Nicholas M. Teets

Physiological mechanisms of seasonal and rapid cold‐hardening in insects

Insects have evolved a number of physiological mechanisms for coping with the detrimental effects of low temperature. As autumn progresses, insects use environmental signals such as shortening day lengths and gradually decreasing temperatures to trigger seasonal cold‐hardening adaptations. These mechanisms include dramatic changes in biochemistry, cell function and gene expression that permit improved cell function and viability at low temperature. Insects are also capable of enhancing cold tolerance on a much shorter time scale, in a process called rapid cold‐hardening (RCH). Rapid cold‐hardening allows insects to improve cold tolerance almost instantaneously (i.e. within minutes to hours) to cope with sudden cold snaps and regularly‐occurring diurnal drops in temperature. Initially, it was assumed that RCH would share many of the same basic mechanisms as seasonal cold‐hardening, albeit on a shorter time scale. Although there is some evidence supporting this, recent work has called into question some of the original hypotheses concerning the mechanisms of RCH. Also, some mechanisms important for seasonal cold‐hardening, such as up‐regulation of stress proteins, are unlikely to function at the temperatures and time scales at which RCH occurs. In the present review, the current understanding of the physiological mechanisms governing both seasonal cold‐hardening and RCH are summarized. A synthesis of the current literature suggests that these two forms of cold‐hardening may be more mechanistically distinct than originally anticipated.

Combined transcriptomic and metabolomic approach uncovers molecular mechanisms of cold tolerance in a temperate flesh fly

The ability to respond rapidly to changes in temperature is critical for insects and other ectotherms living in variable environments. In a physiological process termed rapid cold-hardening (RCH), exposure to nonlethal low temperature allows many insects to significantly increase their cold tolerance in a matter of minutes to hours. Additionally, there are rapid changes in gene expression and cell physiology during recovery from cold injury, and we hypothesize that RCH may modulate some of these processes during recovery. In this study, we used a combination of transcriptomics and metabolomics to examine the molecular mechanisms of RCH and cold shock recovery in the flesh fly, Sarcophaga bullata. Surprisingly, out of ∼15,000 expressed sequence tags (ESTs) measured, no transcripts were upregulated during RCH, and likewise RCH had a minimal effect on the transcript signature during recovery from cold shock. However, during recovery from cold shock, we observed differential expression of ∼1,400 ESTs, including a number of heat shock proteins, cytoskeletal components, and genes from several cell signaling pathways. In the metabolome, RCH had a slight yet significant effect on several metabolic pathways, while cold shock resulted in dramatic increases in gluconeogenesis, amino acid synthesis, and cryoprotective polyol synthesis. Several biochemical pathways showed congruence at both the transcript and metabolite levels, indicating that coordinated changes in gene expression and metabolism contribute to recovery from cold shock. Thus, while RCH had very minor effects on gene expression, recovery from cold shock elicits sweeping changes in gene expression and metabolism along numerous cell signaling and biochemical pathways.

Gene expression changes governing extreme dehydration tolerance in an Antarctic insect

Among terrestrial organisms, arthropods are especially susceptible to dehydration, given their small body size and high surface area to volume ratio. This challenge is particularly acute for polar arthropods that face near-constant desiccating conditions, as water is frozen and thus unavailable for much of the year. The molecular mechanisms that govern extreme dehydration tolerance in insects remain largely undefined. In this study, we used RNA sequencing to quantify transcriptional mechanisms of extreme dehydration tolerance in the Antarctic midge, Belgica antarctica, the world’s southernmost insect and only insect endemic to Antarctica. Larvae of B. antarctica are remarkably tolerant of dehydration, surviving losses up to 70% of their body water. Gene expression changes in response to dehydration indicated up-regulation of cellular recycling pathways including the ubiquitin-mediated proteasome and autophagy, with concurrent down-regulation of genes involved in general metabolism and ATP production. Metabolomics results revealed shifts in metabolite pools that correlated closely with changes in gene expression, indicating that coordinated changes in gene expression and metabolism are a critical component of the dehydration response. Finally, using comparative genomics, we compared our gene expression results with a transcriptomic dataset for the Arctic collembolan, Megaphorura arctica. Although B. antarctica and M. arctica are adapted to similar environments, our analysis indicated very little overlap in expression profiles between these two arthropods. Whereas several orthologous genes showed similar expression patterns, transcriptional changes were largely species specific, indicating these polar arthropods have developed distinct transcriptional mechanisms to cope with similar desiccating conditions.

