Effects of developmental state on low-temperature physiology of the alfalfa leafcutting bee, Megachile rotundata.

The success of agriculture relies on healthy bees to pollinate crops. Commercially managed pollinators are often kept under temperature-controlled conditions to better control development and optimize field performance. One such pollinator, the alfalfa leafcutting bee, Megachile rotundata, is the most widely used solitary bee in agriculture. Problematically, very little is known about the thermal physiology of M. rotundata or the consequences of artificial thermal regimes used in commercial management practices. Therefore, we took a broad look at the thermal performance of M. rotundata across development and the effects of commonly used commercial thermal regimes on adult bee physiology. After the termination of diapause, we hypothesized thermal sensitivity would vary across pupal metamorphosis. Our data show that bees in the post-diapause quiescent stage were more tolerant of low temperatures compared to bees in active development. We found that commercial practices applied during development decrease the likelihood of a bee recovering from another bout of thermal stress in adulthood, thereby decreasing their resilience. Lastly, commercial regimes applied during development affected the number of days to adult emergence, but the time of day that adults emerged was unaffected. Our data demonstrate the complex interactions between bee development and thermal regimes used in management. This knowledge can help improve the commercial management of these bees by optimizing the thermal regimes used and the timing of their application to alleviate negative downstream effects on adult performance.

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.

Overwintering conditions impact insulin pathway gene expression in diapausing Megachile rotundata.

Diapause is a non-feeding state that many insects undergo to survive the winter months. With fixed resources, overall metabolism and insulin signaling (IIS) are maintained at low levels, but whether those change in response to seasonal temperature fluctuations remains unknown. The focus of this study was to determine 1) how genes in the insulin signaling pathway vary throughout diapause and 2) if that variation changes in response to temperature. To test the hypothesis that expression of IIS pathway genes vary in response to temperature fluctuations during overwintering, alfalfa leafcutting bees, Megachile rotundata, were overwintered at either a constant 4 °C in the lab or in naturally fluctuating temperatures in the field. Expression levels of genes in the IIS pathway, cell cycle regulators, and transcription factors were measured. Overall our findings showed that a few key targets of the insulin signaling pathway, along with growth regulators, change during overwintering, suggesting that only cell cycle regulators, and not the IIS pathway as a whole, change across the phases of diapause. To answer our second question, we compared gene expression levels between temperature treatments at each month for a given gene. We observed significantly more differences in expression of IIS pathway targets, indicating that overwintering conditions impact insulin pathway gene expression and leads to altered expression profiles. With differences seen between temperature treatment groups, these findings indicate that constant temperatures like those used in agricultural storage protocols, lead to different expression profiles and possibly different diapause phenotypes for alfalfa leafcutting bees.

Body and Wing Allometries Reveal Flight-Fecundity Tradeoff in Response to Larval Provisioning in Osmia lignaria (Hymenoptera: Megachilidae).

Variation in body size has important implications for physical performance and fitness. For insects, adult size and morphology are determined by larval growth and metamorphosis. Female blue orchard bees, Osmia lignaria, (Say) provision a finite quantity of food to their offspring. In this study, we asked how provision-dependent variation in size changes adult morphology. We performed a diet manipulation in which some larvae were starved in the final instar and some were given unlimited food. We examined the consequences on adult morphology in two ways. First, allometric relationships between major body regions (head, thorax, abdomen) and total body mass were measured to determine relative growth of these structures. Second, morphometrics that are critical for flight (wing area, wing loading, and extra flight power index) were quantified. Head and thorax mass had hyperallometric relationships with body size, indicating these parts become disproportionately large in adults when larvae are given copious provisions. However, abdominal mass and wing area increased hypoallometrically with body size. Thus, large adults had disproportionately lighter abdomens and smaller wing areas than smaller adults. Though both males and females followed these general patterns, allometric patterns were affected by sex. For flight metrics, small adults had reduced wing loading and an increased extra flight power index. These results suggest that diet quantity alters development in ways that affect the morphometric trait relationships in adult O. lignaria and may lead to functional differences in performance.

Diet quantity influences caste determination in honeybees (Apis mellifera).

