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Insects 2020, 11, 470; doi:10.3390/insects11080470 www.mdpi.com/journal/insects
Article
Effects of Temperature on Lifespan of Drosophila
melanogaster from Different Genetic Backgrounds:
Links between Metabolic Rate and Longevity
Mateusz Mołoń 1,*, Jan Dampc 2, Monika Kula-Maximenko 3, Jacek Zebrowski 4,
Agnieszka Mołoń 2, Ralph Dobler 5, Roma Durak 2 and Andrzej Skoczowski 6
1 Department of Biochemistry and Cell Biology, University of Rzeszow, 35-601 Rzeszów, Poland
2 Department of Experimental Biology and Chemistry, University of Rzeszów, 35-310 Rzeszów, Poland;
dampcjan@gmail.com (J.D.); agnieszkamaslowska@o2.pl (A.M.); rdurak@univ.rzeszow.pl (R.D.)
3 The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, 30-239 Krakow, Poland;
kula.monika@gmail.com
4 Department of Plant Physiology and Ecology, University of Rzeszow, 35-310 Rzeszów, Poland;
jaze28@interia.pl
5 Applied Zoology, Faculty of Biology, TU Dresden, 01062 Dresden, Germany; ralph.dobler@tu-dresden.de
6 Institute of Biology, Pedagogical University of Krakow, 30-084 Krakow, Poland;
andrzej.skoczowski@up.krakow.pl
* Correspondence: mateuszmolon@univ.rzeszow.pl
Received: 25 June 2020; Accepted: 24 July 2020; Published: 25 July 2020
Abstract: Despite many studies of the aging process, questions about key factors ensuring longevity
have not yet found clear answers. Temperature seems to be one of the most important factors
regulating lifespan. However, the genetic background may also play a key role in determining
longevity. The aim of this study was to investigate the relationship between the temperature, genetic
background (fruit fly origin), and metabolic rate on lifespan. Experiments were performed with the
use of the wild type Drosophila melanogaster fruit flies originating from Australia, Canada, and Benin
and the reference OregonR strain. The metabolic rate of D. melanogaster was measured at 20 °C , 25 °C ,
and 28 °C in an isothermal calorimeter. We found a strong negative relationship between the total
heat flow and longevity. A high metabolic rate leads to increased aging in males and females in all
strains. Furthermore, our results showed that temperature has a significant effect on fecundity and
body weight. We also showed the usefulness of the isothermal calorimetry method to study the
effect of environmental stress conditions on the metabolic activity of insects. This may be
particularly important for the forecasting of impact of global warming on metabolic activity and
lifespan of various insects.
Keywords: aging; fruit fly; lifespan; microcalorimetry
1. Introduction
Aging is defined as any age-specific decline in traits associated with individual fitness;
specifically, an increased mortality and lower reproduction and physiological performance [1] has
been viewed as a natural process [2,3]. Aging is also a universal process that has been identified in
organisms ranging from Prokaryotes to Eukaryotes [4]. To gain knowledge about basic biological
phenomena, such as senescence, studies on model organisms are used, ranging from single-cell yeast,
through the nematode and fruit fly, to the rat and mouse. In this study, we focused on the fruit fly
Drosophila melanogaster to investigate aging.
Insects 2020, 11, 470 2 of 18
The fruit fly is one of the most commonly used model organisms to study aging, as it has many
benefits (e.g., short lifespan, completely sequenced genome, simple strain maintenance, ease of
environmental and genetic manipulations to affect its lifespan, and powerful molecular techniques)
[4]. It has, therefore, a long tradition in aging research, initiated by Loeb and Northrop [5] in 1916. As
with other model organisms, many genetic and non-genetic factors may affect the lifespan of D.
melanogaster. Among genetic manipulations are many pathways associated with oxidative stress
defense [6,7], DNA repair [8], single gene expression [9], and Insulin/IGF-like signaling [10]. On the
other hand, ambient temperature, mild stress, and diet are often discussed as non-genetic factors
determining aging [11,12]. The reproductive status may also affect lifespan. Virgin Drosophila females
have their lifespans extended twice in comparison to mated females [13]. In turn, Partridge and
Farquhar [14] showed that male virgins also live longer which led to the conclusion that sexual
activity reduces the lifespan of male fruit flies. Dietary status may determine lifespan in two ways.
On the one hand, the length of life may be extended by dietary or caloric restriction; on the other
hand, it may be reduced by a fat-rich diet [14,15]. Also, many biologically active compounds (e.g.,
naringenin or curcumin) determine lifespan [15,16]. Temperature may also affect lifespan,
presumably by changing energy status [17]. It has been long speculated that it may influence aging
due to the more molecular damage being generated [18].
