Trade-offs between winter survival and reproduction in female insects.

In temperate environments most species of insects enter an arrested state of development, known as diapause, that enables them to survive the adverse environmental conditions associated with winter. Although diapause is restricted to a single life stage within species of insects, there are examples of insects that overwinter in the egg, larval, pupal and adult stages. Here we offer a targeted, non-systematic literature review examining how overwintering impacts subsequent reproduction in female insects. Several factors, including the lifestage at which insects overwinter, the type of energy investment strategy females use for breeding, elements of the winter environment, and contributions from male insects can influence trade-offs that female insects face between overwintering survival and post-diapause reproduction. Additionally, climate change and elements of the urban environment, including light pollution and higher temperatures in cities, can exacerbate or ameliorate trade-offs faced by reproducing female insects. Better understanding trade-offs between overwintering survival and reproduction in insects not only enhances our understanding of the underlying physiological mechanisms and ecological processes governing diapause and reproduction, but also provide opportunities to better manage insect pests and/or support beneficial insects.

Reduced male fertility of an Antarctic mite following extreme heat stress could prompt localized population declines.

Climate change is leading to substantial global thermal changes, which are particularly pronounced in polar regions. Therefore, it is important to examine the impact of heat stress on the reproduction of polar terrestrial arthropods, specifically, how brief extreme events may alter survival. We observed that sublethal heat stress reduces male fecundity in an Antarctic mite, yielding females that produced fewer viable eggs. Females and males collected from microhabitats with high temperatures showed a similar reduction in fertility. This impact is temporary, as indicated by recovery of male fecundity following return to cooler, stable conditions. The diminished fecundity is likely due to a drastic reduction in the expression of male-associated factors that occur in tandem with a substantial increase in the expression of heat shock proteins. Cross-mating between mites from different sites confirmed that heat-exposed populations have impaired male fertility. However, the negative impacts are transient as the effect on fertility declines with recovery time under less stressful conditions. Modeling indicated that heat stress is likely to reduce population growth and that short bouts of non-lethal heat stress could have substantial reproductive effects on local populations of Antarctic arthropods.

Insect diapause: from a rich history to an exciting future.

Diapause, a stage-specific developmental arrest, is widely exploited by insects to bridge unfavorable seasons. Considerable progress has been made in understanding the ecology, physiology and evolutionary implications of insect diapause, yet intriguing questions remain. A more complete understanding of diapause processes on Earth requires a better geographic spread of investigations, including more work in the tropics and at high latitudes. Questions surrounding energy management and trade-offs between diapause and non-diapause remain understudied. We know little about how maternal effects direct the diapause response, and regulators of prolonged diapause are also poorly understood. Numerous factors that were recently linked to diapause are still waiting to be placed in the regulatory network leading from photoreception to engagement of the diapause program. These factors include epigenetic processes and small noncoding RNAs, and emerging data also suggest a role for the microbiome in diapause regulation. Another intriguing feature of diapause is the complexity of the response, resulting in a diverse suite of responses that comprise the diapause syndrome. Select transcription factors likely serve as master switches turning on these diverse responses, but we are far from understanding the full complexity. The richness of species displaying diapause offers a platform for seeking common components of a 'diapause toolbox'. Across latitudes, during invasion events and in a changing climate, diapause offers grand opportunities to probe evolutionary change and speciation. At a practical level, diapause responses can be manipulated for insect control and long-term storage. Diapausing insects also contain a treasure trove of pharmacological compounds and offer promising models for human health.

Exploiting tools for manipulating insect diapause.

Tools that could be used to subvert the insect diapause response offer potential for insect pest management as well as for the experimental manipulation of insects and the facilitation of mass rearing procedures. In some cases, it is desirable to break diapause on demand and in other cases, it may be attractive to exploit diapause for long-term storage of biocontrol agents or valuable experimental lines. This review highlights some of the diapause disruptors reported in the literature, as well as chemical and physical manipulations that can be used to extend diapause or even induce diapause in an insect not programmed for diapause. The insect hormones are quite effective agents for breaking diapause and in some cases for extending the duration of diapause, but a collection of other chemical agents can also act as potent diapause disruptors, e.g. organic solvents, weak acids and bases, carbon dioxide, imidazole compounds, LSD, deuterium oxide, DMSO, ouabain, cholera toxin, cyclic GMP, heavy metals, and hydrogen peroxide. Physical manipulations such as artificial light at night, anoxia, shaking and heat shock are also known diapause disruptors. Some of these documented manipulations prevent diapause, others terminate diapause immediately, others alter the duration of diapause, and a few compounds can induce a diapause-like state in insects that are not programmed for diapause. The diversity of tools noted in the literature offers promise for the development of new tools or manipulations that possibly could be used to disrupt diapause or manage diapause in controlled laboratory experiments and in mass-rearing facilities.

