Identification of sex-specific markers based on gene expression in adult Colorado potato beetles.

The Colorado potato beetle, Leptinotarsa decemlineata, (Coleoptera: Chrysomelidae) is an economically important pest insect of potatoes. Understanding how the mechanisms driving its invasiveness vary between sexes will be critical for developing modern control methods. However, the currently available methods for sexing adult Colorado potato beetles are either inefficient or unsuitable for projects that require RNA as an input, like those measuring gene expression. Therefore, the development of simple molecular tools that are tailored to these studies is important. In this study, we used publicly available RNA-seq data to select 5 candidate genes for sex-specific markers in adult Colorado potato beetles. We confirmed that our 5 marker candidates exhibit a sex-specific expression pattern and can be used as PCR markers for sex determination. This method of sex detection will allow researchers to distinguish the sex of the individual with a simple PCR reaction using cDNA as the template and assign sex to RNA-seq samples post hoc.

Plasticity of nutrient accumulation patterns in diapausing fall webworm pupae.

The accumulation of nutrients during diapause preparation is crucial because any lack of nutrition will reduce the likelihood of insects completing diapause, thereby decreasing their chances of survival and reproduction. The fall webworm, Hyphantria cunea, diapause as overwintering pupae and their diapause incidence and diapause intensity are regulated by the photoperiod. In this study, we test the hypothesis that photoperiod influences energy reserve accumulation during diapause preparation in fall webworm. We found that the body size and mass, lipid and carbohydrate content of pupae with a short photoperiod during the diapause induction phase were significantly greater than those of pupae with a relatively short photoperiod, and the efficiency of converting digested food and ingested food into body matter was greater in the short-photoperiod diapause-destined larvae than the relatively short-photoperiod diapause-destined larvae. We also observed higher lipase and amylase activities in short-photoperiod diapause-destined larvae relative to the counterparts. However, no obvious difference was found in protein and protease in the pupae with a short photoperiod during the diapause induction phase and short-photoperiod diapause-destined larvae compared with the counterparts. Therefore, we conclude that the energy reserve patterns of diapausing fall webworm pupae are plastic and that short-photoperiod diapause-destined larvae increase their energy reserves by improving their feeding efficiency and increase their lipid and carbohydrate stores by increasing the lipase and amylase activities in the midgut.

Cold tolerance of laboratory-reared Asian longhorned beetles.

Low winter temperatures in temperate climates can limit the success of non-native species. The Asian longhorned beetle, Anoplophora glabripennis, is an invasive wood-boring pest of hardwood trees in North America and Europe. Native A. glabripennis populations are spread across several climate zones in China and the Korean Peninsula and are likely to encounter low temperatures in at least some of this range. Understanding the lethal limits of the overwintering life stages of A. glabripennis is essential for accurately modeling the risk that invasive populations pose to non-native environments. In this study, we provide the first systematic characterization of the cold tolerance strategy and lower lethal limits of A. glabripennis eggs, larvae, and pupae. In diapausing larvae, the most common overwintering stage in this species, we measure hemolymph glycerol and osmolality and identify the effects of prolonged low temperature exposure. In developing pupae, we identify sublethal effects caused by low temperature exposure before freezing. Eggs and larvae were the most cold-tolerant life stages; eggs were freeze-avoidant with an average supercooling point of -25.8 °C and larvae were freeze tolerant with an LT90 of -25 °C. Hemolymph osmolality of freeze-tolerant larvae, on average, increased to 811 mOsm during chilling. This increase was primarily driven by a concurrent, average increase of 232 mM hemolymph glycerol. Pupae died upon exposure to freezing temperatures, but accumulate strong sublethal effects prior to freezing, indicating that they are chill susceptible. Taken together, these data will be useful to inform species distribution modeling in A. glabripennis.

Help, there are 'omics' in my comparative physiology!

'Omics' methods, such as transcriptomics, proteomics, lipidomics or metabolomics, yield simultaneous measurements of many related molecules in a sample. These approaches have opened new opportunities to generate and test hypotheses about the mechanisms underlying biochemical and physiological phenotypes. In this Commentary, we discuss general approaches and considerations for successfully integrating omics into comparative physiology. The choice of omics approach will be guided by the availability of existing resources and the time scale of the process being studied. We discuss the use of whole-organism extracts (common in omics experiments on small invertebrates) because such an approach may mask underlying physiological mechanisms, and we consider the advantages and disadvantages of pooling samples within biological replicates. These methods can bring analytical challenges, so we describe the most easily analyzed omics experimental designs. We address the propensity of omics studies to digress into 'fishing expeditions' and show how omics can be used within the hypothetico-deductive framework. With this Commentary, we hope to provide a roadmap that will help newcomers approach omics in comparative physiology while avoiding some of the potential pitfalls, which include ambiguous experiments, long lists of candidate molecules and vague conclusions.