The longevity of animals depends on the metabolic rate according to what is known as the rate
of metabolism theory [19]. In this context, longevity is inversely proportional to the level of the basal
metabolic rate measured as oxygen consumption. This theory is connected with mammals [20] but is
also supported by studies on D. melanogaster [17] and yeast [21]. Fruit flies as poikilothermic
organisms live shorter lifespans at higher temperatures because their metabolic rate is higher [17].
Similarly, aphids have shorter lifespans at higher temperatures [22,23]. More recent data showed that
an increase in temperature to 28 °C increased metabolism in Aphis pomi five-fold, measured as heat
energy flow, which caused shortened aphid longevity [24]. The effect of temperature on insect
metabolism has been investigated in many species. Correlation between temperature and metabolic
rate was shown previously in studies on pupae of Platynota stultana [25,26], Musca domestica [27], and
Cydia pomonella [28]. Interestingly, the metabolic rate resulting from temperature changes may
depend on species or geographical coverage. A good example is the comparison of metabolic activity
of the Egyptian bee Apis mellifera lamarckii and the European Apis mellifera carnica. Both of these
populations show a different way of reacting to increases in the temperature. The European
population at high temperatures show slight changes in calorimetric measurement, while the African
population shows a reduction in the heat flow [29]. Similarly, in unicellular organisms more intense
biosynthesis and reproduction processes generate a higher metabolic rate, resulting in a reduction of
lifespan [21]. As shown by the previous data, temperature can modulate lifespan in unicellular
organisms without affecting their fertility [30]. Additionally, genetic background may play a key role
in determining lifespan [31,32]. It is yet unknown which of these two factors is more important in
determining longevity: that is environmental conditions, such as temperature, or genetic background.
Therefore, the aim of this study was to investigate the relationship between temperature (fruit fly
origin) and metabolic activity of the fruit fly measured as a quantity of the produced heat. We
measured the amount of the heat produced by flies using a new generation isothermal calorimeter
which allowed for performing measurement in a non-invasive manner. Here, we also asked whether
the fruit fly’s origins have an impact on aging.
2. Materials and Methods
2.1. Fly Stock and Rearing
Experiments were performed on the wild-type D. melanogaster fruit flies originating from
Australia, Canada, and Benin, and the OregonR strain (Bloomington Drosophila Stock Center).
The population from Australia had originally been sourced from Coffs Harbour and was
collected in 2010 [33]. The population from Benin, Africa, was originally collected in 1970, is routinely
Insects 2020, 11, 470 3 of 18
used for Drosophila genetics research, and is called the Dahomey population [34,35]. The population
from Dundas, Canada, was originally collected in 2005 [36].
Flies were kept in the laboratory in 100 mL bottles at 20 °C, 25 °C, and 28 °C; the photoperiod
was 12/12 h (day/night, respectively) with 60% relative humidity on standard fly medium consisting
of brewer’s yeast (80 g/L), sucrose (50 g/L), agar (15 g/L), and Nipagin (Sigma–Aldrich) (8 mL/L). To
generate the flies for the experiments, groups of 10 mated females were allowed to lay eggs in 100
mL rearing bottles during a restricted period of 6 h maximum under their common laboratory
conditions. No CO2 anesthesia was used, as it affects metabolic traits [37].
2.2. Measurement of Lifespan in Drosophila melanogaster
To measure the lifespan of the flies, virgin flies separated by sex were transferred into vials
containing a standard diet as described elsewhere [38]. Flies lived under standard conditions
(humidity and light/dark rhythm) at three different temperatures: 20 °C, 25 °C , and 28 °C. For each
sex and line, 10 replicate vials were set-up with 30 flies in each vial. Flies were transferred with CO2
anesthesia to vials with fresh food every two days, during which the number of dead flies was
recorded. Flies continued to be transferred until all flies were dead.
2.3. Measurement of Fly Fecundity
Virgin flies were kept at two males and two virgin females on a standard diet as described
elsewhere [11]. Flies were transferred to fresh medium every day, and the number of laid eggs was
counted every day for 15 days. For each temperature, 10 replicates were set-up for each fly strain.
2.4. Body Weight
Newly emerged flies were separated by sex and allowed to feed on standard medium for 15
days at each temperature. Flies were then subjected to mild etherisation followed by immediate
measurement of body weight in batches of 10 flies. For each temperature, 100 flies were measured for
each fly strain and sex.
