Journal of Insect Physiology 56 (2010) 1679–1684

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Journal of Insect Physiology

journal homepage: www.elsevier.com/locate/jinsphys

Ontogenetic stage-dependent effect of temperature on developmental and

metabolic rates in a holometabolous insect
Guillermo Folguera a,b,*, Julián Mensch a, José L. Muñoz b,1, Santiago G. Ceballos a,2,
Esteban Hasson a, Francisco Bozinovic b
a
Departamento de Ecologı´a, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C 1428EHA Buenos Aires, Argentina
b
Centro de Estudios Avanzados en Ecologı´a y Biodiversidad (CASEB) and Departamento de Ecologı´a, Facultad de Ciencias Biológicas,
Pontificia Universidad Católica de Chile, Santiago 6513677, Chile

A R T I C L E I N F O A B S T R A C T

Article history: Different hypotheses attempt to explain how different stages of organisms with complex life cycles
Received 15 April 2010 respond to environmental changes. Most studies have focused at the among-species level showing
Received in revised form 9 June 2010 similar responses to temperature throughout ontogeny. However, there is no agreement about the
Accepted 29 June 2010
pattern expected at the intraspecific scale where a strong selective effect is expected. In this paper, we
studied the effects of thermal treatments on a life history trait (developmental rate) and a physiological
Keywords: trait (metabolic rate) during development in the fruitfly Drosophila buzzatii. First, we estimated the rate
Allometry
of development during larval life (LDR) and the pupal stage (PDR) in flies derived from two natural
Developmental rate
Drosophila
populations exposed to several thermal treatments. Our results showed that the developmental rate
Evolution ratio, LDR/PDR, did not vary between populations, and that the effects of thermal treatments were stage
Isomorphic hypothesis specific. Second, we studied the relationship between developmental rate (DR) and metabolic rate (MR)
Life-history metamorphosis in each life cycle stage. We found that allometric relationships between DR and MR varied throughout
ontogeny, a pattern that shed light on the mechanisms responsible for thermal plasticity. We conclude
that, although different populations may show developmental rate isomorphy; larvae and pupae may
choose alternative ‘‘decisions’’ in terms of life-history evolution and physiological traits when
confronted to different thermal environments.
ß 2010 Elsevier Ltd. All rights reserved.

1. Introduction and is known to be influenced by temperature (Rombough, 2003).

Organisms have different developmental strategies of internal
control to cope with environmental changes in temperature.
Interestingly, strategies appear to be diverse not only among
organisms of different populations and species, but also along
different stages in the same organism. Comparisons among life
history and physiological responses of organisms to thermal
changes during different periods of the life cycle may be a suitable
approach to gain a deeper understanding vis-à-vis this issue
(Dahlgaard and Loeschcke, 1997). The rate at which organisms
develop (developmental rate [DR]) is an important life history trait
* Corresponding author at: Laboratorio de Evolución, Departamento de Ecologı́a,
Genética y Evolución, Facultad de Ciencias Biológicas, Universidad de Buenos Aires,
Espinosa 423 1586, Buenos Aires, Argentina. Tel.: +54 1144323449.
E-mail address: guillefolguera@yahoo.com.ar (G. Folguera).
1
Present address: Departamento de Bioloxı́a Funcional e Ciencias da Saúde,
Universidad de Vigo, 36310 Vigo (Pontevedra), Spain.
2
Present address: Centro Austral de Investigaciones Cientı́ficas, (V9410BRL)
Ushuaia, Tierra del Fuego, Argentina.
Fast developing individuals may gain a fitness advantage, either
through its positive effect on survival under conditions of crowding
or through its putative demographic advantage for early repro-
duction in expanding populations (Stearns, 1992; Chippindale
et al., 1997). Likewise, a number of studies have shown that
ambient temperature determines DR in ectothermic animals, low
temperatures often lengthens development (Partridge et al., 1994;
Prasad et al., 2000; Gibert and De Jong, 2001; Pétavy et al., 2001;
Angiletta et al., 2002). In this context, if the timing of
developmental events is regulated by basic processes such as cell
division, the proportion of the life cycle spent in a particular
developmental stage should not change with temperature (Fig. 1).
The latter is known as the hypothesis of developmental isomorphy
and predicts proportional responses among temperatures of the
rate of development in different phases of an organism’ life cycle
(Gillooly et al., 2002; Jarosik et al., 2002, 2004). Furthermore, it has
been proposed that developmental isomorphy may constrain the
evolution of life history strategies in ectotherms and facilitate the
precise timing of life history events (Jarosik et al., 2004). In other
words, a particular stage cannot adapt to environmental tempera-

