Comparison of the Chemical Compositions of the Cuticle and Dufour's Gland of Two Solitary Bee Species from Laboratory and Field Conditions.

Species-specific biochemistry, morphology, and function of the Dufour's gland have been investigated for social bees and some non-social bee families. Most of the solitary bees previously examined are ground-nesting bees that use Dufour's gland secretions to line brood chambers. This study examines the chemistry of the cuticle and Dufour's gland of cavity-nesting Megachile rotundata and Osmia lignaria, which are species managed for crop pollination. Glandular and cuticular lipid compositions were characterized and compared to each other and according to the nesting experience of adult females. Major lipid classes found were hydrocarbons, free fatty acids, and wax esters. Many components were common to the cuticle and Dufour's glands of each species, yet not identical in number or relative composition. Wax esters and fatty acids were more prevalent in Dufour's glands of M. rotundata than on cuticles. Wax esters were more abundant on cuticles of O. lignaria than in Dufour's glands. In both species, fatty acids were more prevalent in glands of field-collected females compared to any other sample type. Chemical profiles of cuticles and glands were distinct from each other, and, for O. lignaria, profiles of laboratory-maintained bees could be distinguished from those of field-collected bees. Comparison of percentiles of individual components of cuticular and glandular profiles of the same bee showed that the proportions of some cuticular components were predictive of the proportion of the same glandular components, especially for nesting females. Lastly, evidence suggested that Dufour's gland is the major source of nest-marking substances in M. rotundata, but evidence for this role in O. lignaria was less conclusive.

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.

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.

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.

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.

A fluctuating thermal regime improves survival of cold-mediated delayed emergence in developing Megachile rotundata (Hymenoptera: Megachilidae).

A significant concern in the commercial application of the alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae), for pollination is synchronizing bee emergence and activity with peak crop bloom. Previous studies have demonstrated that the commercial spring incubation of this species can be successfully interrupted by low temperature incubation, thereby slowing development and giving producers flexibility in timing emergence to weather conditions or crop bloom. In this study, we demonstrate that the implementation of a fluctuating thermal regime, during which bees are given a daily one hour pulse of high temperature, markedly increases the "shelf-life" of individuals of this species. Although constant temperatures can be used to store bees for up to 1 wk with no decrease in survival, properly staged bees can be stored for up to 6 wk without a significant decrease in percentage of emergence. Hence, we expect fluctuating thermal regime protocols to become a valuable tool for M. rotundata managers, especially when timing nesting activity with peak bloom to maximize effectiveness.

Interrupted incubation and short-term storage of the alfalfa pollinator Megachile rotundata (Hymenoptera: Megachilidae): a potential tool for synchronizing bees with bloom.

A useful technique for synchronizing pollinators with the alfalfa, Medicago savita L. (Fabaceae), bloom is to interrupt the late spring incubation of developing bee pupae and pharate adults of Megachile rotundata (F.) (Hymenoptera: Megachilidae) with short-term low-temperature storage. However, low-temperature exposure can be stressful depending on the temperature, duration of exposure, and the developmental stage exposed. To evaluate the effect of low-temperature storage after development had already been initiated by exposure to 29 degrees C, three developmental stages (eye pigment pupae, body pigment pupae, and pharate adults ready to emerge) were exposed to 6, 12, or 18 degrees C for durations up to 28 d. The effect of delaying the termination of overwintering storage (6 degrees C) from April to July ("April bees" and "July bees," respectively) also was examined. The following observations were made: (1) All developmental stages of the April bees and July bees examined can be stored without harmful effects, as measured by percentage of survival, for 14 d at 12 degrees C and above. (2) Postponing the termination of overwintering storage at 6 degrees C from April until July significantly decreased the mean number of days to 95% adult emergence in the July ready-to-emerge bees across the three temperatures tested. (3) The increase of overwintering duration also caused a decrease in the postemergence longevity of the ready-to-emerge adults stored at 6 degrees C for 14 d. (4) Of the three storage temperatures examined, 18 degrees C seems to be the optimal storage temperature for short-term storage of developing bees because of their slow but continuing development without increased mortality either during storage or after emergence.