2.5. Determination of the Heat Flow Using Isothermal Calorimetry
Metabolic activity of D. melanogaster was measured at 20 °C , 25 °C , and 28 °C in a TAM III
isothermal calorimeter equipped with TAM Assistant Software (TA Instruments, Lindon, UT). Five
newly emerged flies of each strain, male and female separately, were placed in 4.0 mL calorimetric
ampoules with 0.5 mL of agar with apple juice. After the 30 min needed for balancing of temperature,
the the heat flow ( curves) was recorded (in µ W) over 24 h. The specific heat flow–time plots (–t)
were obtained by normalization of the particular curves to the mass of the flies (µ W mg−1). The
metabolic activity of D. melanogaster was determined by integrating the heat flow curves over a time
interval of 24 h to give the total heat flow (TQ-metabolic activity) in joules per gram of body weight
of flies and per hour (J g−1 h−1). The data presented are the average values of 9 independent repetitions
for each object. The degree of impact of the origin of flies on thermal energy production in individual
common rearing temperatures was determined (separately for males and females) using the two-way
ANOVA (p ≤ 0.05).
2.6. Statistical Analysis
The survival analysis [39,40] was used to analyze longevity data. Kaplan–Meier curves with 95%
confidence intervals were calculated and plotted for non-censored data using the package survival in
R. The log-rank test (chi-square statistic) [41] was applied with the same weight to all events to assess
whether overall differences existed among the survival curves. The effects of temperature and the
place of origin were analyzed as described below. Because of violation of the normality and
homoscedasticity assumptions (Shapiro–Wilk’s and Bartlett tests, respectively) as well as due to the
presence of outliers, the statistical tests were performed with robust two-way ANOVA methods for
medians.
Insects 2020, 11, 470 4 of 18
The pbad2way (2000 bootstrap samples) and the med2way functions (R, package WRS2) [42]
were used to test the equality of medians across various groups (origin, temperature) as well as to
estimate the interactions between both factors. In the case of rejection of the null hypothesis of equal
medians, a robust post-hoc test (pairwiseRobustMatrix function, WRS2 package in R) was employed
for pairwise comparison of the median of the groups. Linear regression analysis (heat energy versus
lifespan) was accomplished with the panel.quantile function (latticeExtra in R). The data are
presented in figures mostly as box plots [43] with median value (horizontal line), 95% confidence
interval of the median (the notch), and the interquartile range containing 50% of data (colored areas).
All figures present raw data. The outliers are marked with different colors. A significance level of
0.05 was assumed for the statistical hypothesis testing and the pairwise group comparison. The
statistical computing and most of data presentation were performed using R programming.
3. Results
3.1. Temperature Effects on Longevity
To examine the impact of temperature on lifespan in different genetic backgrounds, we used fly
populations originated from Australia, Benin, and Canada and the widely used laboratory OregonR
strain. To investigate potential dependencies between the genetic background and temperature, we
used three different temperature conditions. We used the temperature of 25 °C as the laboratory
optimum for fruit fly life, 20 °C as a low temperature, and 28 °C as a heat stress. First, we analyzed
lifespan in different temperatures according to the standard procedure. We found a significant effect
of temperature on the maximum lifespan and mean lifespan (p < 0.05). In general, the high
temperature (28 °C) caused a decrease in lifespan for all strains and both sexes (Table S1).
Low temperature conditions generally caused an increase in longevity. Differences among
strains with different origins were most pronounced at 20 °C for both sexes (Figure 1a,b). At 20 °C,
the mean lifespan of Australian males was significantly higher compared to other male genotypes (p
< 0.05). The Canadian flies showed a slightly shorter mean lifespan, while the values for the Benin
and OregonR strains were comparatively low (Figure 2a) (p < 0.05). Among the female genotypes, the
Canadian and Australian flies had the longest lifespans, while the Benin and OregonR flies had the
shortest lifespans (p < 0.05). For males and females, the Australian and the Canadian flies had the
longest lifespans, while flies from Benin and OregonR had the shortest lifespans.