In species that care for their young, provisioning has profound effects on offspring fitness. Provisioning is important in honeybees because nutritional cues determine whether a female becomes a reproductive queen or sterile worker. A qualitative difference between the larval diets of queens and workers is thought to drive this divergence; however, no single compound seems to be responsible. Diet quantity may have a role during honeybee caste determination yet has never been formally studied. Our goal was to determine the relative contributions of diet quantity and quality to queen development. Larvae were reared in vitro on nine diets varying in the amount of royal jelly and sugars, which were fed to larvae in eight different quantities. For the middle diet, an ad libitum quantity treatment was included. Once adults eclosed, the queenliness was determined using principal component analysis on seven morphological measurements. We found that larvae fed an ad libitum quantity of diet were indistinguishable from commercially reared queens, and that queenliness was independent of the proportion of protein and carbohydrate in the diet. Neither protein nor carbohydrate content had a significant influence on the first principle component 1 (PC1), which explained 64.4% of the difference between queens and workers. Instead, the total quantity of diet explained a significant amount of the variation in PC1. Large amounts of diet in the final instar were capable of inducing queen traits, contrary to the received wisdom that queen determination can only occur in the third instar. These results indicate that total diet quantity fed to larvae may regulate the difference between queen and worker castes in honeybees.

Metamorphosis is induced by food absence rather than a critical weight in the solitary bee, Osmia lignaria.

Body size is an important phenotypic trait that correlates with performance and fitness. For determinate growing insects, body size variation is determined by growth rate and the mechanisms that stop growth at the end of juvenile growth. Endocrine mechanisms regulate growth cessation, and their relative timing along development shapes phenotypic variation in body size and development time. Larval insects are generally hypothesized to initiate metamorphosis once they attain a critical weight. However, the mechanisms underlying the critical weight have not been resolved even for well-studied insect species. More importantly, critical weights may or may not be generalizable across species. In this study, we characterized the developmental aspects of size regulation in the solitary bee, Osmia lignaria We demonstrate that starvation cues metamorphosis in O. lignaria and that a critical weight does not exist in this species. Larvae initiated pupation <24 h after food was absent. However, even larvae fed ad libitum eventually underwent metamorphosis, suggesting that some secondary mechanism regulates metamorphosis when provisions are not completely consumed. We show that metamorphosis could be induced by precocene treatment in the presence of food, which suggests that this decision is regulated through juvenile hormone signaling. Removing food at different larval masses produced a 10-fold difference in mass between smallest and largest adults. We discuss the implications of body size variation for insect species that are provided with a fixed quantity of provisions, including many bees which have economic value as pollinators.

Mechanisms underpinning the beneficial effects of fluctuating thermal regimes in insect cold tolerance.

Insects exposed to low temperature often have high mortality or exhibit sublethal effects. A growing number of recent studies have shown beneficial effects of exposing insects to recurrent brief warm pulses during low-temperature stress (fluctuating thermal regime, FTR). The physiological underpinnings of the beneficial effects of FTR on cold survival have been extensively studied over the past few years. Profiling with various '-omics' techniques has provided supporting evidence for different physiological responses between insects exposed to FTR and constant low temperature. Evidence from transcriptomic, metabolomic and lipidomic studies points to a system-wide loss of homeostasis at low temperature that can be counterbalanced by repair mechanisms under FTR. Although there has been considerable progress in understanding the physiological mechanisms underlying the beneficial effects of FTR, here we discuss how many areas still lack clarity, such as the precise role(s) of heat shock proteins, compatible solutes or the identification of regulators and key players involved in the observed homeostatic responses. FTR can be particularly beneficial in applied settings, such as for model insects used in research, integrated pest management and pollination services. We also explain how the application of FTR techniques in large-scale facilities may require overcoming some logistical and technical constraints. FTR definitively enhances survival at low temperature in insects, but before it can be widely used, we suggest that the possible fitness and energy costs of FTR must be explored more thoroughly. Although FTR is not ecologically relevant, similar processes may operate in settings where temperatures fluctuate naturally.

