val between moult (Donahaye 1990, Greenberg & Amos 1996).Interestingly, dif ferences in metabolic rate did not af fect developmental time in lar vae of T. molitor which implies a trade-off with other organismal functions or some traits.Insect pupae grow rapidly and derive energy and materials used in development from fi xed internal stores (Odell 1998), while exchanging only oxygen, carbon dioxide, water, and heat with the environment.Tenebrio molitor pupae showed the lower metabolic rate in all diet treatments, a pattern found in other insects (Harak et al. 1996).In our study the metabolic rate increased rapidly in adult stage without the dif ferences that were present in lar val stage.An explanation to the previous pattern is that insects begin metamorphosis as a large, complex organism that breaks down certain tissues and reforms others form new structures and may reset most metabolic functions (Collatz 2003).

Fig. 1 :
Fig. 1: Body mass of Tenebrio molitor through entire life cycle in different experimental diets.The mean body mass is indicated every 14 days.Abbreviations are; HPC = High Protein Control, HP = High Protein, LPC = Low Protein Control, and LP = Low Protein.Masa corporal de Tenebrio molitor a través del ciclo de vida completo en las diferentes dietas experimentales.Se indica la masa corporal promedio en intervalos de 14 días.Las abreviaciones utilizadas son: HPC = Control Alta Proteína, HP = Alta Proteína, LPC = Control Baja Proteína, y LP = Baja Proteína.

Fig. 2 :
Fig. 2: Effect of dietary protein on (A) larval and (B) adult in growth rate of Tenebrio molitor.The mean is indicated.(HPC = High Protein Control, HP = High Protein, LPC = Low Protein Control, LP = Low Protein).The mean and ± SE is indicated in Fig. 2B.See results for statistics.Efecto de la proteína en la dieta sobre (A) tasa de crecimiento de la larva y (B) tasa de crecimiento del adulto en Tenebrio molitor.(HPC = Control Alta Proteína, HP = Alta Proteína, LPC = Control Baja Proteína, LP = Baja Proteína).Se indica la media ± EE en la Fig. 2B.Ver resultados para la estadística.

Fig. 3 :
Fig. 3: Metabolic rate of different stages in the complex life cycle (L = Larvae, P = Pupae, A = Adult) of Tenebrio molitor maintained in different protein treatments (HP = High Protein, HPC = High Protein Control, LP = Low Protein, and LPC = Low Protein Control).The mean and ± SE is indicated.Signifi cant differences among protein treatments are highlighted by different letters.Tasa metabólica de los diferentes estadios (L = Larva, P = Pupa, A = Adulto) en el ciclo de vida complejo de Tenebrio molitor mantenidos en diferentes tratamientos de proteína (HP = Alta Proteína, HPC = Control Alta Proteína, LP = Baja Proteína y LPC = Control Baja Proteína).Se indica la media ± EE.Las diferencias signifi cativas entre los tratamientos de proteínas son resaltadas por diferentes letras.

TABLE 1
Composition of experimental high and low protein diets.Values are mean ± (SE).