At the optimum rearing temperature of 25 °C, the shape of the survival curves among the fly
populations were more consistent than at 20 °C. It is likely that the temperature effect begins to be
more pronounced, while the effect of the genotype is less evident. At 25 °C, males from Australia and
Oregon had the longest lifespan, while we observed the shortest lifespan for the Benin and Canada
males (Figure 1c and Figure 2c) (p < 0.05). We observed the same pattern for females with the longest
lifespan for the Australian flies and the shortest lifespan for the Benin and OregonR flies (Figure 1d
and Figure 2d) (p < 0.05). At the highest temperature of 28 °C, in the male genotype, the shortest
lifespan was recorded for the OregonR strains, while the longest were for the strains originating from
Canada, Benin, and Australia (Figure 1e and Figure 2e) (p < 0.05). As for the males, we found that the
OregonR females had the shortest lifespan, and the Canadian females had the longest lifespan (p <
0.05). The Australian and Benin females lived longer than the OregonR females but shorter than the
Canadian females (Figure 1f and Figure 2f).
3.2. Temperature Effect on Fertility
Temperature largely determines lifespan regardless of the genotype. Therefore, we subsequently
examined whether the temperature would also affect other parameters of the fruit fly’s life such as
fertility. Our data show that temperature had a significant effect on fertility expressed as the number
of eggs laid within 24 h (p < 0.05) (Figure 3). In general, females laid more eggs at higher temperature.
At 20 °C, the Canadian females laid the highest number of eggs, while females with other genotypes
were at a statistically similar level of fertility (Figure 3a) (p < 0.05). At 25 °C and 20 °C, we observed
the highest fertility in the Canadian female’s genotype and the lowest in the Australian females
Insects 2020, 11, 470 5 of 18
(Figure 3a,b) (p < 0.05). At 28 °C, females from Australia had the highest fertility. Females from Benin
and Canada had significantly lower fertilities, while we observed the lowest fertility in the OregonR
females (Figure 3c) (p < 0.05).
Figure 1. Survival curves showing the origin and temperature modulation of longevity in Drosophila
melanogaster. Lifespans of adult male and female fruit flies fed a standard diet and grown at 20 °C
(a,b), 25 °C (c,d), and 28 °C (e,f). Filled colored ribbons show 95% confidence intervals. The log-rank
test (chi-squared statistics) was used to evaluate whether differences existed among overall curves
and to provide p-values. For each sex, 10 replicate vials were set up with 30 flies in each vial. Aus –
Australian population; Ben – Benin population; Can – Canadian population; Ore – OregonR
laboratory strain.
20 °C
25 °C
28 °C
20 °C
25 °C
28 °C
Log-rank p < 0.001
Log-rank p < 0.001
Log-rank p < 0.001
Log-rank p < 0.001
Log-rank p < 0.001
Log-rank p < 0.001
Aus
Ben
Aus
Aus
Aus
Aus
Aus
Ben
Ben
Ben
Ben
Ben
Can
Can
Can
Can
Can
Can
Ore
Ore
Ore
Ore
Ore
Ore
Insects 2020, 11, 470 6 of 18
Figure 2. Lifespans of adult male and female flies fed a standard diet and grown at 20 °C (a,b), 25 °C
(c,d), and 28 °C (e,f). The box plots (median, interquartile range, and 95% confidence interval) describe
variability of the strains in the order of increasing median values. Robust two-way ANOVA
(pbad2way function, R package WRS2) was followed by a robust post-hoc test (pairwise Robust
Matrix function, WRS2 package in R) for pairwise comparison of the median of groups. Statistical (p
< 0.05) differences among the groups of various origin (across all the temperature levels) are indicated
by different letters. The letters are alphabetically ordered according to the ascending median values.
The outliers in the plot are marked by the shaded squares. The measurements (10 replicate vials with
30 flies in each vial) and statistical analysis were performed separately for each sex. Aus – Australian
population; Ben – Benin population; Can – Canadian population; Ore – OregonR laboratory strain.
20 °C
20 °C
25 °C
25 °C
28 °C
28 °C
Insects 2020, 11, 470 7 of 18
Figure 3. Effect of origin of strain and temperature on fecundity. Number of eggs laid by a female on
a standard diet at 20 °C (a), 25 °C (b), and 28 °C (c). The box plots (median, interquartile range, and
95% confidence interval) describe the variability of the strains in the order of increasing median
values. Robust two-way ANOVA (pbad2way function, R package WRS2) was followed by a robust
post-hoc test (pairwise Robust Matrix function, WRS2 package in R) for pairwise comparison of the
median of groups. Statistical (p < 0.05) differences among the groups of various origin (across all the
temperature levels) are indicated by different letters. The letters are alphabetically ordered according
to the ascending median values. The outliers in the plot are marked by the shaded squares. The
measurements are based on 10 replicate vials with 30 flies in each vial. Aus – Australian population;
Ben – Benin population; Can – Canadian population; Ore – OregonR laboratory strain.