Cues for cavity nesters: investigating relevant zeitgebers for emerging leafcutting bees, Megachile rotundata.

Photoperiod is considered the universal zeitgeber, regulating physiological processes in numerous animals. However, for animals in light-restricted habitats (e.g. burrows or cavities), thermoperiod may be a more important cue. Our study tested this hypothesis in the alfalfa leafcutting bee, Megachile rotundata, which nests in cavities and undergoes development within a brood cell. We assessed the role of environmental cues (thermoperiod and photoperiod) on the process of adult emergence by examining: (1) whether those cues direct circadian rhythms, (2) which cue is more dominant and (3) how sensitive developing bees and emergence-ready adults are to cues. Although we found that 20% of light penetrates the brood cell, and bees respond to photoperiod by synchronizing emergence, thermoperiod is the dominant cue. When presented with a conflicting zeitgeber, bees entrained to the thermophase instead of the photophase. When temperature cues were removed, we observed free-running of emergence, indicating that underlying circadian mechanisms can be synchronized by daily fluctuations in temperature. We also found that emerging bees were highly sensitive to even small increases in temperature, entraining to a ramp speed of 0.33°C h-1 The response and sensitivity to temperature cues suggest that M. rotundata evolved a temperature-mediated clock to time emergence from light-restricted cavities.

Environmental history impacts gene expression during diapause development in the alfalfa leafcutting bee, Megachile rotundata.

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNA sequencing (RNA-seq). However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause development because previous studies have focused on laboratory-reared and -maintained insects. To determine whether gene expression differs between laboratory and field conditions, prepupae of the alfalfa leafcutting bee, Megachile rotundata, entering diapause early or late in the growing season were collected. These two groups were further subdivided in early autumn into laboratory- and field-maintained groups, resulting in four experimental treatments of diapausing prepupae: early and late field, and early and late laboratory. RNA-seq and differential expression analyses were performed on bees from the four treatment groups in November, January, March and May. The number of treatment-specific differentially expressed genes (97 to 1249) outnumbered the number of differentially regulated genes common to all four treatments (14 to 229), indicating that exposure to laboratory or field conditions had a major impact on gene expression during diapause development. Principle component analysis and hierarchical cluster analysis yielded similar grouping of treatments, confirming that the treatments form distinct clusters. Our results support the conclusion that gene expression during the course of diapause development is not a simple ordered sequence, but rather a highly plastic response determined primarily by the environmental history of the individual insect.

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.

Nonconstant Thermal Regimes Enhance Overwintering Success and Accelerate Diapause Development for Smicronyx fulvus (Coleoptera: Curculionidae).

Recent populations of the red sunflower seed weevil, Smicronyx fulvus LeConte (Coleoptera: Curculionidae), have been inconsistent or declining, particularly in North Dakota. Consequently, research on weevil biology, including development of resistant germplasm, has been limited. To determine whether cold storage of diapausing larvae could be improved, nonconstant temperature treatments (fluctuating thermal regime [FTR] and thermoperiod [TP]) were tested versus a constant 6°C for storage up to 365 d. Both alternate temperature treatments produced more adult weevils than constant 6°C for short (42, 91 d) storage, while all temperature treatments were good (≥60% adult emergence) at moderate term (182 d) cold storage, and FTR was best for long (365 d) periods. Excluding the 14-d storage period, which produced too few weevils for comparison, each doubling of cold storage time (e.g., from 42 to 91 d, 91 to 182 d), usually decreased the number of days to 50% relative emergence by ∼10 d. After 365 d of larval storage, emerged S. fulvus adults successfully infested sunflowers in a plant growth chamber, with damage per female similar to that observed in field trials. Compared with previous efforts to store weevil larvae, the method of collection and FTR storage is either more effective (greater adult emergence and reduced parasitism) or more time-efficient, and should permit year-round research using S. fulvus adults. Because successful emergence under FTR was >75% after 365 d, additional research would be required to determine the maximum effective duration of cold storage for S. fulvus.

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.

A fluctuating thermal regime improves long-term survival of quiescent prepupal Megachile rotundata (Hymenoptera: Megachilidae).

