high-throughput-assays-of-critical-thermal-limits-in-insects

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Pepck is a metabolic enzyme that participates in gluconeogenesis through the conversion of oxaloacetate into phosphoenol pyruvate. Numerous transcriptomic studies have identified Pepck as a potential key player during diapause and various stresses responses. Here, we describe expression patterns of both cytosolic and mitochondrial isoforms of Pepck throughout development, during diapause, and in response to starvation and cold shock in the flesh fly, Sarcophaga bullata. We cloned full-length transcripts for both Pepck isoforms and observed that expression of both genes varied throughout development. Diapausing pupae have the highest relative expression of both isoforms, suggesting participation in the anticipatory production of sugars and sugar alcohols that occurs during this overwintering stage. In response to acute stress, the cytosolic isoform was upregulated whereas the mitochondrial variant remained unchanged. Cytosolic Pepck was strongly upregulated after 2 h recovery from cold shock and returned to baseline levels within 8 h. In response to 24 h of starvation, the cytosolic isoform was similarly upregulated and returned to control levels after 24 h of recovery. Acute stress is known to incur a metabolic cost, and Pepck could be a key player in this response. Although it remains unclear why there is such a dramatic divergence in the expression of the two isoforms, the distinction suggests specific roles for the two isoforms that depend on the developmental status of the fly and the stress conditions to which it is exposed.

Two isoforms of Pepck in Sarcophaga bullata and their distinct expression profiles through development, diapause, and in response to stresses of cold and starvation.

Tolerance of climatic stressors is an important predictor of the current distribution of insect species, their potential to invade new environments, and their responses to rapid climate change. Cold stress causes acute injury to nerves and muscles, and here we tested the hypothesis that low temperature causes sublethal deficits in locomotor behaviors that are dependent on neuromuscular function. To do so, we applied a previously developed assay, the rapid iterative negative geotaxis (RING) assay, to investigate behavioral consequences of cold stress in Drosophila melanogaster. The RING assay allows for rapid assessment of negative geotaxis behavior by quantifying climbing height and willingness to climb after cold stress. We exposed flies to cold stress at 0°C and assessed the extent to which duration of cold stress, recovery time, and cold acclimation influenced climbing performance. There was a clear dose-response relationship between cold exposure and performance deficits, with climbing height and willingness decreasing as cold exposure increased from 2 to 24 hr. Following cold exposure of an intermediate duration (12 hr), climbing height and willingness gradually improved as recovery time increased from 4 to 72 hr but flies never fully recovered. Finally, cold acclimation improved overall climbing height and willingness in both untreated and cold-stressed flies but did not prevent a reduction in climbing performance. Thus, cold stress causes deficits in locomotor and behavior that are dependent on the dose of cold exposure and persist long after the stress subsides. These results likely have implications for the ecological and evolutionary responses of insect populations to thermally variable environments.

Cold stress results in sustained locomotor and behavioral deficits in Drosophila melanogaster.

In many species, courtship displays are reliable signals of male quality, and current hypotheses suggest that these displays allow females to choose males with high cellular function. Environmental stressors generate excess reactive oxygen species (ROS) that impair cellular function, and thus antioxidant pathways that remove ROS are probably critical for preserving complex sexual behaviours. Here, we test the hypothesis that enhanced antioxidant activity in mitochondria preserves mating performance following oxidative stress. Using a transgenic approach, we directly manipulated mitochondrial antioxidant activity in the Caribbean fruit fly, Anastrepha suspensa, a lek-mating species with elaborate sexual displays and intense sexual selection that is also a model for sterile insect technique programmes. We generated seven transgenic lines that overexpress mitochondrial superoxide dismutase (MnSOD). Radiation is a severe oxidative stressor used to induce sterility for sterile insect programmes. After radiation treatment, two lines with intermediate MnSOD overexpression showed enhanced mating performance relative to wild-type males. These improvements in mating corresponded with reduced oxidative damage to lipids, demonstrating that MnSOD overexpression protects flies from oxidative stress at the cellular level. For lines with improved mating performance, overexpression also preserved locomotor activity, as indicated by a laboratory climbing assay. Our results show a clear link between oxidative stress, antioxidant capacity and male performance. Our work has implications for fundamentally understanding the role of antioxidants in sexual selection, and shows promise for using transgenic approaches to enhance the field performance of insects released for area-wide pest management strategies and improving performance of biological control agents in general.

Overexpression of an antioxidant enzyme improves male mating performance after stress in a lek-mating fruit fly.

