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Keywords:

  • Acclimation;
  • chill coma;
  • experimental evolution;
  • storage pests;
  • stress;
  • Tribolium

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cold tolerance is an important trait directly related to survival and hence fitness. In the present study, the link is addressed between cold tolerance and body size, which is associated with many key fitness traits, at both the intra- and interspecific levels. Specifically, chill coma recovery time, as a metric of cold tolerance, is examined in five related flour beetle species (four of them belonging to the genus Tribolium), two additional Tribolium castaneum Herbst populations selected for higher temperatures, and a mutant showing reduced body size. Recovery times are negatively correlated with the species average body size but not within each species. Females usually recover faster than males, although this difference is significant in only a single species, and is unrelated to body size. Repeating the experimental procedure with the same individuals, after 2 days in isolation with a limited amount of food, results in longer recovery times. Therefore, even if cold acclimation takes place, its influence appears to be diminished by the deleterious effects associated with the experimental procedure. Hence, the findings provide evidence for an association between body size and cold tolerance in the genus Tribolium, with larger species recovering faster from chill than smaller species. By contrast, the smalleyed flour beetle Palorus ratzeburgii Wissmann does not follow this pattern. Additionally, a population of T. castaneum selected for the highest temperature takes longer to recover from chill coma, indicating a trade-off between cold and heat adaptations and not to a cross-protection effect, as sometimes demonstrated.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Many ectotherms experience fluctuating temperatures and need to cope with such changes even on a daily scale. Thus, the ability to cope well with unfavourable climatic conditions is important for reducing associated costs, such as the loss of cell membrane integrity in mild cold temperatures, and ice crystallization and shrinkage of cells in colder temperatures (Lee, 1989; Ramløv, 2000). This has led to the evolution of protective physiological mechanisms, such as heat-shock proteins or the accumulation of anti-freeze materials, such as glycerol (Clark & Worland, 2008; Teets & Denlinger, 2013). At the population level, knowledge of the individual thermal ranges is vital for understanding species distribution ranges (Kimura, 2004).

Chill coma recovery time is a common metric of cold tolerance in insects that correlates well with low temperature tolerance and survival (Gibert et al., 2001; MacMillan & Sinclair, 2011). It is defined as the time needed to regain muscle control after losing the righting response and the ability to stand and move (Hazell & Bale, 2011; MacMillan & Sinclair, 2011). Chill coma recovery is determined by a combination of internal and external factors and insects demonstrate high levels of plasticity as a consequence of ontogeny and external conditions (Bowler & Terblanche, 2008; Angilletta, 2009). For example, seasonal acclimation and rapid-cold hardening are two common ways for insects to improve their cold tolerance (Sinclair & Roberts, 2005; Teets & Denlinger, 2013).

Intra-specifically, larger insects generally survive longer under harsh conditions (Renault et al., 2003; Scharf et al., 2009; Modlmeier et al., 2013). This can be generalized inter-specifically to a positive (though moderating) association between body size and starvation or desiccation endurance (Terblanche et al., 2011). It is logical to expect a similar positive association between body size and chill coma recovery time because colder growth temperatures or habitats of origin select for both larger bodies and faster chill coma recovery times (Hallas et al., 2002). Nevertheless, the intra-specific association between body size and recovery time is complex and system-specific. In ants, for example, contradictory evidence comes from different study systems (Modlmeier et al., 2012). The effects of sex are also inconclusive, and perhaps depend on the sexual differences in body size and life cycle (David et al., 1998; Salin et al., 2000; Zeilstra & Fischer, 2005).

Comparative inter-specific studies regarding cold- or heat-tolerance across related species differing in a limited set of traits are rare. Notable exceptions are comparisons of cold tolerance of related Drosophila species from different climatic regions. Such studies show that flies originating from cooler regions resist low temperatures better than those from warmer regions (Gibert et al., 2001; Hallas et al., 2002; Kimura, 2004). Drosophila from colder regions are usually larger (Hallas et al., 2002; Gilchrist et al., 2004). However, heat tolerance is uncorrelated with the climatic region (Kimura, 2004) and Nyamukondiwa et al. (2011) detect no correlation in the laboratory between high and low thermal tolerances of Drosophila species.

