Residual force depression is not related to positive muscle fascicle work during submaximal voluntary dorsiflexion contractions in humans.

Residual force depression (rFD) following active muscle shortening is assumed to correlate most strongly with muscle work, but this has not been tested during voluntary contractions in humans. Using dynamometry, we compared steady-state ankle joint torques (N = 16) following tibialis anterior (TA) muscle-tendon unit (MTU) lengthening and shortening to the time-matched torque during submaximal voluntary fixed-end dorsiflexion reference contractions (REF) at a matched MTU length and EMG amplitude. Ultrasound revealed significantly reduced (P < 0.001) TA fascicle shortening amplitudes during MTU lengthening without a preload over small and medium amplitudes, respectively, relative to REF. MTU lengthening with a preload over a large amplitude significantly (P < 0.001) increased fascicle shortening relative to REF, as well as stretch amplitudes relative to MTU lengthening without a preload (P = 0.001). Significant (P = 0.028) steady-state fascicle force enhancement relative to REF was observed following MTU lengthening, and was similar among MTU lengthening-hold conditions (3-5%). MTU shortening with and without a preload over small and large amplitudes significantly (P < 0.001) increased positive fascicle and MTU work relative to REF, but significant (P = 0.006) rFD was observed following MTU shortening with a preload (7-10%) only. rFD was linearly related to positive MTU work [rrm (47) = 0.48, P < 0.001], but not positive fascicle work [rrm (47) = 0.16, P = 0.277]. Our findings indicate that MTU lengthening without substantial fascicle stretch enhances steady-state force output, which might arise from less shortening-induced rFD. Our findings also indicate similar rFD following different amounts of positive fascicle/MTU work, which cautions against using work to predict rFD during submaximal voluntary contractions. KEY POINTS: Accurately predicting muscle force is challenging because active muscle shortening depresses force output. The residual force depression (rFD) that exists following active muscle shortening is commonly assumed to correlate strongly and positively with muscle work. We found that tibialis anterior muscle fascicle work and muscle-tendon unit work did not accurately predict rFD during submaximal voluntary dorsiflexion contractions. Fascicle shortening during fixed-end reference contractions also potentially induced rFD of 3-5%, which was similar to the rFD following muscle-tendon unit shortening without a preload. A higher number of active muscle fibres during shortening probably increased rFD, which suggests that motor unit recruitment during shortening might predict rFD.

ROS and hypoxia signaling regulate periodic metabolic arousal during insect dormancy to coordinate glucose, amino acid, and lipid metabolism.

Metabolic suppression is a hallmark of animal dormancy that promotes overall energy savings. Some diapausing insects and some mammalian hibernators have regular cyclic patterns of substantial metabolic depression alternating with periodic arousal where metabolic rates increase dramatically. Previous studies, largely in mammalian hibernators, have shown that periodic arousal is driven by an increase in aerobic mitochondrial metabolism and that many molecules related to energy metabolism fluctuate predictably across periodic arousal cycles. However, it is still not clear how these rapid metabolic shifts are regulated. We first found that diapausing flesh fly pupae primarily use anaerobic glycolysis during metabolic depression but engage in aerobic respiration through the tricarboxylic acid cycle during periodic arousal. Diapausing pupae also clear anaerobic by-products and regenerate many metabolic intermediates depleted in metabolic depression during arousal, consistent with patterns in mammalian hibernators. We found that decreased levels of reactive oxygen species (ROS) induced metabolic arousal and elevated ROS extended the duration of metabolic depression. Our data suggest ROS regulates the timing of metabolic arousal by changing the activity of two critical metabolic enzymes, pyruvate dehydrogenase and carnitine palmitoyltransferase I by modulating the levels of hypoxia inducible transcription factor (HIF) and phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Our study shows that ROS signaling regulates periodic arousal in our insect diapasue system, suggesting the possible importance ROS for regulating other types of of metabolic cycles in dormancy as well.

Simulated climate warming causes asymmetric responses in insect life-history timing potentially disrupting a classic ecological speciation system.