0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2010.06.015
[(Fig._1)TD$IG]
1680 G. Folguera et al. / Journal of Insect Physiology 56 (2010) 1679–1684
Fig. 1. Hypothetical growth trajectories under different thermal conditions during
development. Comparisons are between developmental stages using the slope of
the LDR/PDR ratio. LDR: larvae developmental rate; PDR: pupae developmental
rate.
ture without affecting thermal adaptation in other developmental
stages.
Most studies performed to date have compared among species
within large arthropod groups and demonstrated the prevalence of
rate isomorphy (Gillooly et al., 2002; Jarosik et al., 2002, 2004). In
contrast, there is no consensus about what to expect at the
intraspecific scale; considering that these general rules of variation
are not necessarily guaranteed within species. The aim of the first
part of our study is to unveil the relationship between DR and
temperature at the intraspecific scale both within and between
populations.
The relationships between metabolic rate (MR) and body mass
(mb), DR and mb (Rhees and Atchley, 2000) and generation time
and mb are well established (Millar and Zammuto, 1983, reviewed
in Ginzburg and Colyvan, 2004). Based on these allometries, we can
infer that generation time (and associated variables such as DR)
and MR should also be correlated. At first, MR would provide the
physiological basis for variation in DR, an issue that has not been
fully investigated (Folguera et al., 2007). Therefore, whether the
different responses of life cycle stages are actual evidence of
evolutionary selection strategies or if they are merely conse-
quences of changes in physiological condition during development
are not trivial questions (Stillwell and Fox, 2005). The main aim of
the second part of our study was to investigate the relationship
between DR and MR in flies exposed to different thermal
conditions during development.
In this paper we compare variation in DR among populations
living at contrasting altitudes (highland vs. lowland populations) in
an ecological specialist, the cactophilic Drosophila buzzatii, exposed
to thermal treatments during development. Two main questions
are addressed: (i) given that evidence is mostly concordant with
the predictions of the hypothesis of DR isomorphy in large groups
of insects (Jarosik et al., 2002), are proportion of total develop-
mental time spent in the larval and pupal stages constant in flies
raised under different thermal regimes (both between and within
populations)? and (ii) since DR and MR are correlated at higher
levels of organization. Does this allometric relationship also apply
at the intraspecific level along development?
2. Materials and methods
2.1. First experiment
2.1.1. Experimental populations
The first experiment was performed with flies derived from
natural populations of D. buzzatii sampled at 798 m.a.s.l. (268280 S,
658220 W) and 1855 m.a.s.l. (268270 S, 668020 W) in north-western
Argentina. These sampling localities, referred from hereafter as
lowland and highland populations, are separated by high
Preandean mountain ranges that preclude gene flow. Flies were
collected by net sweeping on fermented banana baits. Once in the
laboratory large numbers of isofemale lines were founded by
raising the progeny of wild inseminated females collected in all
sampling localities. All cultures were maintained at low density (to