Fine-scale variation in microhabitat conditions influences physiology and metabolism in an Antarctic insect.

Microhabitats with distinct biotic and abiotic properties exist within landscapes, and this microhabitat variation can have dramatic impacts on the phenology and physiology of the organisms occupying them. The Antarctic midge Belgica antarctica inhabits diverse microhabitats along the Western Antarctic Peninsula that vary in macrophyte composition, hygric qualities, nutrient input, and thermal patterns. Here, we compare seasonal physiological changes in five populations of B. antarctica living in close proximity but in different microhabitats in the vicinity of Palmer Station, Antarctica. Thermal regimes among our sample locations differed in both mean temperature and thermal stability. Between the warmest and coldest sites, seasonal mean temperatures differed by 2.6˚C and degree day accumulations above freezing differed by a factor of 1.7. Larval metabolic and growth rates varied among the sites, and adult emergence occurred at different times. Distinct microhabitats also corresponded with differences in body composition, as lipid and carbohydrate content of larvae differed across sites. Further, seasonal changes in carbohydrate and protein content were dependent on site, indicating fine-scale variation in the biochemical composition of larvae as they prepare for winter. Together, these results demonstrate that variation in microhabitat properties influences the ontogeny, phenology, physiology, and biochemical makeup of midge populations living in close proximity. These results have implications for predicting responses of Antarctic ecosystems to environmental change.

Rapid stress hardening in the Antarctic midge improves male fertility by increasing courtship success and preventing decline of accessory gland proteins following cold exposure.

Rapid hardening is a process that quickly improves an animal's performance following exposure to potentially damaging stress. In this study of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), we examined how rapid hardening in response to dehydration (RDH) or cold (RCH) improves male pre- and post-copulatory function when the insects are subsequently subjected to a damaging cold exposure. Neither RDH nor RCH improved survival in response to lethal cold stress, but male activity and mating success following sublethal cold exposure were enhanced. Egg viability decreased following direct exposure of the mating males to sublethal cold but improved following RCH and RDH. Sublethal cold exposure reduced the expression of four accessory gland proteins, while expression remained high in males exposed to RCH. Though rapid hardening may be cryptic in males, this study shows that it can be revealed by pre- and post-copulatory interactions with females.

Expression of aquaporins in response to distinct dehydration stresses that confer stress tolerance in the Antarctic midge Belgica antarctica.

Larvae of the Antarctic midge Belgica antarctica Jacobs (Diptera: Chironomidae) are highly tolerant of diverse environmental stresses, including freezing, severe desiccation, and osmotic extremes. Furthermore, dehydration confers subsequent desiccation and freeze tolerance. While a role for aquaporins-channels for water and other solutes-has been proposed in these dehydration processes, the types of aquaporins involved in dehydration-driven stress tolerance remain unknown. In the present study, we investigated expression of six aquaporins (Drip, Prip, Eglp1, Eglp2, Aqp12L, and Bib) in larvae of B. antarctica subjected to three different dehydration conditions: desiccation, cryoprotective dehydration, and osmotic dehydration. The expression of Drip and Prip was up-regulated under desiccation and cryoprotective dehydration, suggesting a role for these aquaporins in efficient water loss under these dehydration conditions. Conversely, expression of Drip and Prip was down-regulated under osmotic dehydration, suggesting that their expression is suppressed in larvae to combat dehydration. Larval water content was similarly decreased under all three dehydration conditions. Differences in responses of the aquaporins to the three forms of dehydration suggests distinct water management strategies associated with different forms of dehydration stress.

ROS and hypoxia signaling regulate periodic metabolic arousal during insect dormancy to coordinate glucose, amino acid, and lipid metabolism.

Metabolic suppression is a hallmark of animal dormancy that promotes overall energy savings. Some diapausing insects and some mammalian hibernators have regular cyclic patterns of substantial metabolic depression alternating with periodic arousal where metabolic rates increase dramatically. Previous studies, largely in mammalian hibernators, have shown that periodic arousal is driven by an increase in aerobic mitochondrial metabolism and that many molecules related to energy metabolism fluctuate predictably across periodic arousal cycles. However, it is still not clear how these rapid metabolic shifts are regulated. We first found that diapausing flesh fly pupae primarily use anaerobic glycolysis during metabolic depression but engage in aerobic respiration through the tricarboxylic acid cycle during periodic arousal. Diapausing pupae also clear anaerobic by-products and regenerate many metabolic intermediates depleted in metabolic depression during arousal, consistent with patterns in mammalian hibernators. We found that decreased levels of reactive oxygen species (ROS) induced metabolic arousal and elevated ROS extended the duration of metabolic depression. Our data suggest ROS regulates the timing of metabolic arousal by changing the activity of two critical metabolic enzymes, pyruvate dehydrogenase and carnitine palmitoyltransferase I by modulating the levels of hypoxia inducible transcription factor (HIF) and phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Our study shows that ROS signaling regulates periodic arousal in our insect diapasue system, suggesting the possible importance ROS for regulating other types of of metabolic cycles in dormancy as well.