3.3. Temperature Effect on Body Weight
At 25 °C, the body weights of males and females were the highest (with exception of the
Canadian males and the OregonR and Canadian females) (p < 0.05). Generally, females were heavier
than males. The mean body weight of males did not exceed 1 mg, while all females (except for the
ones from Australia living at 20 °C ) had a body weight well above 1 mg (Figure 4). In the case of
females, the temperature had the highest impact on the Australia and Benin genotypes (Figure 4b,d,f).
20 °C
25 °C
28 °C
Insects 2020, 11, 470 8 of 18
Females from Benin had the highest body weight at all analyzed temperatures (p < 0.05). The lowest
temperature effect on body weight was observed for the OregonR strain females (p < 0.05). Males
originating from Australia were the lightest at 20 °C , and the Canadian were the heaviest (p < 0.05).
As seen in Figure 4b, males from Benin had the highest body weight, while the Canadian and
OregonR males were the lightest (p < 0.05). At 28 °C, the body weight of males was the lightest
compare with 20 °C and 25 °C (p < 0.05).
Figure 4. Effect of temperature on the body weight in male (A) and female (B) flies on a standard diet
at 20 °C, 25 °C, and 28 °C. The box plots (median, interquartile range, and 95% confidence interval)
describe variability of the strains in the order of increasing median values. Robust two-way ANOVA
(pbad2way function, R package WRS2) was followed by a robust post-hoc test (pairwise Robust
Matrix function, WRS2 package in R) for pairwise comparison of the median of groups. Statistical (p
< 0.05) differences among the groups of various origin (across all the temperature levels) are indicated
by different letters. The letters are alphabetically ordered according to the ascending median values.
The outliers in the plot are marked by the shaded squares. The measurements (10 replicate vials with
30 flies in each vial) and statistical analysis were performed separately for each sex.
20 °C
20 °C
25 °C
25 °C
28 °C
28 °C
Insects 2020, 11, 470 9 of 18
3.4. Calorimetric Results
Changes in the metabolic heat produced by flies (), measured in microwatts and depending on
the ambient temperature may be seen in Figure 1a–f. Analysis of the –t plots (µ W mg−1) showed a
significant effect of culture temperature on the metabolic heat flow of the flies. Both males and
females were characterized by higher
values at higher temperatures (Figure 5a–f). We therefore
observed the lowest
values in flies reared at 20 °C and the highest at 28 °C (Figure 5a,b,e,f). No
significant differences in heat flow were found between males and females within the same
temperature limits. However, the daily course of the –t plots was more varied for male genotypes
than for female ones.
For the Benin males, the
values at 20 °C were significantly higher than for males from all other
strains (Figure 5a) (p < 0.05). For the Benin females, we only found a higher thermal power emission
at the initial stages of the measurement (between 2 and 6 h). Moreover, we found an increase of the
value from hour 14 of the measurement for the Australia females’ strain (Figure 5b) (p < 0.05). At 25
°C, the highest
values were observed for males and females from Benin. However, for males the –
t curve fluctuated with the heat rate higher compared to other genotypes in the first 4 h and from
hour 11 to the end of the 24 h cycle (Figure 5c) (p < 0.05). For the Benin females, the heat flow gently
dropped until hour 11 of the cycle and was similar to the
values of the other genotypes from this
time point onward (Figure 5d) (p < 0.05). At 28 °C, we found the lowest values of heat flow for males
and females of the OregonR strain (Figure 6e,f). The low survivorship of the OregonR genotype at 28
°C was confirmed by the data presented in Figure 2e,f.
Mobility of the flies can be determined by the analysis of the –t curves [44]. Jagged time course
of the curves for the male flies (Figure 5a,c,e) is explained by the flying activity of the flies in the
ampoule during the measurement. On the other hand, the smoother course of the curves with less
amplitude of changes for females indicates movement of the flies around the whole ampoule with no
flying (Figure 5b,d,f). However, some increase in the activity and a tendency to fly during the first 12
h of the cycle can be observed for the female Benin genotype at 25 °C (Figure 5d).
The total heat flow (TQ) produced by males and females in all genotypes increased with
increasing temperature from approximately 1.0 (J mg−1 h−1) at 20 °C to approximately 2.1 (J mg−1 h−1)
at 28 °C (Figure 6a–f).
The largest TQ at 20 °C and 25 °C was recorded for the Benin males and females (Figure 6a–d).