Climate change may alter phenology within populations with cascading consequences for community interactions and on-going evolutionary processes. Here, we measured the response to climate warming in two sympatric, recently diverged (~170 years) populations of Rhagoletis pomonella flies specialized on different host fruits (hawthorn and apple) and their parasitoid wasp communities. We tested whether warmer temperatures affect dormancy regulation and its consequences for synchrony across trophic levels and temporal isolation between divergent populations. Under warmer temperatures, both fly populations developed earlier. However, warming significantly increased the proportion of maladaptive pre-winter development in apple, but not hawthorn, flies. Parasitoid phenology was less affected, potentially generating ecological asynchrony. Observed shifts in fly phenology under warming may decrease temporal isolation, potentially limiting on-going divergence. Our findings of complex sensitivity of life-history timing to changing temperatures predict that coming decades may see multifaceted ecological and evolutionary changes in temporal specialist communities.

Sequence and expression analysis of the spermatogenesis-specific gene cognates, wampa and Prosα6T, in Drosophila suzukii.

The sterile insect technique (SIT) is a highly effective biologically-based method for the population suppression of highly invasive insect pests of medical and agricultural importance. The efficacy of SIT could be significantly enhanced, however, by improved methods of male sterilization that avoid the fitness costs of irradiation. An alternative sterilization method is possible by gene-editing that targets genes essential for sperm maturation and motility, rendering them nonfunctional, similar to the CRISPR-Cas9 targeting of ß2-tubulin in the genetic model system, Drosophila melanogaster. However, since genetic strategies for sterility are susceptible to breakdown or resistance in mass-reared populations, alternative targets for sterility are important for redundancy or strain replacement. Here we have identified and characterized the sequence and transcriptional expression of two genes in a Florida strain of Drosophila suzukii, that are cognates of the D. melanogaster spermatocyte-specific genes wampa and Prosalpha6T. Wampa encodes a coiled-coil dynein subunit required for axonemal assembly, and the proteasome subunit gene, Prosalpha6T, is required for spermatid individualization and nuclear maturation. The reading frames of these genes differed from their NCBI database entries derived from a D. suzukii California strain by 44 and 8 nucleotide substitutions/polymorphisms, respectively, though all substitutions were synonymous resulting in identical peptide sequences. Expression of both genes is predominant in the male testis, and they share similar transcriptional profiles in adult males with ß2-tubulin. Their amino acid sequences are highly conserved in dipteran species, including pest species subject to SIT control, supporting their potential use in targeted male sterilization strategies.

Divergence in cell cycle progression is associated with shifted phenology in a multivoltine moth: the European corn borer, Ostrinia nubilalis.

Evolutionary change in diapause timing can be an adaptive response to changing seasonality, and even result in ecological speciation. However, the molecular and cellular mechanisms regulating shifts in diapause timing remain poorly understood. One of the hallmarks of diapause is a massive slowdown in the cell cycle of target organs such as the brain and primordial imaginal structures, and resumption of cell cycle proliferation is an indication of diapause termination and resumption of development. Characterizing cell cycle parameters between lineages differing in diapause life history timing may help identify molecular mechanisms associated with alterations of diapause timing. We tested the extent to which progression of the cell cycle differs across diapause between two genetically distinct European corn borer strains that differ in their seasonal diapause timing. We show the cell cycle slows down during larval diapause with a significant decrease in the proportion of cells in S phase. Brain-subesophageal complex cells slow primarily in G0/G1 phase whereas most wing disc cells are in G2 phase. Diapausing larvae of the earlier emerging, bivoltine E-strain (BE) suppressed cell cycle progression less than the later emerging, univoltine Z-strain (UZ) individuals, with a greater proportion of cells in S phase across both tissues during diapause. Additionally, resumption of cell cycle proliferation occurred earlier in the BE strain than in the UZ strain after exposure to diapause-terminating conditions. We propose that regulation of cell cycle progression rates ultimately drives differences in larval diapause termination, and adult emergence timing, between early- and late-emerging European corn borer strains.

