A Caenorhabditis elegans Wild Type Defies the Temperature–Size Rule Owing to a Single Nucleotide Polymorphism in tra-3

Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature–size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature–size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature–size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 × CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature–size rule, which has puzzled biologists for decades.     

The temperature-size rule in ectotherms: may a general explanation exist after all?

The majority of ectotherms mature at a larger size at lower rearing temperatures. Although this temperature-size rule is well established, a general explanation for this phenomenon has remained elusive. In this article, we address the problem by exploring the proximate and ultimate reasons for why a temperate grasshopper, Chorthippus brunneus, is an exception to the temperature-size rule. Using a complete set of life-history data to parameterize an established life-history model, we show that it is optimal for this species to mature at a larger size at higher temperatures. We also show that plasticity in adult size is determined by the relative difference between the minimum temperature thresholds for growth and development rates. The mechanism relates to aspects of the biophysical model of van der Have and de Jong. Ectotherms that obey the temperature-size rule are identified as having a higher temperature threshold for development rate than for growth rate; exceptions are identified as having a lower temperature threshold for development rate than for growth rate. The latter scenario may arise broadly in two ways. These are discussed in reference to the thermal biology of temperate grasshoppers and ectotherms in general.     

Effects of temperature on cell size and number in the yellow dung fly Scathophaga stercoraria

(1) A number of hypotheses suggest that the temperature-size rule (larger at cooler temperatures), and consequently Bergmann clines in whole-organism body size (larger at higher latitudes), may be a mere consequence of processes at the cellular level, i.e., a physiological constraint.(2) We show that in the yellow dung fly, Scathophaga stercoraria (Diptera: Scathophagidae), the temperature-size rule holds for wing cell and ommatidia size. Increases in cell number made up two-thirds (eye) to three-quarters (wing) of the increase in organ size. Temperature effects on body size can be fully explained by its effects on cell size and number.(3) Our study adds to the generality of previous results in Drosophila spp. The physiological constraint hypothesis remains viable as a proximate, non-adaptive explanation for the temperature-size rule in ectotherms.     

A genome-wide library of CB4856/N2 introgression lines of Caenorhabditis elegans

Recombinant inbred lines (RILs) derived from Caenorhabditis elegans wild-type N2 and CB4856 are increasingly being used for mapping genes underlying complex traits. To speed up mapping and gene discovery, introgression lines (ILs) offer a powerful tool for more efficient QTL identification. We constructed a library of 90 ILs, each carrying a single homozygous CB4856 genomic segment introgressed into the genetic background of N2. The ILs were genotyped by 123 single-nucleotide polymorphism (SNP) markers. The proportion of the CB4856 segments in most lines does not exceed 3%, and together the introgressions cover 96% of the CB4856 genome. The value of the IL library was demonstrated by identifying novel loci underlying natural variation in two ageing-related traits, i.e. lifespan and pharyngeal pumping rate. Bin mapping of lifespan resulted in six QTLs, which all have a lifespan-shortening effect on the CB4856 allele. We found five QTLs for the decrease in pumping rate, of which four colocated with QTLs found for average lifespan. This suggests pleiotropic or closely linked QTL associated with lifespan and pumping rate. Overall, the presented IL library provides a versatile resource toward easier and efficient fine mapping and functional analyses of loci and genes underlying complex traits in C. elegans.     

The temperature-size rule in ectotherms: simple evolutionary explanations may not be general

In many organisms, individuals in colder environments grow more slowly but are larger as adults. This widespread pattern is embodied by two well-established rules: Bergmann's rule, which describes the association between temperature and body size in natural environments, and the temperature-size rule, which describes reaction norms relating temperature to body size in laboratory experiments. Theory predicts that organisms should grow to be larger in colder environments when growth efficiency decreases with increasing environmental temperature. Using data from 97 laboratory experiments, including 58 species of ectotherms, we found little evidence that growth efficiency is negatively related to environmental temperature within the thermal range that is relevant to the temperature-size rule. Instead, growth efficiency was either positively related or insensitive to environmental temperature in the majority of cases (73 of 89 cases for gross growth efficiency and 18 of 24 cases for net growth efficiency). Two possibilities merit consideration. First, high temperatures may impose constraints on growth that only arise late during ontogeny; this simple and potentially general explanation is supported by the fact that thermal optima for growth efficiency and growth rate decrease as individuals grow. Alternatively, the general explanation for relationships between temperature and body size may not be simple. If the latter view is correct, the best approach might be to generate and test theories that are tailored specifically to organisms with similar behavior and physiology.     