At 28 °C, the OregonR males showed a significantly lower TQ (1.9 J mg−1 h−1), while the Australian
strain showed the highest TQ (Figure 6e) (p < 0.05). As seen in Figure 6e, the OregonR females showed
a significantly lower TQ (1.8 J mg−1 h−1), while for the other genotypes the TQ did not significantly
differ (p < 0.05). Generally, a higher rearing temperature increased the metabolic activity of D.
melanogaster (measured as the total heat flow—TQ) and, consequently, reduced survivorship of the
flies. Survivorship for male and female genotypes can be prolonged by lowering their metabolic
activity (elongation of straight line at 1.00 on the survivorship curves). A strong negative correlation
between heat rate and mean lifespan is evident, although the tested fruit flies strongly differed in
their geographic origin. Spearman’s rank rho correlation coefficient values were −0.819 for female
and −0.867 for male D. melanogaster (p < 0.01) (Figure 7).
Insects 2020, 11, 470 10 of 18
Figure 5. Specific thermal power: time curves representing the heat flow for male and female
Drosophila melanogaster flies of different origin of strain and at different growth temperatures.
20 °C
20 °C
25 °C
25 °C
28 °C
28 °C
Insects 2020, 11, 470 11 of 18
Figure 6. Total heat flow (TQ - metabolic activity in J∙mg−1∙h−1) for male and female flies of Drosophila
melanogaster for three different growth temperatures and various origin of strain. Robust two-way
ANOVA (pbad2way function, R package WRS2) was followed by a robust post-hoc test (pairwise
Robust Matrix function, WRS2 package in R) for pairwise comparison of the median of groups.
Statistical (p < 0.05) differences among the groups of various origin (across all the temperature levels)
are indicated by different letters. The letters are alphabetically ordered according to the ascending
median values. The outliers in the plot are marked by the shaded squares. The measurements (10
replicate vials with 30 flies in each vial) and statistical analysis were performed separately for each
sex.
Insects 2020, 11, 470 12 of 18
Figure 7. Scatter plots presenting the relationship between lifespan and total heat flow values (TQ -
metabolic activity) for females and males of the Drosophila melanogaster fruit fly. The straight lines
depict a linear trend in the data. The 95% confidence intervals are given as a shaded area. Spearman’s
rank rho correlation coefficients for medians and p-value are also given.
4. Discussion
Aging is a universal process that occurs in all living organisms from the simplest bacteria to
plants and animals [4]. It is interesting that despite many studies and much effort put into research
on that complex process, there is no unambiguous answer to the following question: What is the main
factor that lengthens or shortens the lifespan? So far, several important factors have been described
as those that may significantly affect lifespan, yet not all mechanisms are thoroughly understood.
Nevertheless, it is clear that the mechanism of aging may be modulated by environmental and genetic
factors [45,46].
In this work, we have shown that significant reduction in the rate of metabolism is one of the
universal mechanisms that can lead to longevity. One of the more vividly discussed factors
modulating the rate of metabolism is temperature. According to van’t Hoff’s rule, an increase in
temperature increases the rate of chemical reaction and the rate of chemical reaction increases
exponentially with temperature. It seems, therefore, that temperature is one of the most important
determinants of lifespan in poikilothermic organisms. Earlier works on the budding yeast as a model
organism led to similar conclusions: a) temperature determines lifespan; and b) the rate of
metabolism is the key to achieving longevity. First of all, temperature significantly modulates
longevity in these unicellular fungi [30]. Moreover, genetic manipulations, such as protein
biosynthesis disorders which lead to a reduction in the rate of metabolism, significantly extend the
lifespan of yeast [21]. Therefore, in this study, we investigated whether the metabolic rate is the most
important factor in determining the aging rate of the fruit fly originating from different continents.
For the purpose of the analysis, we used four fly strains originated from Australia, Africa
(Benin), North America (Canada), and the OregonR laboratory strain [33,36,47,48]. We assumed that
TQ / J mg-1 h-1
Lifespan/days
Spearman’s rank correlation
rho = −0.819 p < 0.01
rho = −0.867 p < 0.01
Female
Male
Insects 2020, 11, 470 13 of 18
in nature, these strains live in different optimum temperatures [49]. Therefore, it was interesting to
test their lifespans at different temperatures. Our lifespan analysis fully confirmed the earlier
extensive data on the effect of temperature [50,51]. In general, the fruit fly is known to exhibit many
genetics differences among geographic location. Additionally, there are some problems concerned
with laboratory storage of wild populations. As was shown previously, a laboratory line subjected to
a constant thermal regime for twelve years showed some thermal selection effects, e.g., higher size,
short lifespan [32]. Interestingly, the threshold of cultured wild populations in a laboratory is
unknown, which allowed for the use of wild populations without a thermal selection effect. In this
study, we used fruit flies cultured in a laboratory over many years, but the results did not show, for
example, that the Benin population reared fifty years ago had drastically impaired viability in higher
temperatures or fecundity in all analyzed temperature. Furthermore, the Australian population
reared ten years ago did not exhibit statistical significance in longevity with a temperature of 28 °C.