The alfalfa leafcutting bee Megachile rotundata (F.) is the primary pollinator for alfalfa seed production. Under standard management conditions, the alfalfa leafcutting bee develops to the diapausing prepupal stage under field conditions, after which they are cold-stored at a static temperature until the following spring, when temperatures are raised and development resumes. We have assessed the effects of a fluctuating thermal regime (FTR) during overwintering cold storage, where bees were exposed to a daily 1 h pulse of 20 degrees C, and compared viability and insect quality to bees stored under a static thermal regime. Our results demonstrate that implementing an FTR protocol dramatically increases the survival of cold-stored alfalfa leafcutting bees, effectively extending their shelf-life into the subsequent growing season. These findings could substantially ameliorate significant obstacles that restrict the more widespread use of this important pollinator, such as the biological constraints that restrict its use in early blooming crops, and yearly fluctuations in bee prices that add significant financial uncertainty to end users. This study also strengthens a growing body of evidence that indicates FTR protocols are superior to static thermal regime protocols for insect cold storage.

Sugar feeding improves survival of nondiapausing cold-stored Culex pipiens.

The continuous culture of mosquitoes is a costly endeavor for vector biology laboratories. In addition to the resources that must be committed to colony maintenance, biological costs, including genetic drift and accidental colony loss, also can occur. Although alternatives do exist, their application to mosquitoes is limited. Mosquito cryopreservation remains elusive, and many important species lack a well-defined diapause. Previously, we demonstrated that cold storing nondiapausing mated adult females of the northern house mosquito, Culex pipiens L. resulted in a nearly four-fold increase in longevity when measured at the LT50, allowing for cold storage for up to 10 wk. In the current study, we used sugar feeding during cold storage to significantly improve cold storage longevity. At 6 degrees C, the LT50 of cold stored females was 23 wk, and 100% mortality was not realized until 43 wk. Cold-stored females did exhibit reduced fecundity, but egg production returned to normal levels within two generations. These results suggest that cold storage without diapause induction is a viable option for Cx. pipiens, and with the addition of sugar feeding, a colony could be maintained with less than two generations per year.

Duration and frequency of a high temperature pulse affect survival of emergence-ready Megachile rotundata (Hymenoptera: Megachilidae) during low-temperature incubation.

Synchronizing Megachile rotundata (F.) nesting activity with alfalfa bloom is essential for ensuring optimal pollination for alfalfa seed production. This is achieved by timing the initiation of spring bee incubation so that adults will emerge -2 wk before peak bloom. If weather conditions change so as to delay the bloom, bee managers will commonly expose the developing bees to a period of low-temperature incubation to slow their development. We have previously demonstrated survival during low-temperature incubation can be significantly increased by using a fluctuating thermal regime (FTR) where the bees receive a daily pulse at 20 degrees C. A FTR incubation protocol is composed of a number of different components, such as the base and pulse temperatures, and the duration and frequency of the pulse. In this investigation, the effect of the duration of the pulse (5-120 min) and the frequency of a pulse (twice daily to weekly) on the survival of developing M. rotundata was examined. A pulse as short as 5 min at 20 degrees C increased survival of the developing bees as compared with the constant 6 degrees C controls. Increasing the pulse duration induced a further increase in tolerance to 6 degrees C. As with the pulse duration, increasing the pulse frequency from once weekly to twice daily had a significant effect on improving the bees tolerance to low-temperature incubation. This investigation further strengthens the argument that a FTR protocol is superior to using a constant low-temperature exposure for interrupting the spring incubation of M. rotundata.

Timing of Diapause Initiation and Overwintering Conditions Alter Gene Expression Profiles in Megachile rotundata.