Bee population returns and cherry yields in an orchard pollinated with Osmia lignaria (Hymenoptera: Megachilidae).

During 1998-2003, we used populations of the solitary bee Osmia lignaria Say to pollinate a commercial sweet cherry orchard in northern Utah. Bee densities released each year ranged from 1290 to 1857 females/ha, with approximately twice as many males. Female progeny produced each year were greater than parental populations released, except in 2003, when nesting was poor due to bird predation. Despite poor weather during bloom, and in contrast to most other local producers, the study orchard produced harvestable crops in 1999 (2,964 kg/ha) and 2001 (3,154 kg/ha). In 1998 and 2000, record yields were obtained (10,625 and 12,096 kg/ha, respectively). Including only those years with harvestable crops, average production was 2.2 times higher in 1998-2003 (when O. lignaria populations were used) compared with 1992-1997 (when 10 Apis mellifera hives were used). This is the first study reporting multiyear cherry yields in an orchard pollinated with O. lignaria in North America.

Effects of three-dimensional and color patterns on nest location and progeny mortality in alfalfa leafcutting bee (Hymenoptera: Megachilidae).

ABSTRACT In alfalfa, Medicago sativa L., seed production where high bee densities are released, alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae), females may enter several nesting holes before locating their nests. Such levels of "wrong hole" visits lead to an increase in the time spent by females locating their own nests, thereby decreasing alfalfa pollination efficiency and possibly healthy brood production. The objectives of this study were to determine the effect of different nesting board configurations in commercial alfalfa leafcutting bee shelters (separating nesting boards, applying a three-dimensional pattern to the boards, applying a color contrast pattern, or applying a combination of three-dimensional and color contrast patterns) on nest location performance, on the incidence of chalkbrood disease, and on the incidence of broodless provisions. Separating the nesting boards inside shelters improved the ability of females to locate their nests. An increase in nest location performance also occurred in boards with the three-dimensional pattern and the combined three-dimensional and color contrast pattern, compared with the uniform board (a standard configuration currently used commercially). The percentage of provisioned cells that were broodless was not statistically different between treatments, but the percentage of larvae infected with chalkbrood decreased by half in the three-dimensional board design, compared with the uniform board.

How Hives Collapse: Allee Effects, Ecological Resilience, and the Honey Bee.

We construct a mathematical model to quantify the loss of resilience in collapsing honey bee colonies due to the presence of a strong Allee effect. In the model, recruitment and mortality of adult bees have substantial social components, with recruitment enhanced and mortality reduced by additional adult bee numbers. The result is an Allee effect, a net per-individual rate of hive increase that increases as a function of adult bee numbers. The Allee effect creates a critical minimum size in adult bee numbers, below which mortality is greater than recruitment, with ensuing loss of viability of the hive. Under ordinary and favorable environmental circumstances, the critical size is low, and hives remain large, sending off viably-sized swarms (naturally or through beekeeping management) when hive numbers approach an upper stable equilibrium size (carrying capacity). However, both the lower critical size and the upper stable size depend on many parameters related to demographic rates and their enhancement by bee sociality. Any environmental factors that increase mortality, decrease recruitment, or interfere with the social moderation of these rates has the effect of exacerbating the Allee effect by increasing the lower critical size and substantially decreasing the upper stable size. As well, the basin of attraction to the upper stable size, defined by the model potential function, becomes narrower and shallower, indicating the loss of resilience as the hive becomes subjected to increased risk of falling below the critical size. Environmental effects of greater severity can cause the two equilibria to merge and the basin of attraction to the upper stable size to disappear, resulting in collapse of the hive from any initial size. The model suggests that multiple proximate causes, among them pesticides, mites, pathogens, and climate change, working singly or in combinations, could trigger hive collapse.