avoid competition) for four generations at 25 8C and a photoperiod
of 12 h light:12 h dark. For the first experiment, six isofemale lines
per locality were randomly chosen from the pools of lines derived
from collections in both localities.
The procedure for the acquisition of experimental flies of each
isofemale line began with the release of 200 flies in egg collecting
chambers with Petri dishes containing an egg-laying medium (agar
2% + 5 ml of a 3:1 solution of ethanol:60% acetic acid). Twelve
hours later Petri dishes were removed and incubated until egg
hatching (36 h). Batches of 40 first instar larvae were transferred
from the dishes to vials containing Drosophila instant medium.
Previous studies have shown that these larval densities are nearly
optimal (Miller, 1964; Tantawy and Soliman, 1967; Barker and
Podger, 1970). Four vials (replicates) of each isofemale line (six of
each population) were randomly assigned to one out of three
thermal treatments: 17, 25 or 30 8C and incubated from the first
instar larval stage until adult emergence.
2.1.2. Life history and physiological traits
In the first study, we estimated DR by scoring two different
variables. First, we estimated larval developmental time (LDT) as
the time elapsed since first instar larvae were seeded in the vials
until pupation (defined as the moment in which third instar larvae
stop wandering and everted the anterior spiracles, referred from
hereafter as larval developmental time) by counting the number of
larvae reaching the third instar every 8 h. Then, we estimated the
weighted average of LDT in each vial. Statistical analysis we
performed using larval developmental rate (LDR) as the dependent
variable calculated as the inverse of developmental time value (1/
LDT).
The second variable scored was the duration of the pupal stage
(referred to as pupal developmental time [PDT]), as the time
elapsed from pupation to the emergence of the imago, by counting
the number of adult flies emerging from the puparium every 8 h.
The variable employed in statistical tests was pupal developmental
rate (PDR) which was calculated as the inverse of PDT (1/PDT). In
each vial we also measured the proportion of individuals that
reached the pupal stage relative to the number of first instar larvae
seeded in the vials, and the number of flies that reached the adult
stage relative to the number of individuals that entered the pupal
stage.
2.1.3. Data analysis
LDR and PDR datasets were analyzed by means of regression
analyses on temperature. In these analyses replicates were
considered as experimental units. We also used analyses of
covariance (ANCOVAs) to test whether rate of development in
highland and lowland population differs in slope or elevation and
whether regression slopes of LDR and PDR on temperature were
parallel. To the end to compare between slopes of regression we
considered multiple comparisons correction.
Since the progeny of wild inseminated females may be
composed of individuals sharing the same mother, a relatively
important fraction of the differences among isofemale lines
(families) may be considered to be of genetic origin. Thus, we
conducted a set of ANOVAs for each combination of population and
thermal treatment with line (random) as the unique source of
variation to estimate the relative contribution of the genetic
 G. Folguera et al. / Journal of Insect Physiology 56 (2010) 1679–1684 1681

