Are infrared thermography, feeding behavior, and heart rate variability measures capable of characterizing group-housed sow social hierarchies?

Group gestation housing is quickly becoming standard practice in commercial swine production. However, poor performance and welfare in group housed sows may result from the formation and maintenance of the social hierarchy within the pen. In the future, the ability to quickly characterize the social hierarchy via precision technologies could be beneficial to producers for identifying animals at risk of poor welfare outcomes. Therefore, the objective of this study was to investigate the use of infrared thermography (IRT), automated electronic sow feeding systems, and heart rate monitors as potential technologies for detecting the social hierarchy within five groups of sows. Behavioral data collection occurred for 12 h after introducing five sow groups (1-5; n = 14, 12, 15, 15, and 17, respectively) to group gestation housing to determine the social hierarchy and allocate individual sows to 1 of 4 rank quartiles (RQ 1-4). Sows within RQ1 were ranked highest while RQ4 sows were ranked lowest within the hierarchy. Infrared thermal images were taken behind the neck at the base of the ear of each sow on days 3, 15, 30, 45, 60, 75, 90, and 105 of the experiment. Two electronic sow feeders tracked feeding behavior throughout the gestation period. Heart rate monitors were worn by 10 randomly selected sows per repetition for 1 h prior to and 4 h after reintroduction to group gestation housing to collect heart rate variability (HRV). No differences were found between RQ for any IRT characteristic. Sows within RQ3 and RQ4 had the greatest number of visits to the electronic sow feeders overall (P < 0.04) but spent shorter time per visit in feeders (P < 0.05) than RQ1 and RQ2 sows. There was an interaction of RQ with hour for feed offered (P = 0.0003), with differences between RQ occurring in hour 0, 1, 2, and 8. Higher-ranked sows (RQ1 and RQ2) occupied the feeder for longer during the first hour than lower ranking sows (RQ3 and RQ4; P < 0.04), while RQ3 sows occupied the feeder longer than RQ1 sows during hour 6, 7, and 8 (P < 0.02). Heart beat interval (RR) collected prior to group housing introduction differed between RQ (P < 0.02 for all), with RQ3 sows exhibiting the lowest RR, followed by RQ4, RQ1, and RQ2. Rank quartile also affected standard deviation of RR (P = 0.0043), with RQ4 sows having the lowest, followed by RQ1, RQ3, and RQ2 sows. Overall, these results indicate that feeding behavior and HRV measures may be capable of characterizing social hierarchy in a group housing system.

Sows that are housed in groups establish a social hierarchy to gain preferential access to needed resources, such as feed. The establishment and maintenance of this social hierarchy may lead to reduced welfare and performance for certain sows. Therefore, the purpose of this study was to determine whether infrared thermography, feeding behavior, and heart rate variability measurement could identify the social hierarchy. In the future, the ability to automatically detect the social hierarchy within group housed pens using technology may give a producer the ability to mitigate any hierarchy-related welfare and performance issues on their own farm. Our results show that feeding behavior collected by an automated feeding system may be a promising tool for future social hierarchy detection. Additionally, measures related to changes in heart rate over time are capable of identifying high- and low-ranked sows when the measures are collected before sows are moved into groups. Therefore, technologies capable of measuring feeding behavior and changes in heart rate over time may be promising for future automated detection of the sow social hierarchy.

Anoxia hormesis improves performance and longevity at the expense of fitness in a classic life history trade-off.

Hormesis occurs as a result of biphasic dose relationship resulting in stimulatory responses at low doses and inhibitory ones at high doses. In this framework, environmental factors are often studied to understand how this exposure benefits the animal. In the current study we used anoxia, the total absence of oxygen, as the most extreme version of low oxygen hormesis. Our goal was to determine the dose, the extent of the effect, and the cost of that response in Tenebrio molitor. We identified that the hormetic range (1 to 3 h of anoxia) was similar to that of other insects. Individuals that were exposed to 3 h had high emergence, increased activity throughout life, and lived longer. Beetles that experienced 1 h of anoxia performed better than the controls while the 6-h group had compromised performance. These boosts in performance at 3 h were accompanied by significant costs. Treated individuals had a delay in development and once matured they had decreased fitness. There were also transgenerational effects of hormesis and F1 beetles also experienced a delay in development. Additionally, the F1 generation had decreased developmental completion (i.e., stress-induced developmental halt). Our data suggests that anoxia hormesis triggers a trade-off where individuals benefiting from improved performance and living longer experience a decrease in reproduction.