Genome-wide variation and transcriptional changes in diverse developmental processes underlie the rapid evolution of seasonal adaptation.

Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. We tested the hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster Our transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drove more rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, our data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella We further discuss patterns that suggest hourglass-like developmental divergence early and late in diapause development and an important role for hub genes in the evolution of transcriptional divergence.

Genomically correlated trait combinations and antagonistic selection contributing to counterintuitive genetic patterns of adaptive diapause divergence in Rhagoletis flies.

Adaptation to novel environments can result in unanticipated genomic responses to selection. Here, we illustrate how multifarious, correlational selection helps explain a counterintuitive pattern of genetic divergence between the recently derived apple- and ancestral hawthorn-infesting host races of Rhagoletis pomonella (Diptera: Tephritidae). The apple host race terminates diapause and emerges as adults earlier in the season than the hawthorn host race, to coincide with the earlier fruiting phenology of their apple hosts. However, alleles at many loci associated with later emergence paradoxically occur at higher frequencies in sympatric populations of the apple compared to the hawthorn race. We present genomic evidence that historical selection over geographically varying environmental gradients across North America generated genetic correlations between two life history traits, diapause intensity and diapause termination, in the hawthorn host race. Moreover, the loci associated with these life history traits are concentrated in genomic regions in high linkage disequilibrium (LD). These genetic correlations are antagonistic to contemporary selection on local apple host race populations that favours increased initial diapause depth and earlier, not later, diapause termination. Thus, the paradox of apple flies appears due, in part, to pleiotropy or linkage of alleles associated with later adult emergence and increased initial diapause intensity, the latter trait strongly selected for by the earlier phenology of apples. Our results demonstrate how understanding of multivariate trait combinations and the correlative nature of selective forces acting on them can improve predictions concerning adaptive evolution and help explain seemingly counterintuitive patterns of genetic diversity in nature.

Protein storage and reproduction increase in grasshoppers on a diet matched to the amino acids of egg yolk protein.

The diets of animals are essential to support development, and protein is key. Accumulation of stored nutrients can support developmental events such as molting and initiation of reproduction. Agricultural studies have addressed how dietary protein quality affects growth, but few studies have addressed the effects of dietary protein quality on developmental transitions. Studies on how dietary quality may affect protein storage and development are possible in arthropods, which store proteins in the hemolymph. We hypothesized that diets with a composition of amino acids that matches the precursor of egg yolk protein (vitellogenin, Vg) will be high quality and support both egg production and accumulation of storage proteins. Grasshoppers were fed one of two isonitrogenous solutions of amino acids daily: Vg-balanced (matched to Vg) or Unbalanced (same total moles of amino acids, but not matched to egg yolk). We measured reproduction and storage protein levels in serial hemolymph samples from individuals. The Vg-balanced group had greater reproduction and greater cumulative levels of storage proteins than did the Unbalanced group. This occurred even though amino acids fed to the Vg-balanced group were not a better match to storage protein than were the amino acids fed to the Unbalanced group. Further, oviposition timing was best explained by a combination of diet, age at the maximum level of storage protein hexamerin-270 and accumulation of hexamerin-90. Our study tightens the link between storage proteins and commitment to reproduction, and shows that dietary protein quality is vital for protein storage and reproduction.

Induced dormancy in Indian meal moth Plodia interpunctella (Hübner) and its impact on the quality improvement for mass rearing in parasitoid Habrobracon hebetor (Say).