Applying experimental evolution to test the coupling of coping with high and low temperatures leads to mixed results. Adaptations to a particular temperature selection regime sometimes come at the expense of performance at the other temperature (Partridge et al., 1994) but not necessarily (Anderson et al., 2005) and may even lead to better functioning in both cold and warm temperatures (Mori & Kimura, 2008). Because most cold tolerance studies focus on Drosophila, there is a need to expand such research to a greater diversity of model organisms (Maysov & Kipyatkov, 2009).

The present study investigates cold tolerance as expressed via chill coma recovery time in five different flour beetle species, which are very similar in their ecology and natural history but differ with respect to body size. Flour beetles of the family Tenebrionidae are cosmopolitan storage pests, which experience a wide range of temperatures (Campbell et al., 2010). The well-studied Tribolium castaneum Herbst (Red flour beetle) has become a major model system in different contexts (Michalczyk et al., 2011; Kerstes et al., 2013). Although other tenebrionid beetles are the subject of research, much less is known about them (Sokoloff, 1974).

In the present study, it is expected that larger species will recover faster from chill coma than smaller species. In addition, it is investigated whether selection for higher temperatures in T. castaneum interferes with its ability to cope with low temperatures. Both negative (i.e. a trade-off) and positive (i.e. a ‘cross-protection effect’) correlations are known between the ability to cope with warm and cold temperatures (Hallas et al., 2002; Sejerkilde et al., 2003). However, the design used in the present study does not separate between the genetic and plastic components of thermal responses because each population is only kept at the temperature it was selected for, without exposing populations to the opposite growth temperatures. It is clear that growth conditions (i.e. the plastic component) contribute to thermal tolerance and the latter does not depend only on long-term genetic adaptation (Colinet et al., 2012). Recovery from cold shock is measured again 2 days after the first trial. The experimental procedure (i.e. exposure to chill, isolation and maintenance in a well of a tissue culture plate for 2 days) may weaken the beetles leading to inferior performance later. Alternatively, recovery could be faster in the second trial, after some acclimation procedure induced by the first exposure to cold temperatures.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study species

Stocks of T. castaneum, Tribolium madens Charpentier, Tribolium brevicornis LeConte, Tribolium freeman Hinton and Palorus ratzeburgii Wissmann were initially supplied by Richard Beeman at USDA and housed continually on organic flour (with 10% brewer's yeast) in a climate chamber at constant temperature (30 °C as standard; Grazer & Martin, 2012) and humidity conditions (approximately 70 % relative humidity). Tribolium castaneum ‘Pygmy’ is a mutant of T. castaneum possessing a recessive or semi-dominant gene that is responsible for reduced body size (Brownlee & Sokoloff, 1988); it is referred to here as a separate T. castaneum population. Tribolium brevicornis is the largest species assessed, followed by T. freemani and T. madens (the former is slightly larger but generally similar in size), T. castaneum and then T. castaneum ‘Pygmy’ (Arnaud et al., 2005; present study). Palorus ratzeburgii is the smallest species in the present study. Two additional T. castaneum stocks had been kept either at 33 °C for several years, or selected at 35 °C for approximately 2 years. Adult flour beetles for the experiments described below were always kept at 30 °C, except individuals from the two T. castaneum elevated temperature stocks that were kept at either 33 or 35 °C during their whole life cycle.

Experimental procedures

Thirty to 40 random individuals were chosen from each species or population to be assessed, and placed individually in a well of a six-well tissue culture plate and covered with a lid (Sarstedt Inc., Newton, North Carolina). The bottom of the wells was lined with a filter paper disc to enable beetles to move without slipping. Six beetles at a time were chosen. They were tested in the six-well plate to determine how they recovered from chill by placing the plates with the beetles in a −20 °C freezer for exactly 5 min (Zeilstra & Fischer, 2005; Karl et al., 2008; Modlmeier et al., 2012). The temperature inside the freezer was relatively constant at −20 °C ± 1 °C. Five minutes of exposure time was chosen based on a preliminary experiment to ensure that beetles would enter a coma but would not die. Only two of 284 beetles assessed in the experiments died during chilling or after chilling as a result of cold injury. After 5 min, beetles were brought back to room temperature (20–21 °C) and two recovery times were observed: (i) the time to first movement (TFM) (almost always of one leg or antenna) and (ii) time to coordinated movement (TCM), with all six legs (usually when the beetle is still turned on its back). The more common ‘righting response’ was not used because the time it took the beetles to turn over varied much among species, in a way unrelated to cold tolerance but probably depending on beetle size, leg size, grasp of the filter paper to turn over and location in the well. Chilling and recovery procedures were similar to those described in Modlmeier et al. (2012).