Temperature, growth rate, and body size in ectotherms: fitting pieces of a life-history puzzle

The majority of ectotherms grow slower but mature at a larger body size in colder environments. This phenomenon has puzzled biologists because classic theories of life-history evolution predict smaller sizes at maturity in environments that retard growth. During the last decade, intensive theoretical and empirical research has generated some plausible explanations based on nonadaptive or adaptive plasticity. Nonadaptive plasticity of body size is hypothesized to result from thermal constraints on cellular growth that cause smaller cells at higher temperatures, but the generality of this theory is poorly supported. Adaptive plasticity is hypothesized to result from greater benefits or lesser costs of delayed maturation in colder environments. These theories seem to apply well to some species but not others. Thus, no single theory has been able to explain the generality of temperature-size relationships in ectotherms. We recommend a multivariate theory that focuses on the coevolution of thermal reaction norms for growth rate and size at maturity. Such a theory should incorporate functional constraints on thermal reaction norms, as well as the natural covariation between temperature and other environmental variables.     

Is the temperature-size rule mediated by oxygen in aquatic ectotherms?

Temperature is an important environmental factor that influences key traits like body size, growth rate and maturity. Ectotherms reared under high temperatures usually show faster growth, but reach a smaller final size, a phenomenon known as the temperature-size rule (TSR). Oxygen may become a limiting resource at high temperatures, when demand for oxygen is high, especially in water as oxygen uptake is far more challenging under water than in air. Therefore, in aquatic ectotherms, the TSR might very well be mediated by temperature effects on oxygen availability and oxygen demand. To distinguish between the direct effects of temperature and oxygen mediated effects, growth rate and final size were measured in the aquatic ectotherm Asellus aquaticus (Linnaeus, 1758) reared under different temperature and oxygen conditions in a factorial design. Growth could be best described by a modified Von Bertalanffy growth function. Both temperature and oxygen affected age at maturity and growth. Growth responses to temperature were dependent on oxygen conditions (interactive effect of temperature and oxygen). Only under hypoxic conditions, when oxygen was most limiting, did we find a classic TSR. Moreover, when comparing treatments differing in temperature, but where the balance between oxygen demand and supply was similar, high temperature increased both growth rate and final size. Thus effects of oxygen may resolve the life-history puzzle of the TSR in aquatic ectotherms.     

Reproductive allometry does not explain the temperature-size rule in water striders (Aquarius remigis)

Most ectotherms follow the temperature-size rule meaning that individuals growing up under cool conditions are larger as adults than those growing up in warm conditions. This pattern is difficult to explain because growth is usually slower in the cold meaning it takes longer to reach a larger adult size. One potential explanation for this pattern is that the typical increase in fecundity with body size is steeper in cool environments than it is in warm environments. As such, the relative gain in fecundity for being larger in the cold would compensate for the extra time it takes to grow to that size. We present the first empirical test of this model using the water strider Aquarius remigis. Individuals were reared at either 20° or 25°C with subsampling at each instar to estimate growth trajectories. At adult eclosion, half were switched to the alternate temperature and all females isolated, mated, and reproductive output measured for 3 weeks. We found that A. remigis does follow the temperature-size rule but that fecundity was highly plastic with respect to laying temperature such that the slightly greater fecundity of those reared at 20°C was due to their larger size. Overall, those laying at 25°C were more fecund and showed a positive relationship between body size and fecundity. Those laying at 20°C did not show a significant relationship between size and fecundity. As such, the reproductive allometry shows a pattern reverse to what would be needed for a larger size in the cold to be adaptive in this species. Although A. remigis follows the temperature-size rule over this temperature range, this is likely due to a constraint on growth and development rather than being adaptive plasticity.     