Rapid cold hardening (RCH) is a type of beneficial phenotypic plasticity that occurs on extremely short time scales (minutes to hours) to enhance insects' ability to cope with cold snaps and diurnal temperature fluctuations. RCH has a well-established role in extending lower lethal limits, but its ability to prevent sublethal cold injury has received less attention. The Antarctic midge, , is Antarctica's only endemic insect and has a well-studied RCH response that extends freeze tolerance in laboratory conditions. However, the discriminating temperatures used in previous studies of RCH are far below those ever experienced in the field. Here, we tested the hypothesis that RCH protects against non-lethal freezing injury. Larvae of  were exposed to control (2°C), direct freezing (-9°C for 24 h) or RCH (-5°C for 2 h followed by -9°C for 24 h). All larvae survived both freezing treatments, but RCH larvae recovered more quickly from freezing stress and had a significantly higher metabolic rate during recovery. RCH larvae also sustained less damage to fat body and midgut tissue and had lower expression of two heat shock protein transcripts ( and ), which is consistent with RCH protecting against protein denaturation. The protection afforded by RCH resulted in energy savings; directly frozen larvae experienced a significant depletion in glycogen energy stores that was not observed in RCH larvae. Together, these results provide strong evidence that RCH protects against a variety of sublethal freezing injuries and allows insects to rapidly fine-tune their performance in thermally variable environments.

Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect.

For insects that overwinter as adults, winter food resources may affect subsequent spring reproduction and abundance. We tested if provision of food supplements to overwintering adult Hippodamia convergens (Guerin) increased energy reserves, winter survival, and spring reproduction. During 2015-2016, H. convergens adults were placed in field cages in December; adults in each cage received water, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs, bee pollen, wheast protein, sugar, honey, or no food (control). In 2016-2017, treatments were reduced to sugar, bee pollen, A. pisum with E. kuehniella eggs, and no food (control). Adults were sampled to quantify weight, lipid, carbohydrate, and protein content. In 2015-2016, A. pisum and E. kuehniella eggs increased adult weight and protein content, but adult carbohydrate content was reduced by A. pisum and wheast protein treatments. Adults receiving honey and sugar supplementation had higher lipid and carbohydrate content relative to controls. The number of live individuals at the end of the experiment in March 2016 did not differ among treatments. In 2016-2017, winter prey supplements had the greatest effect on protein content, weight, and number of live adults recovered, whereas sugar supplementation increased lipid and carbohydrate content, and number of live adults recovered. Spring reproduction of surviving pairs was evaluated among treatments in March 2017. Prey supplementation in 2016-2017 increased the number of eggs laid and decreased preoviposition period, and food treatment did not affect fertility. Our results indicate that prey and sugar resources improve the overwintering success and spring reproduction of H. convergens.

Supplemental Foods Affect Energetic Reserves, Survival, and Spring Reproduction in Overwintering Adult Hippodamia convergens (Coleoptera: Coccinellidae).

The ability to quickly respond to changes in environmental temperature is critical for organisms living in thermally variable environments. To cope with sudden drops in temperature, insects and other ectotherms are capable of rapid cold hardening (RCH), in which mild chilling significantly enhances cold tolerance within minutes. While the ecological significance of RCH is well established, the mechanisms underlying RCH are still poorly understood. Previous work has demonstrated that RCH is regulated at the cellular level by post-translational signaling mechanisms, and here we tested the hypothesis that cultured cells are capable of RCH. A 2 h cold shock at -8°C significantly reduced the metabolic viability of  S2 cells, but pre-treatment with RCH at 4°C for 2 h prevented this decrease in viability. Thus, S2 cells are capable of RCH in a similar manner to whole insects and provide a new system for investigating the cell biology of RCH.

Evidence for a rapid cold hardening response in cultured  S2 cells.

Freeze-tolerance, or the ability to survive internal ice formation, is relatively rare among insects. Larvae of the Antarctic midge  are freeze-tolerant year-round, but in dry environments, the larvae can remain supercooled (i.e., unfrozen) at subzero temperatures. In previous work with summer-acclimatized larvae, we showed that freezing is considerably more stressful than remaining supercooled. Here, these findings are extended by comparing survival, tissue damage, energetic costs, and stress gene expression in larvae that have undergone an artificial winter acclimation regime and are either frozen or supercooled at -5 °C. In contrast to summer larvae, winter larvae survive at -5 °C equally well for up to 14 days, whether frozen or supercooled, and there is no tissue damage at these conditions. In subsequent experiments, we measured energy stores and stress gene expression following cold exposure at -5 °C for either 24 h or 14 days, with and without a 12 h recovery period. We observed slight energetic costs to freezing, as frozen larvae tended to have lower glycogen stores across all groups. In addition, the abundance of two heat shock protein transcripts,  and , tended to be higher in frozen larvae, indicating higher levels of protein damage following freezing. Together, these results indicate a slight cost to being frozen relative to remaining supercooled, which may have implications for the selection of hibernacula and responses to climate change.