After their recovery, beetles stayed in the cell culture plates and were kept at 30 °C for 2 days (except the two T. castaneum populations at 33 and 35 °C, which were maintained at their selection temperatures). The three largest species were kept in the same six-well plates, although the small ones were moved during these 2 days to 24-well plates. A small amount of flour was added to each well. On the third day, the chilling and recovery procedure was repeated to obtain the TCM and TFM. Between the first and third days, 15 beetles escaped (approximately 5% of the initial sample size). Those beetles were removed from all analyses. After the second assay, all beetles were killed by freezing at −20 °C. Subsequently, the sex of all beetles was determined, and the length of the elytra of each beetle was measured under a binocular microscope as a proxy of body size (as often performed in beetles and Tenebrionidae in particular; Krasnov et al., 1996).

Statistical analysis

Inter-specific analysis

The first analysis investigated whether the species would differ in their cold shock recovery behaviour. Because the TFM and TCM of the first and second recovery after chilling events were positively correlated, a principal component analysis (PCA) was performed on the four traits. Before the PCA, a Z-score transformation was applied to control for the variance of the variables and the difference in magnitude (TCM was naturally longer than TFM; Gotelli & Ellison, 2004). The PC loadings and eigenvalues are presented, as well as the percentage of the variance explained for all axes (Table 1). Reference is made only to PCs demonstrating eigenvalues > 1 (Gotelli & Ellison, 2004).

Table 1. Results of the principal component (PC) analysis of chill coma recovery traits for the inter-specific analysis including four Tribolium species, Tribolium castaneum ‘Pygmy’ as a separate population, and one Palorus species, as well as the intra-specific analysis of T. castaneum including three temperature selected populations
 Inter-specificTribolium castaneum
PC1PC1PC2
  1. TCM, time to coordinated movement; TFM, time to first movement. The last four lines represent the PC loadings of the recovery times.

λ2.6211.8531.004
Percentage of variance65.5546.3325.09
TFM, assay 10.44250.17700.9325
TCM, assay 10.53780.4988−0.3557
TFM, assay 20.50560.56620.0552
TCM, assay 20.50930.6319−0.0299

A linear model was performed with the relevant PCs as response variables and species/populations (six levels), sex and elytra length (EL) deviations (see below) as explanatory variables. EL deviations (i.e. the difference between the size of each individual and the mean species size divided by this mean) were used instead of ELs themselves because general size was confounded within species and the size relative to the species (or sample) average is of more interest. A saturated model was used, with all possible interactions and interactions gradually removed, followed by main effects based on low F-values, until a single explanatory variable remained. A model selection procedure (Akaike information criterion corrected for small sample size; AICc) was used to choose the model that best explained the current data (Johnson & Omland, 2004). There was interest also in the general effect of size, irrespective of species, on recovery time. Because the two latter factors were tightly correlated, PC1 on body size was regressed, without including species as a factor.

Next, it was of interest to determine the difference in recovery times between two successive assays, with 2 days in between. Two repeated-measures analysis of covariance (ancova) were used, with TFMt=1 day and TFMt=3 day as response variables in the first test and TCMt=1 day and TCMt=3 day in the second. Species, sex and EL deviations were used as the between-subject factors. Analysis started with a fully saturated mode, and interactions and main effects were gradually removed. The best model was chosen using a model selection procedure.

Intra-specific analysis of T. castaneum

The cold shock recovery times were studied of the T. castaneum populations selected for three temperatures in two ways. First, and similar to the inter-specific analysis, a PCA was performed on the four cold shock recovery traits (TFMt=1day, TCMt=1day, TFMt=3day and TFMt=3day), after applying a Z-score transformation (Table 1). A linear model was performed with the PCs having eigenvalues larger than one as response variables and populations (three levels), sex and EL deviations (see below) as explanatory variables. EL deviations were used because the populations differed in their body size. Next, it was of interest to examine the difference in recovery time between the two assays, similar to the inter-specific analysis. Two repeated-measures ancovas were used, with the two TFM and TCM as response variables. Species, sex and EL deviations were used as the between-subject factors. Analysis was started with a fully saturated model and nonsignificant interactions and main effects gradually removed (the best model was selected using AICc). The TFM and TCM usually deviated from normality and were log transformed before analysis (although these are still presented in the units measured to ease comparisons).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Inter-specific analysis