Physiological responses to fluctuating temperatures are characterized by distinct transcriptional profiles in a solitary bee.

Exposure to stressful low temperatures during development can result in the accumulation of deleterious physiological effects called chill injury. Metabolic imbalances, disruptions in ion homeostasis and oxidative stress contribute to the increased mortality of chill-injured insects. Interestingly, survival can be significantly increased when chill-susceptible insects are exposed to a daily warm-temperature pulse during chilling. We hypothesize that warm pulses allow for the repair of damage associated with chill injury. Here, we describe transcriptional responses during exposure to a fluctuating thermal regime, relative to constant chilled temperatures, during pupal development in the alfalfa leafcutting bee, Megachile rotundata, using a combination of RNA-seq and qPCR. Pupae were exposed to either a constant, chilled temperature of 6°C, or 6°C with a daily pulse of 20°C for 7 days. RNA-seq after experimental treatment revealed differential expression of transcripts involved in construction of cell membranes, oxidation-reduction and various metabolic processes. These mechanisms provide support for shared physiological responses to chill injury across taxa. The large number of differentially expressed transcripts observed after 7 days of treatment suggests that the initial divergence in expression profiles between the two treatments occurred upstream of the time point sampled. Additionally, the differential expression profiles observed in this study show little overlap with those differentially expressed during temperature stress in the diapause state of M. rotundata While the mechanisms governing the physiological response to low-temperature stress are shared, the specific transcripts associated with the response differ between life stages.

Transcriptional responses to fluctuating thermal regimes underpinning differences in survival in the solitary bee Megachile rotundata.

The transcriptional responses of insects to long-term, ecologically relevant temperature stress are poorly understood. Long-term exposure to low temperatures, commonly referred to as chilling, can lead to physiological effects collectively known as chill injury. Periodically increasing temperatures during long-term chilling has been shown to increase survival in many insects. However, the transcripts responsible for this increase in survival have never been characterized. Here, we present the first transcriptome-level analysis of increased longevity under fluctuating temperatures during chilling. Overwintering post-diapause quiescent alfalfa leafcutting bees (Megachile rotundata) were exposed to a constant temperature of 6°C, or 6°C with a daily fluctuation to 20°C. RNA was collected at two different time points, before and after mortality rates began to diverge between temperature treatments. Expression analysis identified differentially regulated transcripts between pairwise comparisons of both treatments and time points. Transcripts functioning in ion homeostasis, metabolic pathways and oxidative stress response were up-regulated in individuals exposed to periodic temperature fluctuations during chilling. The differential expression of these transcripts provides support for the hypotheses that fluctuating temperatures protect against chill injury by reducing oxidative stress and returning ion concentrations and metabolic function to more favorable levels. Additionally, exposure to fluctuating temperatures leads to increased expression of transcripts functioning in the immune response and neurogenesis, providing evidence for additional mechanisms associated with increased survival during chilling in M. rotundata.

A pipeline for the de novo assembly of the Themira biloba (Sepsidae: Diptera) transcriptome using a multiple k-mer length approach.

BACKGROUND: The Sepsidae family of flies is a model for investigating how sexual selection shapes courtship and sexual dimorphism in a comparative framework. However, like many non-model systems, there are few molecular resources available. Large-scale sequencing and assembly have not been performed in any sepsid, and the lack of a closely related genome makes investigation of gene expression challenging. Our goal was to develop an automated pipeline for de novo transcriptome assembly, and to use that pipeline to assemble and analyze the transcriptome of the sepsid Themira biloba. RESULTS: Our bioinformatics pipeline uses cloud computing services to assemble and analyze the transcriptome with off-site data management, processing, and backup. It uses a multiple k-mer length approach combined with a second meta-assembly to extend transcripts and recover more bases of transcript sequences than standard single k-mer assembly. We used 454 sequencing to generate 1.48 million reads from cDNA generated from embryo, larva, and pupae of T. biloba and assembled a transcriptome consisting of 24,495 contigs. Annotation identified 16,705 transcripts, including those involved in embryogenesis and limb patterning. We assembled transcriptomes from an additional three non-model organisms to demonstrate that our pipeline assembled a higher-quality transcriptome than single k-mer approaches across multiple species. CONCLUSIONS: The pipeline we have developed for assembly and analysis increases contig length, recovers unique transcripts, and assembles more base pairs than other methods through the use of a meta-assembly. The T. biloba transcriptome is a critical resource for performing large-scale RNA-Seq investigations of gene expression patterns, and is the first transcriptome sequenced in this Dipteran family.

