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Mass Versus Stage Effects on Oxygen Consumption of Rana pipiens Larvae During Spontaneous Metamorphosis

by Anonymous student

Submitted February 2014

Abstract

Rates of minimal oxygen uptake (VO₂), measured with an oxygen sensitive electrode, were determined using the general allometric expression VO₂ = AM^b. Using partial correlation coefficients we demonstrated that VO₂ variance was due to changes in larval mass independent of developmental stage.

Introduction

Minimal, or resting oxygen consumption rates (VO₂), have been determined for many anuran species during spontaneous metamorphosis. Deviations from established VO₂ values may reflect the effects of ecological, physiological, and morphological factors (Feder 1981). Boell proposed that VO₂ variation in anuran larvae was due to the intense metabolic activity accompanying growth and differentiation (Boell 1948). For free-swimming larvae mass-specific VO₂ is thought to remain constant or decline, depending on the species, throughout the remainder of the metamorphic period despite the correlation of substantial growth and differentiation (Feder 1982). Measurements of VO₂ for amphibian larvae have generally been used to distinguish body mass and developmental stage effects on the pattern of VO₂ variation. Failure to provide appropriate controls and inadequate statistical designs have frustrated clear, absolute interpretations (Feder 1982). While the literature describing VO₂ values for amphibian larvae is extensive, the discrepancy between the influence of mass and/or developmental stage has yet to be resolved (Beck and Congdon 2003, Vladimirova et al 2012).

Feder found that larvae of Rana berlandieri and Hymenochirus boettgeri resemble most other animals in their VO₂-M relationship; that is, VO₂ rates, measured in volumes O₂ STP hr^-1, scale with body mass according to the equation VO₂ = AM^b denotes body mass, A and b are constants, and 0 < b < 1 (Feder 1982). Therefore, an increase in body mass correlates with a decrease in mass correlates with a decrease in mass-specific VO₂. Multivariant analyses demonstrated that developmental stage had little or no effect on VO₂ throughout metamorphosis, while changes in body mass correlated strongly with VO₂ variation during metamorphosis (Feder 1982).

Our study sought to extend Feder’s work to free-swimming larvae of the anuran species Rana pipiens. Using a standard allometric equation (Schmidt-Nielsen 1977), body mass and developmental stage effects on VO₂ variation were examined during spontaneous metamorphosis. Because larval body mass during growth is variable among individuals independent of developmental stage, we expected to verify Feder’s findings that changes in VO₂ during metamorphosis were due primarily to variation in body mass as opposed to developmental stage. Metamorphosis will hereafter refer to all manifest changes during the transformation of tadpole to metamorphic climax, defined as stage number 45 (Gosner 1960).

Materials and methods

Care of animals. Rana pipiens were induced to spawn in the laboratory and the larvae were reared in aerated tap water. Larvae were fed a commercial tropical fish food twice daily (Hartz). Photoperiod was roughly maintained at 12:12. Water temperatures ranged with room temperature, from 20 to 24 °C, throughout the period of study.

VO₂ measurements: We measured VO₂ with a YSI meter, model 51B, and a YSI oxygen – temperature probe (Yellow Spring Instruments Co., Yellow Springs, Ohio). We equipped a mason jar (volume 885 ml), with a magnetic stir bar housed in a slotted Petri dish glued to the bottom of the jar. Water samples from the aquarium were used and petroleum jelly served as a sealant once the vessel had been fitted with the probe.

Oxygen consumption was recorded in ppm and VO₂ values (ml O₂ STP · hr-1), were determined for n=13 larvae, stages 31-45 (protocol established by Gosner 1960). Mass-specific VO₂ was determined with the following relationship: ml O₂ STP · (hr · M)^-1, where M is larval wet mass in mg. Statistical methods: Chi-squared analysis, correlation coefficients, and partial correlation coefficients were used to determine mass versus developmental stage effects on VO₂ variation (Freund 1984).