Breaking the temperature-size rule: Thermal effects on growth,development and fecundity of a crustacean from temporary waters

The temperature-size rule (TSR) is a well-established phenomenon to describe the growth response of ectotherms to temperature by which individuals maintained at low temperatures grow more slowly, but attain a larger size upon maturity. Although there are adaptive and non-adaptive theories about the plasticity of body size in response to temperature, these cannot be applied to all ectotherms, and little is known about the changes in growth and development rates through ontogeny. The ostracod species Heterocypris bosniaca, an inhabitant of freshwater temporary ponds, was used to examine the growth and development rates of its nine growth stages and female fecundity at four different temperatures (15 °C, 20 °C, 25 °C and 30 °C). The development rate of this species accelerates with increasing temperature, reaching a maximum value at 25 °C. The growth factor has a reverse-TSR in younger instars, and the typical TSR is followed only in the last two moults, resulting in non-monotonic response of adult size to temperature. Fecundity (total offspring per female) was not directly related to adult size and was generally higher at lower temperatures. Our results agree with recent research showing that the TSR may vary during ontogeny, and may not be a general trend in ostracod species from temporary waters. Indeed, adult carapace size seems to follow the pattern of a thermal reaction norm, probably influenced by the reduction of oxygen bioavailability at low temperature and the drastic increase in metabolic demand at the upper extreme of the thermal gradient.     

Environmental influence on the genetic correlations between life-history traits in Caenorhabditis elegans

Empirical evidence is mounting to suggesting that genetic correlations between life-history traits are environment specific. However, detailed knowledge about the loci underlying genetic correlations in different environments is scant. Here, we studied the influence of temperature (12 degrees C and 24 degrees C) on the genetic correlations between egg size, egg number and body mass in the nematode Caenorhabditis elegans. We used a quantitative trait loci (QTL) approach based on a genetic map with evenly spaced single nucleotide polymorphism markers in an N2 x CB4856 recombinant inbred panel. Significant genetic correlations between various traits were found at both temperatures. We detected pleiotropic or closely linked QTL, which supported the negative correlation between egg size and egg number at 12 degrees C, the positive correlation across temperatures for body mass, and the positive correlation between body mass and egg size at 12 degrees C. The results indicate that specific loci control the covariation in these life-history traits and the locus control is prone to environmental conditions.     

Does temperature and oxygen affect duration of intramarsupial development and juvenile growth in the terrestrial isopod Porcellio scaber (Crustacea,Malacostraca)?

According to the temperature-size rule (TSR), ectotherms developing under cold conditions experience slower growth as juveniles but reach a larger size at maturity. Whether temperature alone causes this phenomenon is unknown, but oxygen limitation can play a role in the temperature-size relationship. Oxygen may become limited under warm conditions when the resulting higher metabolism creates a greater demand for oxygen, especially in larger individuals. We examined the independent effects of oxygen concentration (10% and 22% O₂) and temperature (15 °C and 22 °C) on duration of ontogenic development, which takes place within the maternal brood pouch (marsupium), and juvenile growth in the terrestrial isopod common rough woodlouse (Porcellio scaber). Individuals inside the marsupium undergo the change from the aqueous to the gaseous environment. Under hypoxia, woodlice hatched from the marsupium sooner, but their subsequent growth was not affected by the level of oxygen. Marsupial development and juvenile growth were almost three times slower at low temperature, and marsupial development was longer in larger females but only in the cold treatment. These results show that temperature and oxygen are important ecological factors affecting developmental time and that the strength of the effect likely depends on the availability of oxygen in the environment.     