Molecular mechanisms and trade-offs underlying fluctuating thermal regimes during low-temperature storage.

Insects exposed to constant low temperatures (CLT) exhibit high rates of mortality as well as a variety of sublethal effects. In many species, interruptions of CLT with brief pulses of warm temperatures (fluctuating thermal regimes, FTR) lead to increases in survival and fewer sublethal effects. However, we still lack a complete understanding of the physiological mechanisms activated during FTR. In this review, we discuss recent advances in understanding FTR's underlying molecular mechanisms. We discuss knowledge gaps related to potential trade-offs between FTR's beneficial effects and the costs of these repairs to overwintering reserves and reproduction. We present the hypothesis that the warm pulse of FTR helps to maintain daily rhythmicity.

Metabolic and transcriptomic characterization of summer and winter dormancy in the solitary bee, Osmia lignaria.

The solitary bee Osmia lignaria is a native pollinator in North America with growing economic importance. The life cycle of O. lignaria provides a unique opportunity to compare the physiological and molecular mechanisms underlying two ecologically contrasting dormancies within the same species. O. lignaria prepupae become dormant during the summer to avoid high temperatures. Shortly after adult eclosion, they enter a second dormancy and overwinter as diapausing adults. To compare these two dormancies, we measured metabolic rates and gene expression across development as bees initiate, maintain, and terminate both prepupal (summer) and adult (overwintering) dormancies. We observed a moderate temperature-independent decrease in gas exchange during both the prepupal dormancy after cocoon spinning (45 %) and during adult diapause after eclosion (60 %). We sequenced and assembled a high-quality reference genome from a single haploid male bee with a contiguous n50 of 5.5 Mbp to facilitate our transcriptomic analysis. The transcriptomes of dormant prepupae and diapausing adults clustered into distinct groups more closely associated with life stage than dormancy status. Membrane transport, membrane-bound cellular components, oxidoreductase activity, glutathione metabolism, and transcription factor activity increased during adult diapause, relative to prepupal dormancy. Further, the transcriptomes of adults in diapause clustered into two groups, supporting multiple phases of diapause during winter. Late adult diapause was associated with gene expression profiles supporting increased insulin/IGF, juvenile hormone, and ecdysone signaling.

Molecular signatures of diapause in the Asian longhorned beetle: Gene expression.

Most previous studies on gene expression during insect diapause do not address among-tissue variation in physiological processes. We measured transcriptomic changes during larval diapause in the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae). We conducted RNA-seq on fat body, the supraesophageal ganglion, midgut, hindgut, and Malpighian tubules during pre-diapause, diapause maintenance, post-diapause quiescence, and post-diapause development. We observed a small, but consistent, proportion of genes within each gene expression profile that were shared among tissues, lending support for a core set of diapause-associated genes whose expression is tissue-independent. We evaluated the overarching hypotheses that diapause would be associated with cell cycle arrest, developmental arrest, and increased stress tolerance and found evidence of repressed TOR and insulin signaling, reduced cell cycle activity and increased capacity of stress response via heat shock protein expression and remodeling of the cytoskeleton. However, these processes varied among tissues, with the brain and fat body appearing to maintain higher levels of cellular activity during diapause than the midgut or Malpighian tubules. We also observed temperature-dependent changes in gene expression during diapause maintenance, particularly in genes related to the heat shock response and MAPK, insulin, and TOR signaling pathways. Additionally, we provide evidence for epigenetic reorganization during the diapause/post-diapause quiescence transition and expression of genes involved in post-translational modification, highlighting the need for investigations of the protein activity of these candidate genes and processes. We conclude that diapause development is coordinated via diverse tissue-specific gene expression profiles and that canonical diapause phenotypes vary among tissues.

Energy Consumption and Cold Hardiness of Diapausing Fall Webworm Pupae.