Results

When plotted against log₁₀ body mass, increases in log₁₀ VO₂ were directly proportional to larval wet mass (Fig. 1). Body mass was a good predictor for increasing VO₂ (correlation coefficient, r = 0.79). Clearly, a significant amount of increased oxygen consumption relates to increased mass (62%). And as Figure 2 demonstrates, mass-specific VO₂ decreases with increasing mass. Values for A and b, which are likely to be species-specific, reflecting intrinsic physiological and ecological differences, were A = 1.03 and b =1.33.

[Insert Figure 1 here]

Figure 1. Effects of body mass, M, on rates of oxygen uptake VO₂ for Rana pipiens larvae. VO₂ = AM^b, where A = 1.03 and b = 1.33. Coefficient of correlation, r = 0.79.

[Insert Figure 2 here]

Figure 2. Mass-specific VO₂, relating increased size to decreased VO₂; also referred to as scaler effect.

If developmental stage also correlated with increased VO₂, then it is possible that the heavier larvae were represented in the more advanced developmental stages. In Table 1, the 13 larvae are grouped based on their developmental stage, where group I reflects larvae of stages 35-39 and group II is represented by larvae of stages greater than 39. Based on this sampling size, a tadpole of, for example, 3.6 grams was as likely to be found in group I as in group II (p < 0.05).

[Insert Table 1 here]

Using developmental stage as a predictor, very little, if any VO₂ variation can be explained by developmental stage (r = 0.085). However, when mass and developmental stage were considered together, 88% of the variation in VO₂ was related to these two predictors (Fig. 3). Partial correlation analysis removes either M or 5 effects on VO₂ (Table 2); body mass was a better Predictor of VO₂ variation than developmental stage (positive versus negative correlation, respectively).

[Insert Figure 3 here]

Figure 3. Mass/developmental stage-specific VO₂. The effects of body mass and developmental stage combined as a predictor of VO₂ variation. Correlation coefficient, r = 0.94.

[Insert Table 2 here]

Discussion

Table 3 summarizes our results on mass and/or stage effects on oxygen consumption for Rana pipiens larvae. The general trend of heavier animals exhibiting lower mass-specific VO₂ holds for this species, therefore scalar effects are relevant to this larval anuran.

[insert Table 3 here]

As a predictor of VO₂ body mass accounted for a significant proportion of VO₂ variation. That mass and stage collectively accounted for more VO₂ variation than mass alone needs to be explored further. Our small sampling size may account for the increased predictor value obtained by considering mass and stage together, since it appears that larval mass and developmental stage is not necessarily linked; Since developmental stage alone cannot be used to predict VO₂ (r = 0.085); such intrinsic factors as increased respiratory enzymes may account for this increased predictor value.

Throughout metamorphosis, there is a general trend towards increased size. Metamorphic climax typically is correlated with decreased body weight. If scalar effects are the primary factor responsible for the changes in VO₂, we would expect an increase in VO₂ at metamorphic climax, or stage 45. A previous study reported a significant decline in mass-specific VO₂ for Bufo boreas at the 45th stage (Sivula et al 1972). Feder points out that their plot is actually curvilinear, suggesting that a polynomial regression would be more appropriate than a least-square analysis (Feder 1982). If polynomial regressions are used on their data, the values for VO₂ increase as predicted by the scalar hypothesis (Feder 1982). He concluded that VO₂ values for later stages were influenced by scalar considerations. We could neither confirm nor refute these findings for R. pipiens due to an inadequate number of late-stage larvae. In addition to these statistical considerations, it may be appropriate to incorporate a longitudinal approach, following individuals to stage 45, in order to truly assess the effects of metabolic climax and body mass on VO₂ variation.

Xenopus laevis larvae seem to represent an exception to the scalar hypothesis and may provide insight on the problems of “gill-to-lung aeration adaptations” (Feder 1982). Feder reported that mass-specific VO₂ at metamorphic climax for this species was, in fact, lower than that predicted under the scalar hypothesis. Several factors have been proposed to cause similar declines in VO₂ thyroid hormone production, growth, or morphological changes are each associated with increased VO₂ (Boell 1948; Frieden 1967; Feder 1982). Decreased mass-specific VO₂ near stage 45 may be related to diminished levels of either one or a combination of the above factors. Based on the statistical approach of Feder, it is unclear whether or not previous studies actually demonstrated decreased mass-specific VO₂ at stage 45; more work is in order to determine whether scalar effects of body mass apply to metabolic climax VO₂ or if the profound changes associated at stage 45 will serve to be an appropriate predictor.