component of variation to total phenotypic variance (David et al.,
2005). In these tests replicates were considered as the experimen-
tal units.
2.2. Second experiment
traits (mb–MR and DR–MR) across and within thermal treatments
for each stage. In addition, when necessary, post hoc comparisons
were performed using Tukey’s method. In all cases the datasets
satisfied the assumptions required for each test. For all tests we
used the statistical package Statistica for Windows (6.0).

2.2.1. Experimental populations

The second set of experiments was performed with flies 3. Results
collected in a second pair of lowland (720 m.a.s.l., 248400 S,
658020 W) and highland (2340 m.a.s.l., 258070 S, 668090 W) localities. 3.1. Test of the developmental rate isomorphy hypothesis at the
Several isofemale lines were maintained as mentioned above, until instraspecific scale
equal numbers of individuals of each isofemale line were pooled to
establish two stocks, one derived from the highland and the Differences among thermal treatments in the duration of the
lowland populations. larval stage were significant. Moreover, regression analyses
Six vials were prepared for each combination of population revealed a positive relationship between LDR and temperature
(highland and lowland) and thermal treatment. In three of these both in highland (b = 1  103, P < 0.0001) and lowland localities
treatments temperature was kept constant at 17, 21 or 25 8C, while (b = 9.6  104, P < 0.0001) (Fig. 2). A similar and significant trend
in the fourth temperature varied during the day, reaching a was observed for PDR in the highland (b = 3.1  103, P < 0.0001)
maximum of 25 8C during the period of light and a minimum of and lowland populations (b = 3.8  103, P < 0.0001) (Fig. 2). We
17 8C during the dark period. In all cases, temperatures were compare the slopes of the regressions of LDR and PDR on
chosen considering climatic data for the localities sampled temperature by means of an ANCOVA to test whether the
(Folguera et al., 2007, 2008) and the thermal range of the species regression slope of LDR on temperature is different from the
(Cohet et al., 1980; Imasheva et al., 1997; David et al., 1998). regression coefficient of PDR on temperature. The results of these
tests showed that differences between the slopes of LDR and PDR
2.2.2. Life history and physiological traits on temperature were highly significant in both populations
One individual was randomly chosen from each vial to measure (P < 0.00001), suggesting that temperature affected larvae and
mb and MR in larvae, pupae and adult flies. Concerning the larval pupae in different ways. In addition, ANCOVAs were performed to
stages, only third instar larvae were scored since measuring first compare slopes across populations for the same life cycle stage.
and second instar larvae was very difficult and subject to great These tests did not reveal significant differences in larvae nor
measuring error. As in the first experiment, an individual was [(Fig._2)TD$IG]
pupae after correction for multiple comparisons.
considered in the third larval stage when it stops wandering and in
the pupal stage when it has everted its anterior spiracles.
Measurements of adult flies were performed within 24 h after
emergence from the puparium. Once MR and mb were registered,
vials were discarded to avoid repeated measurements.
For the measurement of MR each individual was placed inside a
glass syringe that was sealed and placed in a temperature-
controlled incubator at 25 8C for 2 h (measurement interval).
Subsequently, MR was measured by means of CO2 production
using a ‘‘closed system’’ (Vleck, 1987) consisting of 2 ml glass
syringes fitted with three-way valves (Chappell, 1983; Ashby,
1997; Chown et al., 1997), see Ligthon (2008) for details. Three
blank syringes served as controls for each batch of measurements.
We injected the air from the syringes to a CO2 analyzer according to
the respirometric design (Fig. 4.8) suggested by Ligthon (2008). At
the end of the measurement interval, CO2 concentrations were
determined using a respirometry system (Sable Systems, Hender-
son, NV). A computer equipped with the program DATACAN
recorded the output of the CO2 analyzer (see Lardies et al., 2004;
Folguera et al., 2007 for methodological details).
We also estimated LDR and PDR in the vials though in a different
way than in the first experiment, due to differences in the
experimental designs. Four vials of each combination of popula-
tion  thermal treatment were used for the estimation of LDR and
four different vials of each combination of population  thermal
treatment for the measurement of PDR. We began to score each vial
for LDR and PDR when at least 20% of the individuals seeded in the
vials reached the pupal or the adult stages, respectively. LDR was
measured as the inverse of the time elapsed from the transfer of
first instar larvae to the vials until pupation and PDR as the inverse
of the time elapsed from pupation until adult emergence.

2.2.3. Data analysis

Fig. 2. Ontogenetic trajectories of larval (LDR) and pupal (PDR) developmental rate
The goal of the second experiment was to investigate static in Drosophila buzzatii exposed to three thermal treatments (17, 25 and 30 8C). LDR/
allometries between pairs of traits within each life cycle stage. To PDR is the ratio between larva development rate and pupa development rate.
this end, we estimated correlation coefficients between pairs of Different curves indicate each locality and stage.
1682 G. Folguera et al. / Journal of Insect Physiology 56 (2010) 1679–1684

Table 1
Percentage of total variance of larval (LDR) and pupal (PDR) developmental rate and the LDR/PDR ratio explained by differences among lines (see text for further explanation)
in flies derived from highland and lowland localities exposed to thermal treatments. NS: P > 0.05.

Stage Larvae Pupae Ratio larvae/pupae

Locality Lowland Highland Lowland Highland Lowland Highland

Temperature (8C)
30–30 55.7 NS NS NS NS NS
25–25 45.4 75.7 90.9 80.1 86.5 53.1
25–17 77.2 47.4 NS 42.3 NS NS
17–17 90.5 34.8 NS 30.2 75.3 NS

