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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. 

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.

 

Megachile rotundata exhibits a facultative prepupal diapause but the cues regulating diapause initiation are not well understood. Possible cues include daylength and temperature. Megachile rotundata females experience changing daylengths over the nesting season that may influence diapause incidence in their offspring through a maternal effect. Juvenile M . rotundata spend their developmental period confined in a nesting cavity, potentially subjected to stressful temperatures that may affect diapause incidence and survival. To estimate the impact of daylength and nest cavity temperature on offspring diapause, we designed a 3D printed box with iButtons that measured nest cavity temperature. We observed nest building throughout the season, monitored nest cavity temperature, and followed offspring through development to measure diapause incidence and mortality. We found that daylength was a cue for diapause, and nest cavity temperature did not influence diapause incidence. Eggs laid during long days had a lower probability of diapause. Siblings tended to have the same diapause status, explaining a lot of the remaining variance in diapause incidence. Some females established nests that contained both diapausing and nondiapausing individuals, which were distributed throughout the nest. Nest cavities reached stressful temperatures, which decreased survival. Mortality was significantly higher in nondiapausing bees and the individuals that were laid first in the nest. In conclusion, we demonstrate a maternal effect for diapause that is mediated by daylength and is independent of nest box temperature. 

Abstract 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. 

Abstract 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.