A steady supply of hosts at the susceptible stage for parasitism is a major component of mass rearing parasitoids for biological control programs. Here we describe the effects of storing 5th instar Plodia interpunctella larvae in dormancy on subsequent host development in the context of host colony maintenance and effects of the duration of host dormancy on the development of Habrobracon hebetor parasitoids reared from dormant hosts. We induced dormancy with a combination of short daylength (12L:12D) and lower temperature (15°C), conditions known to induce diapause in this species, and held 5th instar larvae of P. interpunctella for a series of dormancy durations ranging from 15 to 105 days. Extended storage of dormant 5th instar larvae had no significant impacts on survival, development, or reproductive potential of P. interpunctella, reinforcing that dormant hosts have a substantial shelf life. This ability to store hosts in dormancy for more than 3 months at a time without strong negative consequences reinforces the promise of using dormancy to maintain host colonies. The proportion of hosts parasitized by H. hebetor did not vary significantly between non-dormant host larvae and dormant host larvae stored for periods as long as 105 days. Concordant with a prior study, H. hebetor adult progeny production from dormant host larvae was higher than the number of progeny produced on non-dormant host larvae. There were no differences in size, sex ratio, or reproductive output of parasitoids reared on dormant hosts compared to non-dormant hosts stored for up to 105 days. Larval development times of H. hebetor were however longer when reared on dormant hosts compared to non-dormant hosts. Our results agree with other studies showing using dormant hosts can improve parasitoid mass rearing, and we show benefits for parasitoid rearing even after 3 months of host dormancy.

Rightward shift of optimal fascicle length with decreasing voluntary activity level in the soleus and lateral gastrocnemius muscles.

Much of our understanding of in vivo skeletal muscle properties is based on studies performed under maximal activation, which is problematic because muscles are rarely activated maximally during movements such as walking. Currently, force-length properties of the human triceps surae at submaximal voluntary muscle activity levels are not characterized. We therefore evaluated plantar flexor torque- and force-ankle angle, and torque- and force-fascicle length properties of the soleus and lateral gastrocnemius muscles during voluntary contractions at three activity levels: 100, 30 and 22% of maximal voluntary contraction. Soleus activity levels were controlled by participants via real-time electromyography feedback and contractions were performed at ankle angles ranging from 10 deg plantar flexion to 35 deg dorsiflexion. Using dynamometry and ultrasound imaging, torque-fascicle length curves of the soleus and lateral gastrocnemius muscles were constructed. The results indicate that small muscle activity reductions shift the torque- and force-angle, and torque- and force-fascicle length curves of these muscles to more dorsiflexed ankle angles and longer fascicle lengths (from 3 to 20% optimal fascicle length, depending on ankle angle). The shift in the torque- and force-fascicle length curves during submaximal voluntary contraction have potential implications for human locomotion (e.g. walking) as the operating range of fascicles shifts to the ascending limb, where muscle force capacity is reduced by at least 15%. These data demonstrate the need to match activity levels during construction of the torque- and force-fascicle length curves to activity levels achieved during movement to better characterize the lengths that muscles operate at relative to their optimum during a specific task.

A Practical and Efficient Synthesis of Uniform Conjugated Rod-Like Oligomers.

Herein, a more practical and efficient synthesis protocol for the preparation of uniform rod-like oligo(1,4-phenylene ethynylene)s (OPE)s is presented. Applying an iterative reaction cycle consisting of a decarboxylative coupling reaction and a saponification of an alkynyl carboxylic ester, a uniform pentamer is obtained in ten steps with 14% overall yield. The copper-free conditions prevent homocoupling until the trimer stage, resulting in a significantly easier work-up of the products. Homocoupling is observed from the tetramer stage on, but a simple variation of the work-up procedure also yields the uniform tetramer and pentamer. A thorough comparison with the commonly used and described Sonogashira approach reveals that with the new presented strategy, OPEs can be built in similar overall yield, but easier purification and in a quarter of the time. All oligomers are fully characterized by proton and carbon nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS), size-exclusion chromatography (SEC), and infrared spectroscopy (IR).

Long-chain acyl-CoA synthetase-1 mediates the palmitic acid-induced inflammatory response in human aortic endothelial cells.

