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.

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.

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.

Divergence of the diapause transcriptome in apple maggot flies: winter regulation and post-winter transcriptional repression.

The duration of dormancy regulates seasonal timing in many organisms and may be modulated by day length and temperature. Though photoperiodic modulation has been well studied, temperature modulation of dormancy has received less attention. Here, we leverage genetic variation in diapause in the apple maggot fly, Rhagoletis pomonella, to test whether gene expression during winter or following spring warming regulates diapause duration. We used RNAseq to compare transcript abundance during and after simulated winter between an apple-infesting population and a hawthorn-infesting population where the apple population ends pupal diapause earlier than the hawthorn-infesting population. Marked differences in transcription between the two populations during winter suggests that the 'early' apple population is developmentally advanced compared with the 'late' hawthorn population prior to spring warming, with transcripts participating in growth and developmental processes relatively up-regulated in apple pupae during the winter cold period. Thus, regulatory differences during winter ultimately drive phenological differences that manifest themselves in the following summer. Expression and polymorphism analysis identify candidate genes in the Wnt and insulin signaling pathways that contribute to population differences in seasonality. Both populations remained in diapause and displayed a pattern of up- and then down-regulation (or vice versa) of growth-related transcripts following warming, consistent with transcriptional repression. The ability to repress growth stimulated by permissive temperatures is likely critical to avoid mismatched phenology and excessive metabolic demand. Compared with diapause studies in other insects, our results suggest some overlap in candidate genes/pathways, though the timing and direction of changes in transcription are likely species specific.

Differences in performance and transcriptome-wide gene expression associated with Rhagoletis (Diptera: Tephritidae) larvae feeding in alternate host fruit environments.

Host race formation, the establishment of new populations using novel resources, is a major hypothesized mechanism of ecological speciation, especially in plant-feeding insects. The initial stages of host race formation will often involve phenotypic plasticity on the novel resource, with subsequent genetically based adaptations enhancing host-associated fitness differences. Several studies have explored the physiology of the plastic responses of insects to novel host environments. However, the mechanisms underlying evolved differences among host races and species remain poorly understood. Here, we demonstrate a reciprocal larval performance difference between two closely related species of Rhagoletis flies, R. pomonella and R. zephyria, specialized for feeding in apple and snowberry fruit, respectively. Microarray analysis of fly larvae feeding in apples versus snowberries revealed patterns of transcriptome-wide differential gene expression consistent with both plastic and evolved responses to the different fruit resources, most notably for detoxification-related genes such as cytochrome p450s. Transcripts exhibiting evolved expression differences between species tended to also demonstrate plastic responses to fruit environment. The observed pattern suggests that Rhagoletis larvae exhibit extensive plasticity in gene expression in response to novel fruit that may potentiate shifts to new hosts. Subsequent selection, particularly selection to suppress initially costly plastic responses, could account for the evolved expression differences observed between R. pomonella and R. zephyria, creating specialized races and new fly species. Thus, genetically based ecological adaptations generating new biodiversity may often evolve from initial plastic responses in gene expression to the challenges posed by novel environments.

Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

Dormancy cues alter insect temperature-size relationships.

Developmental temperatures can have dramatic effects on body size in ectotherms. Thermal plasticity in body size is often viewed in the context of seasonality, but the role of seasonal dormancy responses in generating temperature-size relationships is underappreciated. We used the moth Helicoverpa zea (corn earworm) to examine how photoperiodic seasonal dormancy programming for pupal diapause affects the temperature-size relationship. Specifically, we partition out the contributions of somatic growth versus nutrient storage as fat to the thermal reaction norm for size. With increasing temperature from 16 °C to 20 °C, dormant pupae were both overall larger and progressively fatter than non-dormant pupae. This body mass response is likely driven by concurrent increases in food consumption and longer development times as temperatures increase. Our results demonstrate that seasonal photoperiodic cues can alter temperature-size relationships during pre-dormancy development. For biologists interested in seasonal effects on temperature-size relationships, our results suggest that the key to fully understanding these relationships may lie in integrating multiple seasonal cues and multiple aspects of body size and composition in a nutrient-allocation framework.

¹³C NMR metabolomics: applications at natural abundance.

(13)C NMR has many advantages for a metabolomics study, including a large spectral dispersion, narrow singlets at natural abundance, and a direct measure of the backbone structures of metabolites. However, it has not had widespread use because of its relatively low sensitivity compounded by low natural abundance. Here we demonstrate the utility of high-quality (13)C NMR spectra obtained using a custom (13)C-optimized probe on metabolomic mixtures. A workflow was developed to use statistical correlations between replicate 1D (13)C and (1)H spectra, leading to composite spin systems that can be used to search publicly available databases for compound identification. This was developed using synthetic mixtures and then applied to two biological samples, Drosophila melanogaster extracts and mouse serum. Using the synthetic mixtures we were able to obtain useful (13)C-(13)C statistical correlations from metabolites with as little as 60 nmol of material. The lower limit of (13)C NMR detection under our experimental conditions is approximately 40 nmol, slightly lower than the requirement for statistical analysis. The (13)C and (1)H data together led to 15 matches in the database compared to just 7 using (1)H alone, and the (13)C correlated peak lists had far fewer false positives than the (1)H generated lists. In addition, the (13)C 1D data provided improved metabolite identification and separation of biologically distinct groups using multivariate statistical analysis in the D. melanogaster extracts and mouse serum.

Low-oxygen atmospheric treatment improves the performance of irradiation-sterilized male cactus moths used in SIT.

As part of sterile insect technique (SIT) programs, irradiation can effectively induce sterility in insects by damaging genomic DNA. However, irradiation also induces other off-target side effects that reduce the quality and performance of sterilized males. Thus, treatments that reduce off-target effects of irradiation on male performance while maintaining sterility can improve the feasibility and economy of SIT programs. Exposure to ionizing radiation induces the formation of damaging free radicals in biological systems that may reduce sterile male performance. Here, we test whether exposure to an anoxic environment for 1 h before and during irradiation improves male performance, while maintaining sterility in males of the cactus moth, Cactoblastis cactorum (Berg). We show that exposure to 1 h of anoxia increases the moth's antioxidant capacity and that irradiation in anoxia after 1 h of anoxic conditioning decreases irradiation-induced oxidative damage to the moth's lipids and proteins. Anoxia treatment that reduced oxidative damage after irradiation also produced moths with greater flight performance, mating success, and longevity, while maintaining F1 male sterility at acceptable levels for SIT. We conclude that anoxia pretreatment followed by irradiation in anoxia is an efficient way to improve the quality of irradiated moths and perhaps lower the number of moths needed for release SIT moth operations.