Diapause and cold hardiness are essential components of winter survival for most insects in temperate zones. The fall webworm, Hyphantria cunea, overwinters in a pupal diapause. In this study, we investigated the energy consumption and cold hardiness of diapausing pupae. We found that lipid content decreased from October to November and stabilized from November to March. Glycogen content decreased by 61.3% and 52.2% for females and males, respectively, from October to November, and decreased slowly from November to March. We also observed a significant increase in trehalose concentrations as ambient temperatures decreased from October to November and a decrease in trehalose as temperatures increased again in March. We did not observe substantial changes in pupal supercooling points among the dates sampled. In addition, prolonged pupal development time reduced their survival rate and had no significant effect on post-diapause adult body mass and fecundity but reduced egg diameter in females. These results suggest that the energy consumption of H. cunea pupae during early diapause depends on lipid and glycogen, while it shifts to depend on glycogen or other energy stores in the mid- and late diapause stages. Our results also suggest that the prolonged development time of diapausing pupae had a negative effect on post-diapause fitness.

The mitochondrial genomes of two Gryllus crickets (Grylloidea: Gryllidae) via RNA-seq.

Here, we used RNA-seq reads to assemble the complete mitochondrial genomes of the spring field cricket, Gryllus veletis, and the variable field cricket, Gryllus lineaticeps. The mitochondrial genomes of G. veletis (15,686 bp, MW322713) and G. lineaticeps (15,607 bp, MW315773) each contain the expected 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a large control (D-loop) region. The arrangements of these features were similar for both species and consistent with other closely related Orthoptera. A phylogenetic analysis of the mitochondrial genome sequences revealed that G. veletis and G. lineaticeps cluster with the other Gryllus species and all reside in a clade with the Gryllidae.

Physiological Mechanisms of Variable Nutrient Accumulation Patterns Between Diapausing and Non-Diapausing Fall Webworm (Lepidoptera: Arctiidae) Pupae.

Diapause is a highly advantageous strategy for winter survival for insects living in temperate environments. However, insects typically do not feed during diapause and are therefore presented with a complicated energetics problem. The fall webworm, Hyphantria cunea, overwinter as diapausing pupae, but adults of this species lose their ability to feed due to the degeneration of their mouthparts. Thus, the energy reserves stored before diapauses contribute to the survival rate and fitness of the adults after emergence. In this study, we tested the hypothesis that diapause-destined larvae of H. cunea reserve more energy by increasing feeding rate, feeding efficiency, or both, during the diapause preparation phase compared with non-diapause-destined larvae. We observed higher digestive efficiency, increased lipase and amylase activity, and lower protease activity in diapause-destined larvae compared to non-diapause-destined larvae. These differences in digestive physiology during diapause preparation lead to greater body size and mass, increased lipid and carbohydrate content, and lower soluble protein content in diapausing pupae, relative to non-diapause pupae - results consistent with our hypothesis. We conclude that diapause-destined fall webworm reserve more energy than non-diapause-destined individuals by increasing feeding efficiency, and that this increase in efficiency is at least partially driven by increased lipase and amylase activities in the midgut. This is in contrast to non-diapause-destined larvae, which likely reserve greater protein than diapause-destined larvae to help maintain their physiological function.

Diapause differentially modulates the transcriptomes of fat body and flight muscle in the Colorado potato beetle.

Many temperate insects, such as the Colorado potato beetle, enter diapause in winter, during which they arrest their development, suppress their metabolic rate and have high stress tolerance. Diapause phenotypes can be transcriptionally regulated, however many studies to date report only whole animal gene expression rather than tissue-specific processes during diapause. We used RNA-seq to measure gene expression in fat body and flight muscle of diapausing and non-diapausing beetles. We used differential expression and GO enrichment analyses to evaluate longstanding hypotheses about the mechanisms that drive arrested development, changes in energy metabolism, and increased stress tolerance during diapause. We found evidence of G2/M cell cycle arrest, juvenile hormone catabolism, increased antioxidant metabolism, epigenetic modification, transposable element regulation, and cytoskeletal remodeling in both the fat body and flight muscle of diapausing beetles. Beetles differentially modulated the fat body and flight muscle transcriptomes during diapause with fat body playing a larger role in the hypoxia response and immunity, whereas flight muscle had higher abundance of transcripts related to the chaperone response and proteostasis. Our transcriptome provides evidence for distinct roles and responses of fat body and flight muscle during diapause in the Colorado potato beetle, and we provide testable hypotheses for biological processes that appear to drive diapause phenotypes in insects.

Dormancy in laboratory-reared Asian longhorned beetles, Anoplophora glabripennis.