Saturated fatty acid (SFA) induces proinflammatory response through a Toll-like receptor (TLR)-mediated mechanism, which is associated with cardiometabolic diseases such as obesity, insulin resistance, and endothelial dysfunction. Consistent with this notion, TLR2 or TLR4 knockout mice are protected from obesity-induced proinflammatory response and endothelial dysfunction. Although SFA causes endothelial dysfunction through TLR-mediated signaling pathways, the mechanisms underlying SFA-stimulated inflammatory response are not completely understood. To understand the proinflammatory response in vascular endothelial cells in high-lipid conditions, we compared the proinflammatory responses stimulated by palmitic acid (PA) and other canonical TLR agonists [lipopolysaccharide (LPS), Pam3-Cys-Ser-Lys4 (Pam3CSK4), or macrophage-activating lipopeptide-2)] in human aortic endothelial cells. The expression profiles of E-selectin and the signal transduction pathways stimulated by PA were distinct from those stimulated by canonical TLR agonists. Inhibition of long-chain acyl-CoA synthetases (ACSL) by a pharmacological inhibitor or knockdown of ACSL1 blunted the PA-stimulated, but not the LPS- or Pam3CSK4-stimulated proinflammatory responses. Furthermore, triacsin C restored the insulin-stimulated vasodilation, which was impaired by PA. From the results, we concluded that PA stimulates the proinflammatory response in the vascular endothelium through an ACSL1-mediated mechanism, which is distinct from LPS- or Pam3CSK4-stimulated responses. The results suggest that endothelial dysfunction caused by PA may require to undergo intracellular metabolism. This expands the understanding of the mechanisms by which TLRs mediate inflammatory responses in endothelial dysfunction and cardiovascular disease.

A rapidly evolved shift in life-history timing during ecological speciation is driven by the transition between developmental phases.

For insect species in temperate environments, seasonal timing is often governed by the regulation of diapause, a complex developmental programme that allows insects to weather unfavourable conditions and synchronize their life cycles with available resources. Diapause development consists of a series of distinct phases including initiation, maintenance, termination and post-diapause development. The evolution of insect seasonal timing depends in part on how these phases of diapause development and post-diapause development interact to affect variation in phenology. Here, we dissect the physiological basis of a recently evolved phenological shift in Rhagoletis pomonella (Diptera: Tephritidae), a model system for ecological divergence. A recently derived population of R. pomonella shifted from specializing on native hawthorn fruit to earlier fruiting introduced apples, resulting in a 3-4 week shift in adult emergence timing. We tracked metabolic rates of individual flies across post-winter development to test which phases of development may act either independently or in combination to contribute to this recently evolved divergence in timing. Apple and hawthorn flies differed in a number of facets of their post-winter developmental trajectories. However, divergent adaptation in adult emergence phenology in these flies was due almost entirely to the end of the pupal diapause maintenance phase, with post-diapause development having a very small effect. The relatively simple underpinnings of variation in adult emergence phenology suggest that further adaptation to seasonal change in these flies for this trait might be largely due to the timing of diapause termination unhindered by strong covariance among different components of post-diapause development.

Force enhancement in the human vastus lateralis is muscle-length-dependent following stretch but not during stretch.

PURPOSE: Force enhancement is the phenomenon of increased forces during (transient force enhancement; tFE) and after (residual force enhancement; rFE) eccentric muscle actions compared with fixed-end contractions. Although tFE and rFE have been observed at short and long muscle lengths, whether both are length-dependent remains unclear in vivo. METHODS: We determined maximal-effort vastus lateralis (VL) force-angle relationships of eleven healthy males and selected one knee joint angle at a short and long muscle lengths where VL produced approximately the same force (85% of maximum). We then examined tFE and rFE at these two lengths during and following the same amount of knee joint rotation. RESULTS: We found tFE at both short (11.7%, P = 0.017) and long (15.2%, P = 0.001) muscle lengths. rFE was only observed at the long (10.6%, P < 0.001; short: 1.3%, P = 0.439) muscle length. Ultrasound imaging revealed that VL muscle fascicle stretch magnitude was greater at long compared with short muscle lengths (mean difference: (tFE) 1.7 mm, (rFE) 1.9 mm, P ≤ 0.046), despite similar isometric VL forces across lengths (P ≥ 0.923). Greater fascicle stretch magnitude was likely to be due to greater preload forces at the long compared with short muscle length (P ≤ 0.001). CONCLUSION: At a similar isometric VL force capacity, tFE was not muscle-length-dependent at the lengths we tested, whereas rFE was greater at longer muscle length. We speculate that the in vivo mechanical factors affecting tFE and rFE are different and that greater stretch of a passive component is likely contributing more to rFE at longer muscle lengths.