Anoxia hormesis following overwintering diapause boosts bee survivorship and adult performance.

Insect pollination is a crucial component of our ecosystems and biodiversity, but our reliance on this ecosystem service has much broader implications. We depend on these pollination services to produce materials and food. But insect pollinators, especially bees, are in strong decline due to a plethora of factors, least of which are environmental abiotic stressors like climate change. The alfalfa leafcutting bee, Megachile rotundata, is the world's most managed solitary bee and is particularly vulnerable to changes in temperature. This species spends up to ten months overwintering while being exposed to the lowest temperatures of winters and the hottest temperatures of late summer. This results in usage of energy reserves prematurely and asynchronous spring emergence with their food resource. To understand the stress response of these bees and potentially boost their performance, we applied a hormetic framework where bees were exposure to different doses of anoxia (the absence of oxygen) to trigger hormesis; a low-dose stimulatory response known to lower damage and improve performance. We used hormesis on immature bees as a post-winter treatment with the goal of improving springtime performance in adults. One hour of anoxia had no negative effect on adult springtime emergence and bees were quick to recover. These bees were more active than untreated bees, as resistant to starvation, and as long-lived. Higher exposure to anoxia (3 h) was found to be mildly hormetic and 6-h exposures were detrimental. Anoxia hormesis did not represent a significant cost on the energy reserve of overwintering bees but we found that the age at which anoxia is applied will affect the effectiveness of treatment. Our data suggest that anoxia hormesis is a viable intervention to improve springtime performance in overwintering bees.

Low-oxygen hormetic conditioning improves field performance of sterile insects by inducing beneficial plasticity.

As part of sterile insect technique (SIT) programs, irradiation can effectively induce sterility in insects by damaging germline genomic DNA. However, irradiation also induces other off-target side effects that reduce the quality and performance of sterilized males, including the formation of damaging free radicals that can reduce sterile male performance. Thus, treatments that reduce off-target effects of irradiation on male performance while maintaining sterility can improve the feasibility and economy of SIT programs. We previously found that inducing a form of rapid, beneficial plasticity with a 1-hr anoxic-conditioning period (physiological conditioning hormesis) prior to and during irradiation improves male field performance in the laboratory while maintaining sterility in males of the cactus moth, Cactoblastis cactorum. Here, we extend this work by testing the extent to which this beneficial plasticity may improve male field performance and longevity in the field. Based on capture rates after a series of mark release-recapture experiments, we found that anoxia-conditioned irradiated moths were active in the field longer than their irradiated counterparts. In addition, anoxia-conditioned moths were captured in traps that were farther away from the release site than unconditioned moths, suggesting greater dispersal. These data confirmed that beneficial plasticity induced by anoxia hormesis prior to irradiation led to lower postirradiation damage and increased flight performance and recapture duration under field conditions. We recommend greater consideration of beneficial plasticity responses in biological control programs and specifically the implementation of anoxia-conditioning treatments applied prior to irradiation in area-wide integrated pest management programs that use SIT.

Resistance and survival to extreme heat shows circadian and sex-specific patterns in A cavity nesting bee.

The pollination services provided by insects have been a crucial part of evolution and survival for many species, including humans. For bees to be efficient pollinators they must survive the environmental insults they face daily. Thus, looking into the short- and long-term effects of heat exposure on bee performance provides us with a foundation for investigating how stress can affect insect pollination. Solitary bees are a great model for investigating the effects of environmental stress on pollinators because the vast majority of insect pollinator species are solitary rather than social. One of the most pervasive environmental stressors to insects is temperature. Here we investigated how a one-hour heat shock affected multiple metrics of performance in the alfalfa leafcutting bee, Megachile rotundata. We found that a short heat shock (1hr at 45°C) can delay adult emergence in males but not females. Bee pupae were rather resilient to a range of high temperature exposures that larvae did not survive. Following heat shock (1hr at 50°C), adult bees were drastically less active than untreated bees, and this reduction in activity was evident over several days. Heat shock also led to a decrease in bee survival and longevity. Additionally, we found a connection between starvation survival after heat shock and time of exposure, where bees exposed in the morning survived longer than those exposed in the afternoon, when they would normally experience heat shock in the field. These data suggest that there is an unexplored daily/circadian component to the stress response in bees likely similar to that seen in flies, nematodes, and plants which is constitutive or preemptive rather than restorative. Taken together our data indicate that single heat shock events have strong potential to negatively impact multiple life history traits correlated with reproduction and fitness.