Achieving temperature-size changes in a unicellular organism

The temperature-size rule (TSR) is an intraspecific phenomenon describing the phenotypic plastic response of an organism size to the temperature: individuals reared at cooler temperatures mature to be larger adults than those reared at warmer temperatures. The TSR is ubiquitous, affecting >80% species including uni- and multicellular groups. How the TSR is established has received attention in multicellular organisms, but not in unicells. Further, conceptual models suggest the mechanism of size change to be different in these two groups. Here, we test these theories using the protist Cyclidium glaucoma. We measure cell sizes, along with population growth during temperature acclimation, to determine how and when the temperature-size changes are achieved. We show that mother and daughter sizes become temporarily decoupled from the ratio 2:1 during acclimation, but these return to their coupled state (where daughter cells are half the size of the mother cell) once acclimated. Thermal acclimation is rapid, being completed within approximately a single generation. Further, we examine the impact of increased temperatures on carrying capacity and total biomass, to investigate potential adaptive strategies of size change. We demonstrate no temperature effect on carrying capacity, but maximum supported biomass to decrease with increasing temperature.     

Can optimal resource allocation models explain why ectotherms grow larger in cold?

Basically all organisms can be classified as determinate growers if their growth stops or almost stops at maturation, or indeterminate growers if growth is still intense after maturation. Adult size for determinate growers is relatively well defined, whereas in indeterminate growers usually two measures are used: size at maturation and asymptotic size. The latter term is in fact not a direct measure but a parameter of a specific growth equation, most often Bertalanffy's growth curve. At a given food level, the growth rate in determinate growers depends under given food level on physiological constraints as well as on investments in repair and other mechanisms that improve future survival. The growth rate in indeterminate growers consists of two phases: juvenile and adult. The mechanisms determining the juvenile growth rate are similar to those in determinate growers, whereas allocation to reproduction (dependent on external mortality rate) seems to be the main factor limiting adult growth. Optimal resource allocation models can explain the temperature-size rule (stating that usually ectotherms grow slower in cold but attain larger size) if the exponents of functions describing the size-dependence of the resource acquisition and metabolic rates change with temperature or mortality increases with temperature. Emerging data support both assumptions. The results obtained with the aid of optimization models represent just a rule and not a law: it is possible to find the ranges of production parameters and mortality rates for which the temperature-size rule does not hold.     

Rapid population divergence in thermal reaction norms for an invading species: breaking the temperature-size rule

The temperature-size rule is a common pattern of phenotypic plasticity in which higher temperature during development results in a smaller adult body size (i.e. a thermal reaction norm with negative slope). Examples and exceptions to the rule are known in multiple groups of organisms, but rapid population differentiation in the temperature-size rule has not been explored. Here we examine the genetic and parental contributions to population differentiation in thermal reaction norms for size, development time and survival in the Cabbage White Butterfly Pieris rapae, for two geographical populations that have likely diverged within the past 150 years. We used split-sibship experiments with two temperature treatments (warm and cool) for P. rapae from Chapel Hill, NC, and from Seattle, WA. Mixed-effect model analyses demonstrate significant genetic differences between NC and WA populations for adult size and for thermal reaction norms for size. Mean adult mass was 12-24% greater in NC than in WA populations for both temperature treatments; mean size was unaffected or decreased with temperature (the temperature-size rule) for the WA population, but size increased with temperature for the NC population. Our study shows that the temperature-size rule and related thermal reaction norms can evolve rapidly within species in natural field conditions. Rapid evolutionary divergence argues against the existence of a simple, general mechanistic constraint as the underlying cause of the temperature-size rule.     

Empirical evidence for fast temperature-dependent body size evolution in rotifers

Organisms tend to decrease in size with increasing temperature by phenotypic plasticity (the temperature-size rule; ectotherms) and/or genetically (Bergmann’s rule; all organisms). In this study, the evolutionary response of body size to temperature was examined in the cyclically parthenogenetic rotifer Brachionus plicatilis. Our aim was to investigate whether this species, already known to decrease in size with increasing temperature by phenotypic plasticity, presents a similar pattern at the genetic level. We exposed a multiclonal mixture of B. plicatilis to experimental evolution at low and high temperature and monitored body size weekly. Within a month, we observed a smaller size at higher temperature, as compared to body size at lower temperature. The pattern was consistent for the size of both mature females and eggs; rotifers kept at high temperature evolved to be on average 14% (after 2 weeks) and 3% (after 3 weeks) smaller than the ones kept at low temperature (10 and 5% in the case of eggs, respectively). We therefore found that B. plicatilis is genetically programmed to adjust its body size-to-environmental temperature.     

Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans

Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20 °C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25 °C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856 > N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856 > N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear–mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.     

Rapid high resolution single nucleotide polymorphism-comparative genome hybridization mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ~200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.     

Postembryonic growth in two species of freshwater Ostracoda (Crustacea) shows a size-age sigmoid model fit and temperature effects on development time, but no clear temperature-size rule (TSR) pattern

Although the temperature-size rule is a widespread phenomenon that describes the impact of temperature on the intraspecific size of ectotherms, what determines the wide intrinsic variation in moult increment and the intermoult period within and between Crustacea species remains unknown. This work characterizes the growth of freshwater ostracods Chlamydotheca incisa and Strandesia bicuspis under different controlled temperatures and identifies growth patterns. Animals were collected from temporary ponds in Argentina. The experiment was done at two constant temperatures: 17 and 23 °C. The intermoult time and the time from hatching to the final moult were calculated. Three different growth regression equations were tested: von Bertalanffy and two sigmoidal models (Sigmoid and Gompertz). For both species, significant differences in the duration of each instar were found by comparing individuals grown at 17 and at 23 °C. A strong temperature effect was noted on intermoult time but not on growth factors. The best model selected for the size-age relationship was a sigmoid growth type, indicating accelerated growth in the earliest juvenile instars. These results challenge the widely-accepted application of nonsigmoidal growth models and are in agreement with recent analyses of growth patterns in aquatic invertebrates when early juvenile instars are considered.     

Effect of temperature on life-history traits and mating calls of a field cricket,Acanthogryllus asiaticus

Ectotherms are sensitive to changes in ambient temperature that impact their physiology and development. To compensate for the effects of variation in temperature, ectotherms exhibit short or long-term physiological plasticity. An extensive body of literature exists towards understanding these effects and the solutions ectotherms have evolved. However, to what extent rearing temperature during early life stages impacts the behaviour expressed in adulthood is less clearly understood. In the present study, we aimed to examine the effects of developmental temperature on life-history traits and mating call features in a tropical field cricket, Acanthogryllus asiaticus. We raised A. asiaticus at two different developmental conditions: 25 °C and 30 °C. We found developmental time and adult lifespan of individuals reared at 30 °C to be shorter than those reared at 25 °C. Increased developmental temperature influenced various body size parameters differentially. Males raised at 30 °C were found to be larger and heavier than those raised at 25 °C, making A. asiaticus an exception to the temperature-size rule. We found a significant effect of change in immediate ambient temperature on different call features of both field-caught and lab-bred individuals. Developmental temperature also affected mating call features wherein individuals raised at higher temperature produced faster calls with a higher peak frequency compared to those raised at lower temperature. In addition, an interactive effect of both developmental and immediate temperature was found on mating call features. Our study highlights the importance of understanding how environmental temperature shapes life-history and sexual communication in crickets.     

Australian Snakes Do Not Follow Bergmann’s Rule

Bergmann’s Rule (i.e., the tendency of body size to increase with decreasing environmental temperature) was originally explained by a mechanism that is unique to endotherms. Nevertheless, geographic variation of body size of ectotherms, including snakes, is increasingly studied, and some claim that the rule should apply to ectotherms, or to thermoregulating ectotherms. Such studies usually focus on assemblages or on species in a region, but mostly ignore species’ ecological and biological traits when seeking biogeographic patterns. We examined the relationship between environmental temperatures and body size of 146 Australian snake species. We examined this relationship while considering the effects of ecological traits (activity time and habitat use), climatic variables which are thought to influence snake body size, and shared ancestry. Our finding suggest that Bergmann’s Rule is not a valid generalization across species of Australian snakes. Furthermore, ecological traits greatly influence the relationship between snake body size and environmental temperature. Body size of fossorial species decreases with environmental temperature, whereas body size of nocturnal, surface active species increases. Body size of diurnal, surface active species is not related to environmental temperature. Our results indicate that lumping all species in a clade together is misleading, and that ecological traits profoundly affect the geographic variation of snake body size. Though environmental temperature generally does not exert a strong selective force on snake body size, this relationship differs for taxa exhibiting different ecological traits.