Overexpression of an antioxidant enzyme improves male mating performance after stress in a lek-mating fruit fly.

In many species, courtship displays are reliable signals of male quality, and current hypotheses suggest that these displays allow females to choose males with high cellular function. Environmental stressors generate excess reactive oxygen species (ROS) that impair cellular function, and thus antioxidant pathways that remove ROS are probably critical for preserving complex sexual behaviours. Here, we test the hypothesis that enhanced antioxidant activity in mitochondria preserves mating performance following oxidative stress. Using a transgenic approach, we directly manipulated mitochondrial antioxidant activity in the Caribbean fruit fly, Anastrepha suspensa, a lek-mating species with elaborate sexual displays and intense sexual selection that is also a model for sterile insect technique programmes. We generated seven transgenic lines that overexpress mitochondrial superoxide dismutase (MnSOD). Radiation is a severe oxidative stressor used to induce sterility for sterile insect programmes. After radiation treatment, two lines with intermediate MnSOD overexpression showed enhanced mating performance relative to wild-type males. These improvements in mating corresponded with reduced oxidative damage to lipids, demonstrating that MnSOD overexpression protects flies from oxidative stress at the cellular level. For lines with improved mating performance, overexpression also preserved locomotor activity, as indicated by a laboratory climbing assay. Our results show a clear link between oxidative stress, antioxidant capacity and male performance. Our work has implications for fundamentally understanding the role of antioxidants in sexual selection, and shows promise for using transgenic approaches to enhance the field performance of insects released for area-wide pest management strategies and improving performance of biological control agents in general.

Hormetic benefits of prior anoxia exposure in buffering anoxia stress in a soil-pupating insect.

Oxygen is essential for most animals, and exposure to a complete lack of oxygen, i.e. anoxia, can result in irreparable damage to cells that can extend up to the organismal level to negatively affect performance. Although it is known that brief anoxia exposure may confer cross-tolerance to other stressors, few data exist on the biochemical and organismal consequences of repeated intermittent bouts of anoxia exposure. In nature, the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae), is frequently exposed to heavy tropical rainfall while pupating in the soil, equating to multiple exposures to hypoxia or anoxia during development. Here, we tested whether prior anoxia exposures during pupal development can induce a beneficial acclimation response, and we explored the consequences of prior exposure for both whole-organism performance and correlated biochemical metrics. Pharate adults (the last developmental stage in the pupal case) were most sensitive to anoxia exposure, showing decreased survival and fertility compared with controls. These negative impacts were ameliorated by exposure to anoxia in earlier pupal developmental stages, indicating a hormetic effect of prior anoxia exposure. Anoxia exposure early in pupal development reduced the oxygen debt repaid after anoxia exposure relative to pharate adults experiencing anoxia for the first time. Lipid levels were highest in all pupal stages when exposed to prior anoxia. Prior anoxia thus benefits organismal performance and relocates resources towards lipid storage throughout pupal-adult development.

Constraints, independence, and evolution of thermal plasticity: probing genetic architecture of long- and short-term thermal acclimation.