Compact genome of the Antarctic midge is likely an adaptation to an extreme environment

The midge, Belgica antarctica, is the only insect endemic to Antarctica, and thus it offers a powerful model for probing responses to extreme temperatures, freeze tolerance, dehydration, osmotic stress, ultraviolet radiation and other forms of environmental stress. Here we present the first genome assembly of an extremophile, the first dipteran in the family Chironomidae, and the first Antarctic eukaryote to be sequenced. At 99 megabases, B. antarctica has the smallest insect genome sequenced thus far. Although it has a similar number of genes as other Diptera, the midge genome has very low repeat density and a reduction in intron length. Environmental extremes appear to constrain genome architecture, not gene content. The few transposable elements present are mainly ancient, inactive retroelements. An abundance of genes associated with development, regulation of metabolism and responses to external stimuli may reflect adaptations for surviving in this harsh environment.

Insect capa neuropeptides impact desiccation and cold tolerance

Significance Insects are among the most robust organisms on the planet, surviving in virtually all environments and capable of surmounting a range of environmental stresses including desiccation and cold. Although desiccation and cold tolerance share many common traits, potential mechanisms for such linked responses remain unclear. Here we show that an insect neuropeptide gene is associated with tolerance of both desiccation and cold in Drosophila melanogaster, suggesting a novel mechanism in renal tubule epithelia that enhances survival of both desiccation and cold. Also, we can reverse RNAi-induced stress tolerance phenotypes in intact flies using rationally designed peptide mimetic analogs. We thus demonstrate the power of intervention in physiological processes controlled by neuropeptides, with potential for insect pest control. The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. Here we show that the capability (capa) neuropeptide gene is a desiccation- and cold stress-responsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, we demonstrate that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that capa accumulates in the capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. Our results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments our understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance.

Survival and energetic costs of repeated cold exposure in the Antarctic midge, Belgica antarctica : a comparison between frozen and supercooled larvae

SUMMARY In this study, we examined the effects of repeated cold exposure (RCE) on the survival, energy content and stress protein expression of larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae). Additionally, we compared results between larvae that were frozen at –5°C in the presence of water during RCE and those that were supercooled at –5°C in a dry environment. Although >95% of larvae survived a single 12 h bout of freezing at –5°C, after five cycles of RCE survival of frozen larvae dropped below 70%. Meanwhile, the survival of control and supercooled larvae was unchanged, remaining around 90% for the duration of the study. At the tissue level, frozen larvae had higher rates of cell mortality in the midgut than control and supercooled larvae. Furthermore, larvae that were frozen during RCE experienced a dramatic reduction in energy reserves; after five cycles, frozen larvae had 25% less lipid, 30% less glycogen and nearly 40% less trehalose than supercooled larvae. Finally, larvae that were frozen during RCE had higher expression of hsp70 than those that were supercooled, indicating a higher degree of protein damage in the frozen group. Results were similar between larvae that had accumulated 60 h of freezing at –5°C over five cycles of RCE and those that were frozen continuously for 60 h, suggesting that the total time spent frozen determines the physiological response. Our results suggest that it is preferable, both from a survival and energetic standpoint, for larvae to seek dry microhabitats where they can avoid inoculative freezing and remain unfrozen during RCE.