Changes in Energy Reserves and Gene Expression Elicited by Freezing and Supercooling in the Antarctic Midge, .

Rapid cold hardening (RCH) is a type of phenotypic plasticity that allows ectotherms to quickly enhance cold tolerance in response to brief chilling (lasting minutes to hours). In this Review, we summarize the current state of knowledge of this important phenotype and provide new directions for research. As one of the fastest adaptive responses to temperature known, RCH allows ectotherms to cope with sudden cold snaps and to optimize their performance during diurnal cooling cycles. RCH and similar phenotypes have been observed across a diversity of ectotherms, including crustaceans, terrestrial arthropods, amphibians, reptiles, and fish. In addition to its well-defined role in enhancing survival to extreme cold, RCH also protects against nonlethal cold injury by preserving essential functions following cold stress, such as locomotion, reproduction, and energy balance. The capacity for RCH varies across species and across genotypes of the same species, indicating that RCH can be shaped by selection and is likely favored in thermally variable environments. Mechanistically, RCH is distinct from other rapid stress responses in that it typically does not involve synthesis of new gene products; rather, the existing cellular machinery regulates RCH through post-translational signaling mechanisms. However, the protective mechanisms that enhance cold hardiness are largely unknown. We provide evidence that RCH can be induced by multiple triggers in addition to low temperature, and that rapidly induced tolerance and cross-tolerance to a variety of environmental stressors may be a general feature of stress responses that requires further investigation.

Rapid cold hardening: ecological relevance, physiological mechanisms and new perspectives.

Cold tolerance, the ability to cope with low temperature stress, is a critical adaptation in thermally variable environments. An individual's cold tolerance comprises several traits including minimum temperatures for growth and activity, ability to survive severe cold, and ability to resume normal function after cold subsides. Across species, these traits are correlated, suggesting they were shaped by shared evolutionary processes or possibly share physiological mechanisms. However, the extent to which cold tolerance traits and their associated mechanisms covary within populations has not been assessed. We measured five cold tolerance traits-critical thermal minimum, chill coma recovery, short- and long-term cold tolerance, and cold-induced changes in locomotor behavior-along with cold-induced expression of two genes with possible roles in cold tolerance (heat shock protein 70 and frost)-across 12 lines of Drosophila melanogaster derived from a single population. We observed significant genetic variation in all traits, but few were correlated across genotypes, and these correlations were sex-specific. Further, cold-induced gene expression varied by genotype, but there was no evidence supporting our hypothesis that cold-hardy lines would have either higher baseline expression or induction of stress genes. These results suggest cold tolerance traits possess unique mechanisms and have the capacity to evolve independently.

Article	Year
Two isoforms of Pepck in Sarcophaga bullata and their distinct expression profiles through development, diapause, and in response to stresses of cold and starvation. Journal of insect physiology\| PubMed ID: 30392850	2018
Cold stress results in sustained locomotor and behavioral deficits in Drosophila melanogaster. Journal of experimental zoology. Part A, Ecological and integrative physiology\| PubMed ID: 30609298	2019
Overexpression of an antioxidant enzyme improves male mating performance after stress in a lek-mating fruit fly. Proceedings. Biological sciences\| PubMed ID: 31185862	2019
Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect. The Journal of experimental biology\| PubMed ID: 31345935	2019
Supplemental Foods Affect Energetic Reserves, Survival, and Spring Reproduction in Overwintering Adult Hippodamia convergens (Coleoptera: Coccinellidae). Environmental entomology\| PubMed ID: 31748809	2020
Evidence for a rapid cold hardening response in cultured S2 cells. The Journal of experimental biology\| PubMed ID: 31862846	2020
Changes in Energy Reserves and Gene Expression Elicited by Freezing and Supercooling in the Antarctic Midge, . Insects\| PubMed ID: 31878219	2019
Rapid cold hardening: ecological relevance, physiological mechanisms and new perspectives. The Journal of experimental biology\| PubMed ID: 32051174	2020
Distinct cold tolerance traits independently vary across genotypes in Drosophila melanogaster. Evolution; international journal of organic evolution\| PubMed ID: 32463118	2020

Nicholas M. Teets

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