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 (FTR), 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 seven 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 seven 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.
more »
« less
Immediate Transcriptional Response to a Temperature Pulse under a Fluctuating Thermal Regime
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.
more »
« less
- NSF-PAR ID:
- 10129461
- Date Published:
- Journal Name:
- Integrative and Comparative Biology
- Volume:
- 59
- Issue:
- 2
- ISSN:
- 1540-7063
- Page Range / eLocation ID:
- 320 to 337
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract Insects exposed to low temperature stress can experience chill injury, but incorporating fluctuating thermoprofiles increases survival and blocks the development of sub-lethal effects. The specific parameters required for a protective thermoprofile are poorly understood, because most studies test a limited range of thermoprofiles. For example, thermoprofiles with a wave profile may perform better than a square profile, but these two profiles are rarely compared. In this study, two developmental stages of the alfalfa leafcutting bee, Megachile rotundata, eye-pigmented pupae, and emergence-ready adults, were exposed to one of eight thermoprofiles for up to 8 weeks. All the thermoprofiles had a base of 6°C and a peak temperature of either 12°C or 18°C. The duration at peak temperature varied depending on the shape of the thermoprofile, either square or wave form. Two other treatments acted as controls, a constant 6°C and a fluctuating thermal regime (FTR) with a base temperature of 6°C that was interrupted daily by a single, 1-h pulse at 20°C. Compared with constant 6°C, all the test thermoprofiles significantly improved survival. Compared with the FTR control, the thermoprofiles with a peak temperature of 18°C outperformed the 12°C profiles. Bees in the eye-pigmented stage exposed to the 18°C profiles separated into two groups based on the shape of the profile, with higher survival in the square profiles compared with the wave profiles. Bees in the emergence-ready stage exposed to 18°C profiles all had significantly higher survival than bees in the FTR controls. Counter to expectations, the least ecologically relevant thermoprofiles (square) had the highest survival rates and blocked the development of sub-lethal effects (delayed emergence).
-
more » « less
Abstract The eastern oyster, Crassostrea virginica, forms reefs that provide critical services to the surrounding ecosystem. These reefs are at risk from climate change, in part because altered rainfall patterns may amplify local fluctuations in salinity, impacting oyster recruitment, survival, and growth. As in other marine organisms, warming water temperatures might interact with these changes in salinity to synergistically influence oyster physiology. In this study, we used comparative transcriptomics, measurements of physiology, and a field assessment to investigate what phenotypic changes C. virginica uses to cope with combined temperature and salinity stress in the Gulf of Mexico. Oysters from a historically low salinity site (Sister Lake, LA) were exposed to fully crossed temperature (20°C and 30°C) and salinity (25, 15, and 7 PSU) treatments. Using comparative transcriptomics on oyster gill tissue, we identified a greater number of genes that were differentially expressed (DE) in response to low salinity at warmer temperatures. Functional enrichment analysis showed low overlap between genes DE in response to thermal stress compared with hypoosmotic stress and identified enrichment for gene ontologies associated with cell adhesion, transmembrane transport, and microtubule-based process. Experiments also showed that oysters changed their physiology at elevated temperatures and lowered salinity, with significantly increased respiration rates between 20°C and 30°C. However, despite the higher energetic demands, oysters did not increase their feeding rate. To investigate transcriptional differences between populations in situ, we collected gill tissue from three locations and two time points across the Louisiana Gulf coast and used quantitative PCR to measure the expression levels of seven target genes. We found an upregulation of genes that function in osmolyte transport, oxidative stress mediation, apoptosis, and protein synthesis at our low salinity site and sampling time point. In summary, oysters altered their phenotype more in response to low salinity at higher temperatures as evidenced by a higher number of DE genes during laboratory exposure, increased respiration (higher energetic demands), and in situ differential expression by season and location. These synergistic effects of hypoosmotic stress and increased temperature suggest that climate change will exacerbate the negative effects of low salinity exposure on eastern oysters.
-
more » « less
SUMMARY Plants respond to low temperatures by altering the mRNA abundance of thousands of genes contributing to numerous physiological and metabolic processes that allow them to adapt. At the post‐transcriptional level, these cold stress‐responsive transcripts undergo alternative splicing, microRNA‐mediated regulation and alternative polyadenylation, amongst others. Recently, m6A, m5C and other mRNA modifications that can affect the regulation and stability of RNA were discovered, thus revealing another layer of post‐transcriptional regulation that plays an important role in modulating gene expression. The importance of m6A in plant growth and development has been appreciated, although its significance under stress conditions is still underexplored. To assess the role of m6A modifications during cold stress responses, methylated RNA immunoprecipitation sequencing was performed in Arabidopsis seedlings esposed to low temperature stress (4°C) for 24 h. This transcriptome‐wide m6A analysis revealed large‐scale shifts in this modification in response to low temperature stress. Because m6A is known to affect transcript stability/degradation and translation, we investigated these possibilities. Interestingly, we found that cold‐enriched m6A‐containing transcripts demonstrated the largest increases in transcript abundance coupled with increased ribosome occupancy under cold stress. The significance of the m6A epitranscriptome on plant cold tolerance was further assessed using the
mta mutant in which the major m6A methyltransferase gene was mutated. Compared to the wild‐type, along with the differences inCBFs andCOR gene expression levels, themta mutant exhibited hypersensitivity to cold treatment as determined by primary root growth, biomass, and reactive oxygen species accumulation. Furthermore, and most importantly, both non‐acclimated and cold‐acclimatedmta mutant demonstrated hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis. -
more » « less
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.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/icb/icz096