Table 2 relationship between temperature and developmental time) in

Correlation coefficients estimated between pairs of traits investigating allometric
relationships between metabolic rate (MR), body mass (mb) and developmental rate
(DR) in different stages of the life cycle.
Correlation analyses/stage Larvae Pupae
DR–mb 0.58** 0.09
MR–DR 0.43** 0.25
**
P < 0.01.
****
P < 0.0001.
Our main objective was to determine whether the duration of
the larval and pupal stages would be differentially affected by the
thermal regime to which flies were exposed during development in
highland and lowland populations. Further analysis showed that
the regression of the LDR/PDR ratio on temperature was negative
and significant in both populations (b = 6.8  102; P < 0.0001
and b = 6.5  102; P < 0.0001 in the lowland and highland
populations, respectively; see also Fig. 2). Moreover, an ANCOVA
revealed that the slopes of the regressions of the LDR/PDR on
temperature did not differ between populations (P > 0.05),
suggesting that thermal treatments affected in similar and
proportional ways the duration of the larval and pupal stages
across populations.
Results of the ANOVAs investigating the patterns of variation of
DR in each combination of population and thermal treatment are
summarized in Table 1. The most relevant features arising from
these ANOVAs are that differences among lines were highly
significant in most cases and that the proportion of total variance
explained by differences among lines (which according to our
experimental design may be considered as an estimate of the
genetic component of trait variation) varied largely among the
different combinations of population and treatments.
3.2. Correlation analyses
We also investigated the patterns of variation in allometric
relationships between pairs of traits (DR, mb and MR) measured in
the same stage of the life cycle (static allometries) for each
temperature  population combination by means of correlation
analysis. Overall, these tests showed that only one correlation
coefficient was significant out of 48 comparisons (not shown) and
that most have the same sign indicating similar trends. Thus, we
decided to perform correlation analyses after pooling across
sampling localities and thermal treatments. In general, patterns of
allometric relationships between traits varied among life cycle
stages (Table 2); all correlation coefficients between traits (MR–DR
and DR–mb) were negative and significant in the larval and adult
stages, whereas correlation coefficients were not significant in the
pupal stage (Table 2).
4. Discussion
A large number of investigations have established a positive
relationship between temperature and DR (and an inverse
insects and other ectotherms (e.g. Partridge et al., 1994; James
et al., 1995; James and Partridge, 1995). The growth and
development of insects is slower at lower than at higher
Adult
temperatures. Considering that one of the proximal causes of this
0.62**** relationship may be the duration of cell division (Van der Have and
0.43**** de Jong, 1996), it might be expected that the duration of all
developmental stages should be equally affected by temperature.
Following this line of reasoning, it has been suggested that the
relative duration of different stages of the life cycle should be
constant independently of ambient temperature. Indeed, there is
abundant evidence supporting the developmental rate isomorphy
hypothesis in insects, suggesting that, at the interspecific scale,
rate isomorphy may a general phenomenon, particularly, in short-
lived organisms (Jarosik et al., 2004).
Nevertheless, similar studies testing the hypothesis of isomor-
phic responses at the intraspecific level are both scarce and
necessary, since different individuals (and/or populations) may
express diverse responses (or strategies) in different stages of the
life cycle when confronted to alternative thermal environments.
Our simultaneous analysis of LDR, PDR and the LDR/PDR ratio
allowed us to acquire a deeper understanding of the factors
involved in the regulation of developmental rate in each stage. In
our analysis, we observed that LDR/PDR ratio present negative
slope suggesting non-isomorphic responses between stages of
development.
Concerning the comparison between highland and lowland
populations, although DR was lower in the former than in the
latter, the population effect was no significant for the LDR/PDR
ratio. In addition, LDR/PDR exhibited similar slopes across
populations. Indeed, the lack of significance of the locali-
ty  thermal treatment interaction for LDR/PDR suggests that
the responses to thermal treatments were independent of the
sampling locality, or in other words a feature that may be
considered as distinctive of the species. Considering that the
populations analyzed are separated by geographical barriers that
prevent gene flow, our present results may be considered as
supporting evidence of the developmental rate isomorphy
hypothesis at least at the interpopulation scale. As mentioned
above the most plausible explanation of the proportional
responses of larvae and pupae to thermal variation may be same
genetic source and a basic common process such as the duration of
cell division (Van der Have and de Jong, 1996). Interestingly, a
recent study of developmental time (the reverse of DR) in D.
melanogaster, revealed a complex genetic architecture that
involves a large number of candidate genes, most of them affecting