Multi-level analysis of reproduction in an Antarctic midge identifies female and male accessory gland products that are altered by larval stress and impact progeny viability.

The Antarctic midge, Belgica antarctica, is a wingless, non-biting midge endemic to Antarctica. Larval development requires at least 2 years, but adults live only 2 weeks. The nonfeeding adults mate in swarms and females die shortly after oviposition. Eggs are suspended in a gel of unknown composition that is expressed from the female accessory gland. This project characterizes molecular mechanisms underlying reproduction in this midge by examining differential gene expression in whole males, females, and larvae, as well as in male and female accessory glands. Functional studies were used to assess the role of the gel encasing the eggs, as well as the impact of stress on reproductive biology. RNA-seq analyses revealed sex- and development-specific gene sets along with those associated with the accessory glands. Proteomic analyses were used to define the composition of the egg-containing gel, which is generated during multiple developmental stages and derived from both the accessory gland and other female organs. Functional studies indicate the gel provides a larval food source as well as a buffer for thermal and dehydration stress. All of these function are critical to juvenile survival. Larval dehydration stress directly reduces production of storage proteins and key accessory gland components, a feature that impacts adult reproductive success. Modeling reveals that bouts of dehydration may have a significant impact on population growth. This work lays a foundation for further examination of reproduction in midges and provides new information related to general reproduction in dipterans. A key aspect of this work is that reproduction and stress dynamics, currently understudied in polar organisms, are likely to prove critical in determining how climate change will alter their survivability.

Environmental factors influencing fine-scale distribution of Antarctica's only endemic insect.

Species distributions are dependent on interactions with abiotic and biotic factors in the environment. Abiotic factors like temperature, moisture, and soil nutrients, along with biotic interactions within and between species, can all have strong influences on spatial distributions of plants and animals. Terrestrial Antarctic habitats are relatively simple and thus good systems to study ecological factors that drive species distributions and abundance. However, these environments are also sensitive to perturbation, and thus understanding the ecological drivers of species distribution is critical for predicting responses to environmental change. The Antarctic midge, Belgica antarctica, is the only endemic insect on the continent and has a patchy distribution along the Antarctic Peninsula. While its life history and physiology are well studied, factors that underlie variation in population density within its range are unknown. Previous work on Antarctic microfauna indicates that distribution over broad scales is primarily regulated by soil moisture, nitrogen content, and the presence of suitable plant life, but whether these patterns are true over smaller spatial scales has not been investigated. Here we sampled midges across five islands on the Antarctic Peninsula and tested a series of hypotheses to determine the relative influences of abiotic and biotic factors on midge abundance. While historical literature suggests that Antarctic organisms are limited by the abiotic environment, our best-supported hypothesis indicated that abundance is predicted by a combination of abiotic and biotic conditions. Our results are consistent with a growing body of literature that biotic interactions are more important in Antarctic ecosystems than historically appreciated.

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

Freeze-tolerance, or the ability to survive internal ice formation, is relatively rare among insects. Larvae of the Antarctic midge Belgica antarctica 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, hsp60 and hsp90, 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.

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

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, Belgica antarctica, 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 B. antarctica 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 (hsp60 and hsp90), 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.

Thermoprotective adaptations are critical for arthropods feeding on warm-blooded hosts.

Blood feeding in arthropods has evolved in multiple lineages. This feeding preference provides a source of ample proteins and lipids for egg production and survival, but ingestion of a large warm blood-meal can boost the arthropod's body temperature 15°-20°C within seconds to minutes. This represents one of, if not the most, rapid thermal change documented under a natural setting. Here, we describe mechanisms of thermoregulation and thermotolerance in arthropods during blood feeding. The ability to prevent blood-induced thermal damage is a fundamental physiological adaptation linked to the use of warm-blooded vertebrates as food sources. Specific functional and comparative studies have identified unique and divergent mechanisms that suppress or repair thermal stress during blood feeding. These mechanisms include countercurrent heat exchange, evaporative cooling, and upregulation of stress associated proteins.

Genome and Ontogenetic-Based Transcriptomic Analyses of the Flesh Fly, Sarcophaga bullata.