Optimizing Fluctuating Thermal Regime Storage of Developing Megachile rotundata (Hymenoptera: Megachilidae).

The alfalfa leafcutting bee, Megachile rotundata (F.), is the primary pollinator for alfalfa seed production in North America. Under current management practice, developing pupae are incubated at 29-30°C until the adults emerge for pollination. If unfavorable spring weather delays peak alfalfa bloom, managers will cool pupae to slow development, which can increase mortality and causes sublethal effects. Previously, we demonstrated that exposure to a fluctuating thermal regime (FTR) increases survival and extends the viable storage period. To determine the optimal conditions for FTR during storage of developing M. rotundata , we examined four variables: temperature of the daily warm pulse, duration of the warm pulse, number of weeks exposed to the FTR treatment, and developmental stage of the bee. Survival was measured by successful eclosion to the adult stage. Under all conditions, exposure to FTR increased survival compared with exposure to a constant 6°C. When the temperature of the daily warm pulse was 20-25°C from a base temperature of 6°C, and the pulse duration was extended to 3 h, survival rates were as high as those observed under standard storage conditions (29°C). Under this FTR storage protocol, bee managers can delay emergence for ∼8 wk without significant decreases in survival. Our findings have substantial economic implications for bee management and alfalfa seed production by increasing the flexibility and efficiency of M. rotundata adult emergence.

Thermoperiodism Synchronizes Emergence in the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae).

Alfalfa seed production in the northwestern United States and western Canada is heavily dependent upon the pollinating services of Megachile rotundata (F.) (Hymenoptera: Megachilidae). M. rotundata females nest in cavities either naturally occurring or in artificial nesting blocks. Because of the physical nature of the nest, M. rotundata brood may have limited to no exposure to photoperiodic cues in order to regulate important circadian functions. Therefore, various thermoperiod regimes were used to characterize the possible role of thermoperiodism in synchronizing M. rotundata adult emergence. Adult emergence was monitored using a microprocessor-controlled event logger. Incubating bees under constant 29°C and darkness resulted in an arhythmic adult emergence pattern. Exposing developing M. rotundata to a thermoperiod synchronized emergence to the beginning of the thermophase and decreased the total number of days required for all adults to emerge. The amplitude of the thermoperiod regulated the timing of peak emergence in relationship to the increase in temperature. A thermoperiod amplitude of only 2°C was sufficient to synchronize peak adult emergence to take place during the rise in temperature. Increasing the amplitude of the thermoperiod to 4 or 8°C caused a positively correlated shift in peak emergence to later in the thermophase. Brood stored under constant 29°C and darkness for different durations (May or June early in the growing season or July or August late in the growing season) or under a fluctuating thermal regime (base temperature of 6°C and daily 1-h pulse of 20°C until September or November) maintained their capacity for entraining emergence timing by thermoperiodism.

Alfalfa leafcutting bee population dynamics, flower availability, and pollination rates in two Oregon alfalfa fields.

Since the 1970s, it has become increasingly difficult for U.S. alfalfa seed producers to maintain Megachile rotundata (F.) populations used for alfalfa, Medicago sativa L., pollination. In 1998, we monitored M. rotundata population dynamics and foraging behavior, as well as alfalfa bloom and pollination rates in two fields in eastern Oregon. Despite marked differences in bee management, establishment was very similar in the two fields (approximately 0.5 females per nesting cavity) and lagged peak bloom by approximately 2 wk. Pollination rates increased from 0-10% in the first 3 wk to 80-90% in week 4-5. By then, M. rotundata females had difficulty finding untripped (nonpollinated) flowers and visited large numbers of already tripped or not fully matured flowers. M. rotundata progeny mortality was very high (54-78%). Estimated seed yields were similar in both fields. We contend similar seed yields, and improved bee production, could be accomplished with smaller bee populations, better timed with alfalfa bloom.