basic cellular metabolic processes (Mensch et al., 2008).
Another relevant point of our study is that the LDR/PDR ratio
varied across thermal treatments, suggesting that temperature had
specific effects on the duration of each life cycle stage. Thus, the
patterns of variation observed within populations are at variance
with those detected between populations, and, thus, in conflict
with the hypothesis of constant rate of development along the
entire life cycle in Drosophila.
 G. Folguera et al. / Journal of Insect Physiology 56 (2010) 1679–1684
DR is a fitness related traits that gains particular relevance in
organisms, as Drosophila, exploiting ephemeral resources. Thus,
genetic variation for this trait may be expected to be reduced as an
outcome of directional selection. However, differences among
lines, which under our experimental design may be construed as
an estimate of the relative contribution of genetic factors
underlying trait variance, accounted for a substantial proportion
of phenotypic variance. In addition, our results showed that
differences among lines for LDR accounted for a considerable larger
proportion of variance than for PDR. These results suggest that LDR
and PDR may have different underlying genetic architectures, and
that genetic variation for LDR can account for the largest
proportion of DR variation in Drosophila (Gibert and De Jong,
2001; Pétavy et al., 2001; Fanara et al., 2004; Folguera et al., 2007,
2008). Furthermore, these results are in agreement with a recent
survey of allelic variation in several candidate genes responsible
for DR variation in natural populations of D. melanogaster that
showed that most genes affected the duration of larval life rather
than the pupal stage (Mensch et al., 2010). Indeed, our results are
also coincident with other studies that proposed that responses to
thermal changes may vary along development, i.e. between life
cycle stages (McNamara and Houston, 1996; Rombough, 2003). In
this sense, we suggest that larvae and pupae may choose
alternative ‘‘decisions’’ when confronted to thermal variation.
These ‘‘decisions’’ are general ‘‘rules’’ that have a genetic basis
which specify how an actor should respond to an environmental
change. Interestingly, the ‘life history dependent on state’ model
may allow to identify and characterize different ‘‘mechanisms of
growth’’ (Gotthard et al., 2000; Gotthard, 2001). However, it is
important to note, regardless which hypotheses is correct, either
rate isomorphy or stage dependence, that the mechanisms
involved are still poorly understood (Peters, 1983; Van Voorhies,
1996; Angiletta and Dunham, 2003). In this sense, our study alerts
against generalizations of hypotheses across life cycle stages in
organisms with complex life histories as Diptera, and, may be taken
as a recommendation to avoid general and simplistic rules for traits
associated with fitness in holometabolous organisms (Sørensen
et al., 2005).
The second part of our study aimed to investigate allometric
relationships (static allometries) between developmental rate
(DR), metabolic rate (MR) and body mass (mb) in larvae, pupae and
adult flies. Our results revealed that allometries varied throughout
ontogeny, all variables were negatively and significantly correlated
in larvae, whereas only the expected correlation between MR and
mb remained significant in pupae. Overall, our results put forward
the hypothesis that each stage represents different physiological
environments that affect allometric relationships in a stage-
specific way. We also analyzed allometric relationships between
the same biological variables in each combination of population
and thermal treatment. These tests showed that correlations
between DR–MR and DR–mb were non-significant in 47 out of 48
comparisons (2 populations  2 allometric relationships  4 ther-
mal treatments  3 life cycle stages). There are two alternative
(non-mutually exclusive) explanations for these results. First, the
lack of significance of the correlations between pairs of traits may
be due to the small sampling size and, thus, the low statistical
power. Second, these results may suggest that the variables are
uncoupled. If the second alternative is true, general allometries
between DR–MR and DR–mb (shown in Table 2) may reflect,
mainly, plastic responses across thermal treatments.
Finally, our results illustrate that traits respond differentially to
thermal changes and that an organism response in terms of DR and
allometric relationships between physiological and life history
traits may depend on the particular internal state of the organism.
However, it is important to note that our work focused on a
holometabolous insect in which deep changes in morphology and
1683
physiology occur throughout the life cycle. Similar studies in
hemimetabolous insects would be helpful in solving such question.
Acknowledgments
This work was supported by grants of Universidad de Buenos
Aires, ANPCyT and CONICET awarded to EH and FONDAP 1501-
0001 (Program 1) to FB. GF acknowledges to Universidad de
Buenos Aires (predoctoral) and CASEB/FONDAP (post-doctoral)
fellowships. GF is grateful to José Rojas for statistic helpful and to
Alicia Folguera for graphic assistance. JM is postdoctoral fellow of
CONICET and EH is member of Carrera del Investigador Cientı́fico
(CONICET). AlmaCata provided stronger support.
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