The PCA on chill coma recovery traits of the six species resulted in a single PC axis with eigenvalue larger than 1 (Table 1), whereas the second PC axis had an eigenvalue of 0.67 (explaining 16.64% of the variance) and was hence ignored. The first PC represented ‘general recovery time’ because it was composed of all four traits with similar loadings. General recovery time (PC1) differed according to species (F5,188 = 92.13, P < 0.0001), sex (females recovered faster than males; F1,188 = 8.48, P = 0.0040) and their interaction (F5,188 = 4.26, P = 0.0011; ΔAICc to the second best model including EL deviations: −2.23) (Fig. 1a). The fastest recovering species (T. brevicornis) recovered approximately two (TFM) to approximately  four (for TCM) times faster than the slowest one (T. castaneum ‘Pygmy’). Tribolium castaneum was the only species in which females recovered more slowly than males (although not significantly) and hence was responsible for driving the significant interaction. Tribolium freemani demonstrated the largest difference between PCs of males and females (28–32% longer TFM and TCM for males), whereas all the other species showed intermediate levels of differences. EL deviations and their interactions with either species or sex were all nonsignificant, and were not included in the best model. Although body size differences within species had no significant effect on recovery times, when excluding species, body size had a strong and negative effect on recovery times: larger species recovered faster (EL = −0.683 ± 0.107SE, t = −6.38, P < 0.0001, n = 200, R2 = 0.170). Palorus ratzeburgii, the only non-Tribolium species, was an exception (i.e. a small species with fast recovery time). Because this species belongs to a different genus, a comparative analysis was made in which it was excluded from the regression, whereupon much stronger results were obtained (EL = −1.300 ± 0.105SE, t = −12.38, P < 0.0001, n = 167, R2 = 0.481) (Fig. 1b).

image

Figure 1. (a) General recovery time from chill coma [principal component (PC)1 on the four recovery time measurements], presented separately for each sex of each of the six flour beetle species/populations. Species in an alphabetical order: brev, Tribolium brevicornis; cast, Tribolium castaneum; free, Tribolium freemani; made, Tribolium madens; pygm, T. castaneum ‘Pygmy’; ratz, Palorus ratzeburgii. F, females; M, males. Data are the mean ± SE. Small letters denote comparisons among species, and asterisks indicate a significant difference within the same species between the sexes. (b) The negative correlation between general recovery time (PC1) and elytra length (EL). All Tribolium species are included (excluding P. ratzeburgii).

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The repeated-measures anova on the TFM demonstrated a strong effect of assay, species and their two-way interaction (Table 2). All species apart from T. castaneum started moving more slowly after chill coma in the second assay (Fig. 2a) and the largest difference was found for T. freemani and T. brevicornis, increasing TFM by 43–44% in the second assay. Tribolium castaneum deviated by recovering approximately 8% faster during the second assay. The second best model included the Assay × EL deviations interaction (P = 0.36, ΔAICc to this second best model: −2.16; not shown). EL deviations, Sex and all their interactions were not significant and were not included in the best model. TCM showed a similar pattern (Fig. 2b and Table 2), although the Sex × Species interaction was significant (discussed for the PC analysis). Assay × Species was significant here similar to the TFM, although Assay × Sex was not. The effect of assay was weaker for TCM than TFM and, although most species recovered more slowly in the second assay, the difference diminished (Fig. 2).

Table 2. Results of the two repeated-measures analysis of variance (the inter-specific dataset) with time to first movement (TFM) and time to coordinated movement (TCM) as response variables
 Fd.f.P
  1. Significant results are marked in bold.

TFM   
Between subjects   
Species34.095194< 0.0001
Within subjects   
Assay18.451194< 0.0001
Assay × Species2.4651940.035
TCM   
Between subjects   
Species103.25188< 0.0001
Sex9.7611880.0021
Sex × Species4.4451880.0008
Within subjects   
Assay8.0211880.0051
Assay × Species4.3751880.0009
Assay × Sex0.9811880.32
Assay × Species × Sex0.8951880.49
image

Figure 2. A comparison between (a) the time to first movement (TFM) and (b) the time to correlated movement (TCM) after chilling for 5 min at −20 °C of the different flour beetle species tested on the first day and 2 days later (first and second assays). Species in an alphabetical order: brev, Tribolium brevicornis; cast, Tribolium castaneum; free, Tribolium freemani; made, Tribolium madens; pygm, T. castaneum ‘Pygmy’; ratz, Palorus ratzeburgii. Lowercase and uppercase letters denote comparisons among species for the first and second recovery times, respectively. Asterisks indicate a significant difference within the same species between the first and second recovery times. In most species, the second recovery time was longer, although this pattern was stronger for TFM. Data are the mean ± SE.