Within the United States and Canada, the primary pollinator of alfalfa is the alfalfa leafcutting bee (ALCB), Megachile rotundata. Our previous findings showed that overwintering conditions impacted gene expression profile in ALCB prepupae that entered diapause early in the season. However, ALCB are a bivoltine species, which begs the question of whether bees entering diapause later in the season also show this trend. To better understand the effects of the timing of diapause initiation, we analyzed mRNA copy number of genes known to be involved in diapause regulation in early and late season diapausing ALCB that were overwintered in field conditions or using current agricultural management conditions. We hypothesized that overwintering conditions for late diapausing bees also affects gene expression profiles. Our results showed that expression profiles were altered by both overwintering condition and timing of diapause initiation, with bees that entered diapause earlier in the season showing different expression patterns than those that entered diapause later in the season. This trend was seen in expression of members of the cyclin family and several targets of the insulin signaling pathway, including forkhead box protein O (FOXO), which is known to be important for diapause regulation and stress responses. But, of the genes screened, the proto-oncogene, Myc, was the most impacted by the timing of diapause initiation. Under field conditions, there were significant differences in Myc expression between the early and late season samples in all months except for November and February. This same general trend in Myc expression was also seen in the laboratory-maintained bees with significant difference in expression in all months except for November, February, and May. These results support previous conclusions from our research showing that the molecular regulation of diapause development in ALCB is not a simple singular cascade of gene expression but a highly plastic response that varies between bees depending upon their environmental history.

Effects of Temperature and Wildflower Strips on Survival and Macronutrient Stores of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae) Under Extended Cold Storage.

Megachile rotundata (F.) is an important pollinator of alfalfa in the United States. Enhancing landscapes with wildflowers is a primary strategy for conserving pollinators and may improve the sustainability of M. rotundata. Changing cold storage temperatures from a traditionally static thermal regime (STR) to a fluctuating thermal regime (FTR) improves overwintering success and extends M. rotundata's shelf life and pollination window. Whether floral resources enhance overwintering survival and/or interact with a thermal regime are unknown. We tested the combined effects of enhancing alfalfa fields with wildflowers and thermal regime on survival and macronutrient stores under extended cold storage (i.e., beyond one season). Megachile rotundata adults were released in alfalfa plots with and without wildflower strips. Completed nests were harvested in September and stored in STR. After a year, cells were randomly assigned to remain in STR for 6 months or in FTR for a year of extended cold storage; emergence rates were observed monthly. Macronutrient levels of emerged females were assessed. FTR improved M. rotundata survival but there was no measurable effect of wildflower strips on overwintering success or nutrient stores. Timing of nest establishment emerged as a key factor: offspring produced late in the season had lower winter survival and dry body mass. Sugars and glycogen stores increased under FTR but not STR. Trehalose levels were similar across treatments. Total lipid stores depleted faster under FTR. While wildflowers did not improve M. rotundata survival, our findings provide mechanistic insight into benefits and potential costs of FTR for this important pollinator.

Cyclic CO(2) emissions during the high temperature pulse of fluctuating thermal regime in eye-pigmented pupae of Megachile rotundata.

Megachile rotundata (Hymenoptera: Megachilidae), the primary pollinator used in alfalfa seed production, may need to be exposed to low-temperature storage to slow the insects' development to better match spring emergence with the alfalfa bloom. It has been demonstrated that using a fluctuating thermal regime (FTR) improves the tolerance of pupae to low temperatures. Carbon dioxide emission rates were compared between four different FTRs, all with a base temperature of 6°C and a daily high-temperature pulse. Four different high-temperature pulses were examined, 15 or 25°C for 2h and 20°C for 1 or 2h. A subset of pupae at the FTR base temperature of 6°C exhibited continuous gas exchange and, once ramped to 20 or 25°C, shifted to cyclic gas exchange. As temperatures were ramped down from the high-temperature pulse to 6°C, the pupae reverted to continuous gas exchange. The following conclusions about the effect of FTR on the CO(2) emissions of M. rotundata pupae exposed to low-temperature storage during the spring incubation were reached: 1) the high temperature component of the FTR was the best predictor of respiratory pattern; 2) neither pupal body mass nor days in FTR significantly affected which respiratory pattern was expressed during FTRs; 3) cyclic gas exchange was induced only in pupae exposed to temperatures greater than 15°C during the FTR high temperature pulse; and 4) a two hour pulse at 25°C doubled the number of CO(2) peaks observed during the FTR pulse as compared to a two hour pulse at 20°C.