One important consideration not discussed previously is the animal’s behavior during development and its effect on VO₂. Feder (1982) reported that Xenopus larvae swim in mid-water while stage 45 larvae rest on the bottom and feed only periodically. Stage 45 larvae, if they feed at all, are microphagus while larvae of earlier stages are more macrophagus. Reduced physical activity and starvation-like conditions may account for the reported low VO₂ at Stage 45 (Feder 1981). Therefore. careful controls designed to account for the trophic state and activity levels must be employed. Some final comments regarding our methodology; while it is true that we were able to extend most of Feder’s results to Rana pipiens, our values for A and b differ significantly from the values reported by Feder for a Rana species: A=2.5 and b=0.878 (Feder 1982). Factors likely to have influenced our results but not accounted for are microbial VO₂, pCO₂, and larval activity during measurements. Of these three, it is likely that the first had the most significant influence; a sample of the aquarium water without the larva showed 4 ppm oxygen consumption over a similar time course. Another contributing factor may have been ineffective mixing of O₂ saturated and depleted water. Dye introduced to the vessel with and without stirring diffused throughout at a similar rate (~20sec). Assuming that the dye mimicked the diffusion rate of dissolved O₂, this would have led to transitional regions of depleted O₂ subject to mixing during transient bursts of activity.

Acknowledgements

The author thanks A. Gauger and V. Schweickert for the Rana pipiens larvae. The author also wishes to thank V. Schweickert for help with data collection.

Literature Cited

Beck, Christopher W., and Justin D. Congdon. 2003. Energetics of metamorphic climax in the southern toad (Bufo terrestris). Oecologia 137: 344-351.

Boell, E.J. 1948. Biochemical differentiation during amphibian development. Ann. NY Acad. Sci. 49:773-800.

Feder, M.E. 1981. Effect of body size, trophic state, time of day, and experimental stress on oxygen consumption of anuran larvae: an experimental assessment of the literature. Comp. Biochem. Physiol. 70A:497-508.

Feder, M.E. 1982. Effect of developmental stage and body size on oxygen consumption of anuran larvae: a reappraisal. J. Exp. Zool. 220:33-42.

Freund, J.E. 1984. Modern Elementary Statistics, 6th edition. Prentice-Hall, Inc., New Jersey, pp 354, 442, & 454.

Frieden, E. 1967. Thyroid hormones and the biochemistry of amphibian metamorphosis. Rec. Progr. Horm. Res. 23:139-186.

Schmidt-Nie1sen, K. 1977. Problems of scaling: locomotion and physiological correlates. In: Scale Effects in Animal Locomotion, ed T.J. Pedley, Academic Press. NY, pp 1-21.

Gosner, K.L. 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16:183-190.

Sivula, J.C., Mix, M.C., and McKenzie, D.S. 1972. Oxygen consumption of Bufo boreas boreas tadpoles during various developmental stages of metamorphosis. Herpetologica 28:309-313.

Vladimirova, I. G., S. Yu Kleimenov, and T. A. Alekseeva. 2012. Dynamics of body mass and oxygen consumption in the ontogeny of the Spanish ribbed newt (Pleurodeles waltl): 2. Larval stage. Biology Bulletin 39: 10-14.

Table 1: Effects of wet body mass, and Gosner (1960) developmental stage, S, on oxygen consumption VO₂.

* Average and expected VO₂ not significant (chi-squared X², p < 0.05). Expected VO₂ based on Mass = 3.6 grams, A = 1.03, and b = 1.33. Mass in units x 1000 mg; VO₂ in units mL O₂ STP · hr^-1).

Table 2. Partial correlation coefficients for log₁₀ VO₂, V, log₁₀ Mass, M, and Gosner (1960) developmental stage, S.

Table 3. Summary of Mass versus Stage effects on VO₂ variation.

V = log₁₀ VO₂; M = log₁₀ Mass; S = Gosner (1960) developmental stage.

Figure 1

Figure 2

Figure 3

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