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Intra-specific analysis of T. castaneum

The PCA on chill coma recovery traits of the three temperature-selected lines of T. castaneum resulted in two PCs with eigenvalue larger than 1 (Table 1). The first PC represented a general increase in chill coma recovery time, whereas the second PC represented a trade-off between the TFM and the TCM. Large values on the second PC mean that it took relatively long to start moving and then the gap to coordinated movement was short. By contrast, small values mean that it took a short time to start moving, although it then took much longer to the coordinated movement. When testing for the effect of selection line, sex and body size deviations on the first PC, only selection line was significant (F2,95 = 11.01, P < 0.0001; ΔAICc to the second best model including Sex: −1.26). Specifically, beetles selected at 35 °C recovered more slowly than the two other populations (Fig. 3). EL deviations, Sex and the interactions of both were not significant, and were not included in the best model.

image

Figure 3. Differences among the three temperature selected populations in principal component (PC)1 (general recovery time from chill coma) and PC2 (the trade-off between the time to first movement and time to correlated movement; Table 1). Ca30, Ca33 and Ca35 stand for Tribolium castaneum populations selected through being maintained for 2+ years at 30, 33 and 35 °C, respectively.

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Similarly, the second PC differed only based on the selection line (F2,95 = 4.72, P = 0.0111; ΔAICc to the second best model including EL deviations: −2.06). Beetles selected for 35 °C showed a relatively long TFM and then a shorter TCM compared with beetles selected for 30 °C (Fig. 3). All other variables were not significant and were not included in the best model.

Regarding the difference in TFM between the first and second assays, none of the main effects or the interactions were significant (P > 0.092 for all), suggesting that response time was constant across assays and did not differ much among populations or sexes. The best model (where still nothing was significant) included only Populations (F2,95 = 2.09, P = 0.13) and the Assay × Population interaction (F2,95 = 2.00, P = 0.14; ΔAICc to the second best model including EL deviations: –2.18). However, TCM differed among the temperature selected populations (similar to the differences in the first PC described above; F2,95 = 16.08, P < 0.0001; AICc to the second best model including EL deviations: −2.17). The Assay × Population interaction was not significant (F2,95 = 1.91, P = 0.15). By contrast to the other Tribolium species, T. castaneum recovered faster from chill coma in the second assay (Assay: F1,95 = 34.58, P < 0.0001). Sex and EL deviations were not significant and were not included in the best model.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Flour beetles differ in their cold tolerance based on species body size (but not intra-specific size differences) and thermal selection regime. The two most important results of the present study are, first, an association between body size and cold tolerance in the genus Tribolium, with larger species recovering faster from chill than smaller species. Palorus ratzeburgii, belonging to another genus, does not follow this pattern. Second, T. castaneum populations selected for the highest temperature (35 °C) demonstrate a lower level of cold tolerance and slower recovery time from chill compared with populations selected for lower temperatures (30 °C). It is important to note that the relative contributions of longer-term genetic adaptation and growth conditions are confounded because beetles are continuously kept at their selection temperature. Future experiments should aim to disentangle genetic and plastic components more precisely.

Larger Tribolium species recover from chill coma faster than smaller ones. Providing a mechanistic explanation for this result is challenging. Some environments (e.g. colder regions) could select simultaneously for better cold tolerance and larger size to resist starvation, probably as a result of more body reserves (Renault et al., 2002). Desiccation resistance and cold tolerance are correlated (Renault et al., 2002) and desiccation resistance and body size are linked as well (Terblanche et al., 2011). Thus, body size and cold tolerance could be correlated through a third factor, as a consequence of selection to improve desiccation/starvation resistance.

Intra-specific body size differences, in addition to the inter-specific differences, do not explain cold tolerance. This was also the case in several other insect systems (Zeilstra & Fischer, 2005; Modlmeier et al., 2012) but not in others in which larger individuals cope better with cold temperatures (Renault et al., 2003; Kovacs & Goodisman, 2012). It is possible that the present study fails to detect such an effect as a result of the stronger inter-specific and inter-sexual effects, although this probably implies that an effect, if it truly exists, is quite small. Also, it is possible that more accurate equipment is needed to detect these differences (such as a more precise difference between the temperature of the freezer and the testing room) or that chilling at −20 °C for short periods, as in the present study, results in fast recovery times, making it more difficult to detect existing (though small) differences. For future studies, it is recommended to use more than one chilling method to reach a better understanding of recovery from chill coma. For example, beetles could be placed on ice for longer periods (e.g. for 2 h) leading to extended recovery times compared with those of the present study.