The flesh fly, Sarcophaga bullata, is a widely-used model for examining the physiology of insect diapause, development, stress tolerance, neurobiology, and host-parasitoid interactions. Flies in this taxon are implicated in myiasis (larval infection of vertebrates) and feed on carrion, aspects that are important in forensic studies. Here we present the genome of S. bullata, along with developmental- and reproduction-based RNA-Seq analyses. We predict 15,768 protein coding genes, identify orthology in relation to closely related flies, and establish sex and developmental-specific gene sets based on our RNA-Seq analyses. Genomic sequences, predicted genes, and sequencing data sets have been deposited at the National Center for Biotechnology Information. Our results provide groundwork for genomic studies that will expand the flesh fly's utility as a model system.

Distinct microRNA and mRNA responses elicited by ecdysone, diapause hormone and a diapause hormone analog at diapause termination in pupae of the corn earworm, Helicoverpa zea.

Ecdysone, diapause hormone and a diapause hormone analog are all capable of breaking pupal diapause and prompting initiation of adult development in the cotton earworm, Helicoverpa zea. In this study we asked whether these three chemically-distinct diapause terminators elicit the same effect on expression of a collection of microRNAs and transcripts encoding components of the ecdysone signaling pathway. Injection of all three endocrine agents resulted in downregulation of one miRNA, miR-277-3p, a miRNA previously linked to the insulin/FOXO signaling pathway, and all three agents promoted upregulation of spook, a member of the ecdysone biosynthesis pathway, and iswi, an ecdysone-responsive transcript. Other miRNA and mRNA responses varied depending on the agent used to terminate diapause, thus suggesting that different endocrine pathways and mechanisms can lead to the same final developmental response.

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

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.

Evolutionary transition from blood feeding to obligate nonbiting in a mosquito.

The spread of blood-borne pathogens by mosquitoes relies on their taking a blood meal; if there is no bite, there is no disease transmission. Although many species of mosquitoes never take a blood meal, identifying genes that distinguish blood feeding from obligate nonbiting is hampered by the fact that these different lifestyles occur in separate, genetically incompatible species. There is, however, one unique extant species with populations that share a common genetic background but blood feed in one region and are obligate nonbiters in the rest of their range: Wyeomyia smithii Contemporary blood-feeding and obligate nonbiting populations represent end points of divergence between fully interfertile southern and northern populations. This divergence has undoubtedly resulted in genetic changes that are unrelated to blood feeding, and the challenge is to winnow out the unrelated genetic factors to identify those related specifically to the evolutionary transition from blood feeding to obligate nonbiting. Herein, we determine differential gene expression resulting from directional selection on blood feeding within a polymorphic population to isolate genetic differences between blood feeding and obligate nonbiting. We show that the evolution of nonbiting has resulted in a greatly reduced metabolic investment compared with biting populations, a greater reliance on opportunistic metabolic pathways, and greater reliance on visual rather than olfactory sensory input. W. smithii provides a unique starting point to determine if there are universal nonbiting genes in mosquitoes that could be manipulated as a means to control vector-borne disease.

Bugs battle stress from hot blood.

A heat exchange mechanism in the head of kissing bugs helps to prevent stress and regulate their temperature while they feed on warm blood.

Keeping time without a spine: what can the insect clock teach us about seasonal adaptation?

Seasonal change in daylength (photoperiod) is widely used by insects to regulate temporal patterns of development and behaviour, including the timing of diapause (dormancy) and migration. Flexibility of the photoperiodic response is critical for rapid shifts to new hosts, survival in the face of global climate change and to reproductive isolation. At the same time, the daily circadian clock is also essential for development, diapause and multiple behaviours, including correct flight orientation during long-distance migration. Although studied for decades, how these two critical biological timing mechanisms are integrated is poorly understood, in part because the core circadian clock genes are all transcription factors or regulators that are able to exert multiple effects throughout the genome. In this chapter, we discuss clocks in the wild from the perspective of diverse insect groups across eco-geographic contexts from the Antarctic to the tropical regions of Earth. Application of the expanding tool box of molecular techniques will lead us to distinguish universal from unique mechanisms underlying the evolution of circadian and photoperiodic timing, and their interaction across taxonomic and ecological contexts represented by insects.This article is part of the themed issue 'Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals'.

Reactive oxygen species extend insect life span using components of the insulin-signaling pathway.

Reactive oxygen species (ROS) are well-known accelerants of aging, but, paradoxically, we show that physiological levels of ROS extend life span in pupae of the moth Helicoverpa armigera, resulting in the dormant state of diapause. This developmental switch appears to operate through a variant of the conventional insulin-signaling pathway, as evidenced by the facts that Akt, p-Akt, and PRMT1 are elevated by ROS, but not insulin, and that high levels of p-Akt fail to phosphorylate FoxO through PRMT1-mediated methylation. These results suggest a distinct signaling pathway culminating in the elevation of FoxO, which in turn promotes the extension of life span characteristic of diapause.