Seasonal and daily thermal variation can limit species distributions because of physiological tolerances. Low temperatures are particularly challenging for ectotherms, which use both basal thermotolerance and acclimation, an adaptive plastic response, to mitigate thermal stress. Both basal thermotolerance and acclimation are thought to be important for local adaptation and persistence in the face of climate change. However, the evolutionary independence of basal and plastic tolerances remains unclear. Acclimation can occur over longer (seasonal) or shorter (hours to days) time scales, and the degree of mechanistic overlap is unresolved. Using a midlatitude population of Drosophila melanogaster, we show substantial heritable variation in both short- and long-term acclimation. Rapid cold hardening (short-term plasticity) and developmental acclimation (long-term plasticity) are positively correlated, suggesting shared mechanisms. However, there are independent components of these traits, because developmentally acclimated flies respond positively to short-term acclimation. A strong negative correlation between basal cold tolerance and developmental acclimation suggests that basal cold tolerance may constrain developmental acclimation, whereas a weaker negative correlation between basal cold tolerance and short-term acclimation suggests less constraint. Using genome-wide association mapping, we show the genetic architecture of rapid cold hardening and developmental acclimation responses are nonoverlapping at the SNP and corresponding gene level. However, genes associated with each trait share functional similarities, including genes involved in apoptosis and autophagy, cytoskeletal and membrane structural components, and ion binding and transport. These results indicate substantial opportunity for short-term and long-term acclimation responses to evolve separately from each other and for short-term acclimation to evolve separately from basal thermotolerance.

A test of genomic modularity among life-history adaptations promoting speciation with gene flow.

Speciation with gene flow may require adaptive divergence of multiple traits to generate strong ecologically based reproductive isolation. Extensive negative pleiotropy or physical linkage of genes in the wrong phase affecting these diverging traits may therefore hinder speciation, while genetic independence or "modularity" among phenotypic traits may reduce constraints and facilitate divergence. Here, we test whether the genetics underlying two components of diapause life history, initial diapause intensity and diapause termination timing, constrain differentiation between sympatric hawthorn and apple-infesting host races of the fly Rhagoletis pomonella through analysis of 10,256 SNPs measured via genotyping-by-sequencing (GBS). Loci genetically associated with diapause termination timing were mainly observed for SNPs mapping to chromosomes 1-3 in the genome, most notably for SNPs displaying higher levels of linkage disequilibrium (LD), likely due to inversions. In contrast, selection on initial diapause intensity affected loci on all five major chromosomes of the genome, specifically those showing low levels of LD. This lack of overlap in genetically associated loci suggests that the two diapause phenotypes are largely modular. On chromosome 2, however, intermediate level LD loci and a subgroup of high LD loci displayed significant negative relationships between initial diapause intensity and diapause termination time. These gene regions on chromosome 2 therefore affected both traits, while most regions were largely independent. Moreover, loci associated with both measured traits also tended to exhibit highly divergent allele frequencies between the host races. Thus, the presence of nonoverlapping genetic modules likely facilitates simultaneous, adaptive divergence for the measured life-history components.

Cold adaptation increases rates of nutrient flow and metabolic plasticity during cold exposure in Drosophila melanogaster.

Metabolic flexibility is an important component of adaptation to stressful environments, including thermal stress and latitudinal adaptation. A long history of population genetic studies suggest that selection on core metabolic enzymes may shape life histories by altering metabolic flux. However, the direct relationship between selection on thermal stress hardiness and metabolic flux has not previously been tested. We investigated flexibility of nutrient catabolism during cold stress in Drosophila melanogaster artificially selected for fast or slow recovery from chill coma (i.e. cold-hardy or -susceptible), specifically testing the hypothesis that stress adaptation increases metabolic turnover. Using (13)C-labelled glucose, we first showed that cold-hardy flies more rapidly incorporate ingested carbon into amino acids and newly synthesized glucose, permitting rapid synthesis of proline, a compound shown elsewhere to improve survival of cold stress. Second, using glucose and leucine tracers we showed that cold-hardy flies had higher oxidation rates than cold-susceptible flies before cold exposure, similar oxidation rates during cold exposure, and returned to higher oxidation rates during recovery. Additionally, cold-hardy flies transferred compounds among body pools more rapidly during cold exposure and recovery. Increased metabolic turnover may allow cold-adapted flies to better prepare for, resist and repair/tolerate cold damage. This work illustrates for the first time differences in nutrient fluxes associated with cold adaptation, suggesting that metabolic costs associated with cold hardiness could invoke resource-based trade-offs that shape life histories.