Females tend to recover faster than males but are not necessarily larger. This trend is especially strong for T. freemani. Sexual differences are clearer for TFM than TCM. Sexual differences in recovery times are species specific but do not depend on sexual size dimorphism (no link is detected between the degree of sexual size dimorphism and sexual differences in recovery times among species; not shown). Sexual differences in cold tolerance are only sporadically reported, with mixed evidence (Renault et al., 2002; no differences: Zeilstra & Fischer, 2005; female-biased: David et al., 1998; male-biased: Edwards, 1958).

When comparing cold tolerance between the first and second assays, performance mostly deteriorates during the second assay, especially when considering the TFM. Rapid-cold hardening is attenuated after returning to favourable temperatures (Coulson & Bale, 1990) and is thus unlikely to occur here after being returned to regular temperatures for 2 days. However, the three populations of T. castaneum recover faster in the second assay, suggesting that they go through some acclimation to cold. This could perhaps also be induced by some adaptive response to stressful conditions, which are specifically induced in this species but not in the others, and this is a subject worthy of further investigation. Beetles probably suffer from a high level of stress during this long interval between assays. The deterioration in performance could be the result of some cold injury during the first assay or the stress of isolation and a limited amount of food (beetles not being totally covered by flour such as they are used to). Both could result in increased activity leading to energy depletion.

Previous experimental evolution procedures selecting for cold or warm temperatures show mixed results regarding the ability of Drosophila melanogaster to cope with the opposite temperature (compare Partridge et al., 1994 and Mori & Kimura, 2008). In the present study, a ‘cross-protection effect’ is not observed but, instead, a trade-off is seen: T. castaneum populations selected for a higher temperature (35 °C) have a worse cold tolerance than the other populations. Nevertheless, it is possible that cold resistance in flour beetles derives from cross-protection to dry microhabitats (grain storages with low humidity). Indeed, the mechanisms involved in cold and desiccation resistance overlap (Lee, 1989; Clark & Worland, 2008). Unexpectedly, strong cold tolerance is demonstrated for tropical insects, although these never experience cold temperatures, and this is explained by possessing anti-desiccation mechanisms (Renault et al., 2002). Note that the experiments of the present study do not separate the consequences of genetic adaptation to high temperatures, those of one-generation phenotypic plasticity (experiencing high temperatures during growth) and maintenance conditions immediately before the experiment or between the two assays. The magnitude of effects is expected to follow the order of presentation (long-term evolution > phenotypic plasticity > acclimation). Testing this remains a target for future research, although it is already known in other insects that growth conditions can induce strong plasticity in various traits, such as body size, development time or thermal tolerance (flies: Scharf et al., 2010; Colinet et al., 2012).

Despite the apparently similar natural history of the flour beetle species of the present study, they probably differ in other factors beyond average body size. Arnaud et al. (2005) provide data concerning body size, development time, fecundity (number of eggs) and egg size for ten Tribolium species/populations (Tables 2 and 4 in Arnaud et al., 2005). It is clear that large body size is correlated with a long development time, low fecundity and large egg size, forming a syndrome of correlated life-history traits. Elaborating on such life-history differences could result in a better understanding of the consequences of body size differences among related species.

Other inter-specific comparisons are constrained by the lack of information available for these species. It would be interesting to compare the occurrence of these species in natural human-unrelated habitats. For example, P. ratzeburgii and T. brevicornis are observed to occur in dead wood under bark (Sokoloff, 1974), whereas other species, such as T. castaneum, are pests that have been associated with humans for much longer. To this end, it will be interesting to examine such inter-specific differences further, with the aim of better defining the shared and distinct traits of related species. There is confidence that much can be learnt from such inter-specific comparisons with respect to how both evolution in general and divergent evolution in particular takes place.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The research leading to this manuscript was funded by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. (333442) to IS. SHS and OYM were supported by the Swiss National Science Foundation (SNF Ambizione grants PZ00P3-121777 and PZ00P3-137514 to OYM). We thank the anonymous reviewers for their very helpful comments.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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