Calcium signaling mediates cold sensing in insect tissues

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

Functional characterization of an aquaporin in the Antarctic midge Belgica antarctica

Aquaporins (AQPs) are water channel proteins facilitating movement of water across the cell membrane. Recent insect studies clearly demonstrate that AQPs are indispensable for cellular water management under normal conditions as well as under stress conditions including dehydration and cold. In the present study we cloned an AQP cDNA from the Antarctic midge Belgica antarctica (Diptera, Chironomidae) and investigated water transport activity of the AQP protein and transcriptional regulation of the gene in response to dehydration and rehydration. The nucleotide sequence and deduced amino acid sequence of the cDNA showed high similarity to AQPs in other insects and also showed characteristic features of orthodox AQPs. Phylogenetic analysis revealed that Belgica AQP is a homolog of dehydration-inducible AQP of another chironomid, Polypedilum vanderplanki. A swelling assay using a Xenopus oocyte expression system verified that Belgica AQP is capable of transporting water, but not glycerol or urea. The AQP mRNA was detected in various organs under non-stressed conditions, suggesting that this AQP plays a fundamental role in cell physiology. In contrast to our expectation, AQP transcriptional expression was not affected by either dehydration or rehydration.

Heat shock response to hypoxia and its attenuation during recovery in the flesh fly, Sarcophaga crassipalpis

In this study, pharate adults of the flesh fly Sarcophaga crassipalpis were exposed to two, four, seven, or ten days of severe hypoxia (3% oxygen) to evaluate its impact on emergence and the expression of genes encoding heat shock proteins (Hsps) and heat shock regulatory elements. A four-day exposure to hypoxia significantly reduced survival, but more than seven days was required to reach the LD(50). Eight genes encoding Hsps, at least one from each major family of Hsps (Hsp90, Hsp70, Hsp60, Hsp40, and sHsps) and two genes encoding proteins involved in Hsp regulation (heat shock factor, hsf, and sirtuin) were cloned, and expression levels were assessed during and after hypoxia using qRT-PCR. Most, but not all hsps studied, were significantly up-regulated during hypoxia, and expression levels for most of the hsps reverted to control levels a few hours after return to normoxia. Hsp70 was the most responsive to hypoxia, increasing expression several hundred fold. By contrast, hsp90 and hsp27 showed little response to hypoxia but did respond to recovery. Neither hsf nor sirtuin were elevated by hypoxia, an observation consistent with their assumed post-transcriptional regulatory roles. These data demonstrate a strong Hsp response to hypoxia, suggesting an important role for Hsps in responding to low oxygen environments.

Surviving in a frozen desert: environmental stress physiology of terrestrial Antarctic arthropods

Abiotic stress is one of the primary constraints limiting the range and success of arthropods, and nowhere is this more apparent than Antarctica. Antarctic arthropods have evolved a suite of adaptations to cope with extremes in temperature and water availability. Here, we review the current state of knowledge regarding the environmental physiology of terrestrial arthropods in Antarctica. To survive low temperatures, mites and Collembola are freeze-intolerant and rely on deep supercooling, in some cases supercooling below −30°C. Also, some of these microarthropods are capable of cryoprotective dehydration to extend their supercooling capacity and reduce the risk of freezing. In contrast, the two best-studied Antarctic insects, the midges Belgica antarctica and Eretmoptera murphyi, are freeze-tolerant year-round and rely on both seasonal and rapid cold-hardening to cope with decreases in temperature. A common theme among Antarctic arthropods is extreme tolerance of dehydration; some accomplish this by cuticular mechanisms to minimize water loss across their cuticle, while a majority have highly permeable cuticles but tolerate upwards of 50–70% loss of body water. Molecular studies of Antarctic arthropod stress physiology are still in their infancy, but several recent studies are beginning to shed light on the underlying mechanisms that govern extreme stress tolerance. Some common themes that are emerging include the importance of cuticular and cytoskeletal rearrangements, heat shock proteins, metabolic restructuring and cell recycling pathways as key mediators of cold and water stress in the Antarctic.