However, those long-term populations cultured in a laboratory, in the case Benin population,
influenced an increased body size which is in line with previous data [32].
More than a century ago, Loeb and Northrop [5] were the first to show that the fruit fly’s lifespan
correlates negatively with temperature. Subsequent data showed that D. melanogaster lives longer at
a low rather than high temperature [17,52]. These results were confirmed by Leiser et al. [50] who
concluded that temperature had a significant effect on lifespan. Interestingly, a recent study has
shown that inducing mild heat stress may also decrease the fruit fly’s aging rate by stimulating
pathways associated with genome stability during hormesis [53]. Hormesis in aging positively
supports life due to the cellular responses to single or multiple rounds of mild stress [54–57].
Temperature determines lifespan also in other invertebrates. Much attention was devoted to studies
involving the nematode Caenorhabditis elegans [51]. As highlighted by Keil et al. [58], there is no
invertebrate species in which longevity has been shown to increase with temperature. Indeed, many
studies in insects noted a negative correlation between temperature and longevity [22,59–61]. In turn,
use of some aphid species seems to contradict this general statement. The data show that a
temperature increase leads to lifespan extension, which suggests that optimum temperature and
genotype may be more important in the case of some species of insects [22,62]. Longevity effects
during temperature manipulation were also demonstrated when using poikilothermic vertebrates,
e.g., fish [63,64]. Munch and Salinas [65] highlighted the fact that there is a clear trend for longer
lifespans at lower temperatures in wild species of ectotherms. Among homoeothermic vertebrates,
the mouse was often examined [66]. In this study, we focused on D. melanogaster as an invertebrate
model organism; further information on other invertebrates as well as poikilothermic and
homoeothermic vertebrates has been extensively reviewed elsewhere [58]. Longevity resulting from
decreased temperature is well known in both endothermic and exothermic species, but the
mechanism underlying this change in aging is still poorly understood. In general, it is thought that
low temperature decreases the metabolic rate thus slowing the rate of damage of macromolecules
(nuclei acids, proteins, lipids) in cells caused by reactive oxygen species. Recently, studies have
suggested that the mechanism of lifespan extension through low temperature is an active genetic
process rather than a passive thermodynamic one and is dependent upon genotype. Additionally, a
probable mechanism of lifespan extension by low temperature may work via non- or partially
overlapping molecular pathways, e.g., by calorie restriction response [67]. Interestingly, in Drosophila
short-term exposures to low temperatures lead to long-term increase in longevity and stress
resistance, suggesting that it is not a short-term decrease in metabolism but rather a long-term
physiological adaptation that controls lifespan [68].
In 1908, Max Rubner [19] announced the “rate of living” theory which assumes that energy
consumption by different species of mammals during their lives based on the unit of volume is
similar despite differences in life expectancy. Therefore, some mammals use energy at a quicker pace
and live shorter, or they can consume it more economically which allows them to live for a longer
period. In 1928, Pearl [69] expanded the theory and hypothesized that longevity is inversely
proportional to the level of basic metabolism. The “rate of living” theory does not apply to mammals
only as originally assumed which was confirmed by research using other animal groups, for example,
Insects 2020, 11, 470 14 of 18
insects D. melanogaster [17]. Our previous data with the use of the budding yeast also confirmed that
the rate of metabolism is the major factor determining longevity [21].
Our results confirm the “rate of living” theory. To express the real rate of metabolism in specific
temperatures, we used a unique method of isothermal calorimetry. This method allowed us to track
the heat flow (metabolic heat production) over the time course of the experiment. Heat flow analyses
are an important tool to estimate parameters that affect the actual rate of metabolism. As we showed,
there is a very strong correlation between the total heat flow (TQ) and longevity. For males and
females, the high rate of metabolism (high TQ) determines faster aging regardless of the genotype,
and lower heat flow resulting from low level of metabolism leads to an increased lifespan. This
suggests that the metabolic rate is a crucial factor in determining longevity.
