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Stress can influence lifespan in both positive and negative ways, depending on exposure intensity and duration. However, mechanisms driving positive stress effects on lifespan remain poorly understood. Prolonged hypoxia extends the lifespan of overwintering prepupal Megachile rotundata. Here, we explored telomere length and reduced oxidative stress as potential mechanisms of this extended lifespan. We hypothesized high antioxidant capacity under hypoxia reduces oxidative damage and telomere loss. We exposed prepupae to 10%, 21% or 24% oxygen for up to 9 months and measured monthly survival, telomere length, antioxidant capacity and lipid peroxidation across treatment duration for prepupae and adults. After 9 months of exposure, survival was highest in hypoxia and lowest in hyperoxia. Telomere length did not differ among oxygen treatments but increased in adults compared with prepupae. Total antioxidant capacity and lipid peroxidation showed no significant differences among oxygen treatments, suggesting compensatory responses to maintain baseline oxidative levels. 

ABSTRACT <p>Social bees have been extensively studied for their gut microbial functions, but the significance of the gut microbiota in solitary bees remains less explored. Solitary bee,<italic>Megachile rotundata</italic>females provision their offspring with pollen from various plant species, harboring a diverse microbial community that colonizes larvae guts. The<italic>Apilactobacillus</italic>is the most abundant microbe, but evidence concerning the effects of<italic>Apilactobacillus</italic>and other provision microbes on growth and survival are lacking. We hypothesized that the presence of<italic>Apilactobacillus</italic>in abundance would enhance larval and prepupal development, weight, and survival, while the absence of intact microbial communities was expected to have a negative impact on bee fitness. We reared larvae on pollen provisions with naturally collected microbial communities (Natural pollen) or devoid of microbial communities (Sterile pollen). We also assessed the impact of introducing<italic>Apilactobacillus micheneri</italic>by adding it to both types of pollen provisions. Feeding larvae with sterile pollen +<italic>A. micheneri</italic>led to the highest mortality rate, followed by natural pollen +<italic>A. micheneri</italic>, and sterile pollen. Larval development was significantly delayed in groups fed with sterile pollen. Interestingly, larval and prepupal weights did not significantly differ across treatments compared to natural pollen-fed larvae. 16S rRNA gene sequencing found a dominance of<italic>Sodalis</italic>, when<italic>A. micheneri</italic>was introduced to natural pollen. The presence of<italic>Sodalis</italic>with abundant<italic>A. michene</italic>ri suggests potential crosstalk between both, shaping bee nutrition and health. Hence, this study highlights that the reliance on nonhost-specific environmental bacteria may not impact fitness of<italic>M. rotundata</italic>.</p><sec><title>IMPORTANCEThis study investigates the impact of environmentally acquired gut microbes of solitary bee fitness with insights into the microbial ecology of bee and their health. While the symbiotic microbiome is well-studied in social bees, the role of environmental acquired microbiota in solitary bees remains unclear. Assessing this relationship in a solitary pollinator, the leaf-cutting bee,Megachile rotundata, we discovered that this bee species does not depend on the diverse environmental bacteria found in pollen for either its larval growth or survival. Surprisingly, high concentrations of the most abundant pollen bacteria, Apilactobacillus micheneridid not consistently benefit bee fitness, but caused larval mortality. Our findings also suggest an interaction betweenApilactobacillusand theSodalisand perhaps their role in bee nutrition. Hence, this study provides significant insights that contribute to understanding the fitness, conservation, and pollination ecology of other solitary bee species in the future. 

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. 

Abstract 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.