Exposure to Suboptimal Temperatures during Metamorphosis Reveals a Critical Developmental Window in the Solitary Bee, Megachile rotundata.

Metamorphosis is an important developmental stage for holometabolous insects, during which adult morphology and physiology are established. Proper development relies on optimal body temperatures, and natural ambient temperature (Ta) fluctuations, especially in spring or in northern latitudes, could result in interruptions to development. It is unclear how low-Ta exposure may affect insects that are actively developing. To understand how suboptimal Ta may affect metamorphosing insects, we used the alfalfa leafcutting bee, Megachile rotundata (Fabricius), a solitary, cavity-nesting bee that spends its juvenile and pupal stages within a brood cell. We characterized suites of physiological traits, rather than just using a singular parameter to determine effects of sublethal Ta stress. Metamorphosing M. rotundata were exposed to either constant or fluctuating low-Ta stress and compared to control bees allowed to develop normally. All bees survived and emerged as adults, but the constant low-Ta-stressed bees were affected most severely. Male constant low-Ta-stressed bees had decreased flight performance (lower metabolic rate, shorter flight bouts, decreased wing length), suggesting that the stress altered muscular or neurological development. Constant low-Ta-stressed bees also had altered activity levels, providing more support for the hypothesis that low-Ta stress causes long-term neurological defects. Exposure to fluctuating low Ta also delayed development time for both sexes; males had decreased adult life span, and both sexes had shortened wings. Together, these results provide evidence for a critical developmental window during metamorphosis and suggest that there may be severe implications for bees in the wild that are exposed to low-Ta stressors.

Social evolution. Genomic signatures of evolutionary transitions from solitary to group living.

The evolution of eusociality is one of the major transitions in evolution, but the underlying genomic changes are unknown. We compared the genomes of 10 bee species that vary in social complexity, representing multiple independent transitions in social evolution, and report three major findings. First, many important genes show evidence of neutral evolution as a consequence of relaxed selection with increasing social complexity. Second, there is no single road map to eusociality; independent evolutionary transitions in sociality have independent genetic underpinnings. Third, though clearly independent in detail, these transitions do have similar general features, including an increase in constrained protein evolution accompanied by increases in the potential for gene regulation and decreases in diversity and abundance of transposable elements. Eusociality may arise through different mechanisms each time, but would likely always involve an increase in the complexity of gene networks.

PHYLOGENETIC RELATIONSHIPS OF SCOTUSSA AND LEIOTETTIX (ORTHOPTERA: ACRIDIDAE).

Abstract- A cladistic analysis of the South American grasshopper generaScotussaandLeiotettixwas performed in order to test the monophyly of these genera.Eurotettix, Chlorusand theDichroplus bergispecies group were included as terminal taxa. The genusAtrachelacriswas used to root the tree. Twenty-nine characters from external morphology, male genitalia and female ovipositor were used in the analysis. In order to test for association between the structural change that occurred in the ovipositor valves ofScotussaand the functional change of the oviposition habits, the data matrix was partitioned and two analyses were performed. Characters from the female ovipositor were excluded from the data set used in the first analysis and another analysis was performed where all the characters were included in the analysis. Information on oviposition habits was then mapped on the cladogram, to determine the transformation for performance. Both analyses yielded only one most parsimonious tree and produced congruent results, confirming the monophyly ofLeiotettixandScotussaand corroborating their close relationship. Characters from the female ovipositor valves were informative not only at the species level but also at higher levels in the cladogram. The results also support the hypothesis of association between the structural change that occurred in the ovipositor valves ofScotussawith the functional change in the oviposition habits. However, this association did not seem to be correlated with the adaptive radiation in the genus.

Density dependence in rangeland grasshoppers (Orthoptera: Acrididae).