An insect's capacity to survive winter is critical for range expansion in temperate regions. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-boring insect native to China and the Korean peninsula and poses a high risk of invasion in North America and Europe. It is unclear whether A. glabripennis enters diapause, which means that diapause cannot be included in assessments of the risk of this species invading forests in temperate regions. Using a laboratory colony, we examine larval developmental arrest, metabolic rates, gas exchange patterns, thermal sensitivity, and body composition to characterize larval dormancy. Chilled larvae entered a temperature-independent developmental arrest which usually required more than four weeks of chilling to break, decreased their metabolic rate by as much as 63%, and maintained energy stores throughout the chilling period - results consistent with an obligate diapause. We also observed a switch to discontinuous gas exchange at low temperatures. Thermal sensitivity of metabolic rate did not differ between chilled and non-chilled larvae. Taken together, we conclude that A. glabripennis enters a larval diapause during chilling and terminates diapause after a requisite chilling period. These results will enhance our ability to predict phenology and potential distribution of current and future invasions of A. glabripennis.

Fluctuating thermal regimes prevent chill injury but do not change patterns of oxidative stress in the alfalfa leafcutting bee, Megachile rotundata.

In insects, prolonged exposure to unseasonably low temperatures can lead to detrimental physiological effects known as chill injury. Changes to active and passive transport across epithelia during chilling likely drive the collapse of ion gradients, metabolic imbalance and potentially oxidative stress. In the alfalfa leafcutting bee, Megachile rotundata transcriptomic evidence provides support for these responses at the level of gene expression, but variable expression profiles between life stages in M. rotundata indicate that different mechanisms could be responsible for repairing and protecting against chill injuries across development. Herein, we test the hypotheses that 1) chill injury leads to oxidative stress and damage in insects and 2) exposure to a fluctuating thermal regime (FTR) promotes an increased oxidative stress response leading to a decrease in damage by reactive oxygen species. We measured the expression of transcripts with products known to have antioxidant properties in overwintering prepupae as well as total antioxidant capacity and lipid peroxidation during both extended overwintering in prepupae and low temperature stress during pupal development. We observed differential gene expression for the antioxidant glutathione peroxidase and several transcripts with putative antioxidant properties including vitellogenin, apolipoprotein D, glutathione S-transferase, and nuclear protein 1. However, the expression of transcripts coding for other enzymatic antioxidants did not change between treatments. Neither life stage varied in their capacity to cope with an induced oxidative stress after FTR exposure and we did not observe evidence of lipid peroxidation in chill injured (STR) prepupae. These results did not support our initial hypotheses and indicate that oxidative-stress-induced damage is neither a causal factor or symptom of chill injury.

Immediate Transcriptional Response to a Temperature Pulse under a Fluctuating Thermal Regime.

The response of ectotherms to temperature stress is complex, non-linear, and is influenced by life stage and previous thermal exposure. Mortality is higher under constant low temperatures than under a fluctuating thermal regime (FTR) that maintains the same low temperature but adds a brief, daily pulse of increased temperature. Long term exposure to FTR has been shown to increase transcription of genes involved in oxidative stress, immune function, and metabolic pathways, which may aid in recovery from chill injury and oxidative damage. Previous research suggests the transcriptional response that protects against sub-lethal damage occurs rapidly under exposure to fluctuating temperatures. However, existing studies have only examined gene expression after a week or over many months. Here we characterize gene expression during a single temperature cycle under FTR. Development of pupating alfalfa leafcutting bees (Megachile rotundata) was interrupted at the red-eye stage and were transferred to 6°C with a 1-h pulse to 20°C and returned to 6°C. RNA was collected before, during, and after the temperature pulse and compared to pupae maintained at a static 6°C. The warm pulse is sufficient to cause expression of transcripts that repair cell membrane damage, modify membrane composition, produce antifreeze proteins, restore ion homeostasis, and respond to oxidative stress. This pattern of expression indicates that even brief exposure to warm temperatures has significant protective effects on insects exposed to stressful cold temperatures that persist beyond the warm pulse. Megachile rotundata's sensitivity to temperature fluctuations indicates that short exposures to temperature changes affect development and physiology. Genes associated with developmental patterning are expressed after the warm pulse, suggesting that 1 h at 20°C was enough to resume development in the pupae. The greatest difference in gene expression occurred between pupae collected after the warm pulse and at constant low temperatures. Although both were collected at the same time and temperature, the transcriptional response to one FTR cycle included multiple transcripts previously identified under long-term FTR exposure associated with recovery from chill injury, indicating that the effects of FTR occur rapidly and are persistent.