It should be mentioned that Pearl’s theory is not universally accepted. Speakman et al. [19] were
the first to provide an important proof that within a population of mice there is a positive correlation
between metabolic rate and longevity. These data are contrary to the theories of aging that inversely
link energy expenditure to aging. Speakman et al. [19] proposed an explanation for why a positive
correlation might exist, suggesting that the increased activation of uncoupling protein may decrease
production of reactive oxygen species and increase the rate of metabolism.
Attention was also focused on the influence of temperature on development. In general, a
negative correlation was noted between the developmental temperature and body size [70]. Previous
studies suggested that the decreased lifespan of flies reared at higher temperatures could be due to
their small body size; conversely, a large body weight correlates with an increased lifespan [52,71,72].
Interestingly, Zwaan et al. [52] suggest that wing size is a better measure of adult body size than body
weight, because larger variability was observed in the female body weight due to the egg production.
In turn, our data showed that there was no correlation between longevity and body weight. In our
analysis, the fruit fly achieved the largest body weight in the optimum temperature of 25 °C yet
rearing them at 20 °C significantly increased the lifespan. Previous studies also noted a lack of
positive correlation between lifespan and body size in various temperature groups [52] which
supports our results. It should be noted that Zwaan et al. [52] demonstrated that fruit fly growth at
25 °C resulted in a longer lifespan than at 20 °C. It seems that the general metabolic activity rather
than body weight generally determines longevity. Our results therefore contradict the earlier report
that suggests that larger fruit flies have a lower energy expenditure per weight unit than the smaller
ones [73]. Here, we demonstrated that larger fruit flies (growth at 25 °C) have higher TQ than the
smaller ones (growth at 20 °C). Also, previous studies show that the respiration rate [74] and activity
of antioxidant enzymes, such as superoxide dismutase and catalase [75], cannot explain the
significant reduction of lifespan during growth at higher temperatures.
To conclude, the investigations that we conducted using D. melanogaster of different origins
suggest that genomic effects (assumed by the different sites of origin of the populations) may explain
most of the observed effect variations—but not all. Although we observed some relationships
between life expectancy and temperature, a clear geographic pattern was not observed. As shown by
Trotta et al. [32], adult longevity was a trait for which there was no significant difference between
tropical and temperate flies living in the experimental thermal range of 11 to 32 °C. Importantly.
longevity in nature depends on season and place [76,77], and what is measured in the lab might be
unrelated to fitness in the wild. Fly strain storage may also be problematic in research, but as was
shown, adaptive responses were observed for the developmental rate, fertility, and body size but not
for longevity [32]. It is possible that our results were affected by the long period of laboratory culture
of our experimental populations, as it was previously emphasized [78]. Differences in lifespan were
most pronounced between the sexes, irrespective of temperature or origin. These differences sharply
decreased when flies were grown at higher temperatures. It is probable that both genome expression
and temperature have an impact on lifespan; however, at high temperatures the genotype effect is
not visible. It seems that a broad analysis of gene expression is necessary to understand the aging
mechanisms that lead to longevity. It also seems important for the future of the research to show
whether the loss of some wild traits in flies is the result of plasticity or accumulation of mutations
resulting from the increasing homozygosity of the strains, as is the case with many laboratory lines.
Insects 2020, 11, 470 15 of 18
Supplementary Materials: The following are available online at www.mdpi.com/2075-4450/11/8/470/s1, Table
S1: Lifespan of wild type Drosophila melanogaster flies of various origin cultured at different temperatures. Values
mean respectively: median, mean ± standard deviation, minimum and maximum values (M – Male; F – Female)
Author Contributions: Conceptualization, M.M.; methodology, M.M., J.D., M.K.; software, J.D., J.Z., M.K.;
validation, M.M., J.D., J.Z. ; formal analysis, M.M., M.K., A.S.; investigation, J.D., M.K., A.M.; resources, R.D.
(Ralph Dobler), M.M.; data curation, J.D., M.M., M.K., A.M., R.D. (Roma Durak); writing—original draft
preparation, M.M.; writing—review and editing, M.M., A.M., R.D. (Roma Durak), A.S.; visualization, J.Z., M.K.;
supervision, M.M.; project administration, M.M.; funding acquisition, M.M., R.D. (Ralph Dobler). All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported by the University of Rzeszow’s task grant to M.M. and financially
supported by the DFG Excellence Initiative (via TU Dresden Zukunftskonzept to R.D.).
Acknowledgments: We are grateful to Howard Rundle (University of Ottawa) for the Canadian flies and
Damian K. Dowling (Monash University) for the Australian and Benin flies.
Conflicts of Interest: The authors declare no conflict of interest
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