We tested for the existence of density dependence in annual adult rangeland grasshopper (Orthoptera: Acrididae) data from Montana, USA (1951-1991). Statistical density dependence was, in the sense of a stochastic equilibrium or return tendency, detected in all of the grasshopper mean density time-series from the three major physiographic regions of the state, Northern Glaciated Plains, Southern Unglaciated Plains, and Western Mountains. Parameters were estimated for a model that described the stochastic equilibrium characteristics of regional mean densities. The analyses showed that rangeland grasshopper regional densities fluctuate according to gamma distribution with a mean of 6.1-6.3 grasshoppers per m2. Further, when regions exhibit outbreaks, the resulting infestation period (duration of outbreak) is short, spanning only a few generations.

Cell position during larval development affects postdiapause development in Megachile rotundata (Hymenoptera: Megachilidae).

Megachile rotundata (F.) (Hymenoptera: Megachilidae) is the primary pollinator of alfalfa in the northwestern United States and western Canada and provides pollination services for onion, carrot, hybrid canola, various legumes, and other specialty crops. M. rotundata females are gregarious, nest in cavities either naturally occurring or in artificial nesting blocks, where they construct a linear series of brood cells. Because of the physical layout of the nest, the age of the larvae within the nest and the microenvironment the individual larvae experience will vary. These interacting factors along with other maternal inputs affect the resulting phenotypes of the nest mates. To further our understanding of in-nest physiology, gender and developmental rates were examined in relationship to cell position within the nest. Eighty-two percent of the females were located within the first three cells, those furthest from the nest entrance. For those individuals developing in cells located in the deepest half of the nest, the sex of the previous bee had a significant effect on the female decision of the gender of the following nest mate. Removing the prepupae from the nest and rearing them under identical conditions demonstrated that position within the nest during larval development had a significant effect on the postdiapause developmental rates, with males whose larval development occurred deeper in the nest developing more slowly than those toward the entrance. No positional effect on postdiapause developmental rates was noted for the females. The cell position effect on male postdiapause developmental rate demonstrates that postdiapause development is not a rigid physiological mechanism uniform in all individuals, but is a dynamic plastic process shaped by past environmental conditions.

Changes in respiratory structure and function during post-diapause development in the alfalfa leafcutting bee, Megachile rotundata.

Megachile rotundata, the alfalfa leafcutting bee, is a solitary, cavity-nesting bee. M. rotundata develop from eggs laid inside brood cells constructed from leaf pieces and placed in series in an existing cavity. Due to the cavity nesting behavior of M. rotundata, developing bees may experience hypoxic conditions. The brood cell itself and the position of cell inside the cavity may impact the rates of oxygen diffusion creating hypoxic conditions for developing animals. We hypothesized that bees would be adapted to living in hypoxia and predicted that they would be highly tolerant of hypoxic conditions. To test the hypothesis, we measured critical PO2 (Pcrit) in pupal M. rotundata of varying ages. Defined as the atmospheric O2 level below which metabolic rate cannot be sustained, Pcrit is a measure of an animal's respiratory capacity. Using flow through respirometry, we measured CO2 emission rates of developing bees exposed to 21, 10, 6, 5, 4, 3, 2, 1, and 0 kPa PO2 and statistically determined Pcrit. Mean Pcrit was 4 kPa PO2 and ranged from 0 to 10 kPa PO2, similar to those of other insects. Pcrit was positively correlated with age, indicating that as pupae aged, they were less tolerant of hypoxia. To determine if there were developmental changes in tracheal structure that accounted for the increase in Pcrit, we used synchrotron X-ray imaging and measured the diameter of several tracheae in the head and abdomen of developing bees. Analyses of tracheal diameters showed that tracheae increased in size as animals aged, but the magnitude of the increase varied depending on which trachea was measured. Tracheal diameters increased as pupae molted and decreased as they neared adult emergence, possibly accounting for the decrease in hypoxia tolerance. Little is known about respiratory structures during metamorphosis in bees, and this study provides the first description of tracheal system structure and function in developing M. rotundata. Studies such as this are important for understanding how basic physiological parameters change throughout development and will help to maintain healthy pollinator populations.