A dose of experimental hormesis: When mild stress protects and improves animal performance.

The adaptive response characterized by a biphasic curve is known as hormesis. In a hormesis framework, exposure to low doses leads to protective and beneficial responses while exposures to high doses are damaging and detrimental. Comparative physiologists have studied hormesis for over a century, but our understanding of hormesis is fragmented due to rifts in consensus and taxonomic-specific terminology. Hormesis has been and is currently known by multiple names; preconditioning, conditioning, pretreatment, cross tolerance, adaptive homeostasis, and rapid stress hardening (mostly low temperature: rapid cold hardening). These are the most common names used to describe adaptive stress responses in animals. These responses are mechanistically similar, while having stress-specific responses, but they all can fall under the umbrella of hormesis. Here we review how hormesis studies have revealed animal performance benefits in response to changes in oxygen, temperature, ionizing radiation, heavy metals, pesticides, dehydration, gravity, and crowding. And how almost universally, hormetic responses are characterized by increases in performance that include either increases in reproduction, longevity, or both. And while the field can benefit from additional mechanistic work, we know that many of these responses are rooted in increases of antioxidants and oxidative stress protective mechanisms; including heat shock proteins. There is a clear, yet not fully elucidated, overlap between hormesis and the preparation for oxidative stress theory; which predicts part of the responses associated with hormesis. We discuss this, and the need for additional work into animal hormetic effects particularly focusing on the cost of hormesis.

Commentary: Ultraviolet radiation triggers "preparation for oxidative stress" antioxidant response in animals: Similarities and interplay with other stressors.

Preparation for oxidative stress (POS), i.e., the upregulation of endogenous antioxidants, is a widespread response of animals exposed to extreme conditions. This response has been described for more than 80 animal species belonging to eight phyla during hypometabolism or situations that limit oxygen availability. The pattern of the typical POS-response, in which a mild redox imbalance triggers antioxidant adjustments that results in increased tolerance to subsequent oxidative insults, roughly follows the curve of hormetic phenomena. A similar pattern has been reported for various animal species exposed to ultraviolet radiation (UVR) - these studies, on animals from six phyla, are discussed herein. In the light of the similarities in the redox-response of animals exposed to either oxygen restriction or UVR, we argue in this essay that UVR elicits a type of response that fits the POS theory. Exposure to UVR induces both reactive species formation and antioxidant adaptation, which is the essence of typical POS-responses. Thus, antioxidant response to UVR in animals can be categorized as a POS-type mechanism. Moreover, considering that animals are exposed to multiple stressors simultaneously in nature, this would represent an ecologically relevant process, by which one stressor (e.g., UV or ionizing radiation) may enhance the tolerance to other. We also discuss a possible role of low doses of ionizing radiation as inductor of POS-like responses in animals.

Hormetic benefits of prior anoxia exposure in buffering anoxia stress in a soil-pupating insect.

Oxygen is essential for most animals, and exposure to a complete lack of oxygen, i.e. anoxia, can result in irreparable damage to cells that can extend up to the organismal level to negatively affect performance. Although it is known that brief anoxia exposure may confer cross-tolerance to other stressors, few data exist on the biochemical and organismal consequences of repeated intermittent bouts of anoxia exposure. In nature, the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae), is frequently exposed to heavy tropical rainfall while pupating in the soil, equating to multiple exposures to hypoxia or anoxia during development. Here, we tested whether prior anoxia exposures during pupal development can induce a beneficial acclimation response, and we explored the consequences of prior exposure for both whole-organism performance and correlated biochemical metrics. Pharate adults (the last developmental stage in the pupal case) were most sensitive to anoxia exposure, showing decreased survival and fertility compared with controls. These negative impacts were ameliorated by exposure to anoxia in earlier pupal developmental stages, indicating a hormetic effect of prior anoxia exposure. Anoxia exposure early in pupal development reduced the oxygen debt repaid after anoxia exposure relative to pharate adults experiencing anoxia for the first time. Lipid levels were highest in all pupal stages when exposed to prior anoxia. Prior anoxia thus benefits organismal performance and relocates resources towards lipid storage throughout pupal-adult development.

Low-oxygen atmospheric treatment improves the performance of irradiation-sterilized male cactus moths used in SIT.

As part of sterile insect technique (SIT) programs, irradiation can effectively induce sterility in insects by damaging genomic DNA. However, irradiation also induces other off-target side effects that reduce the quality and performance of sterilized males. Thus, treatments that reduce off-target effects of irradiation on male performance while maintaining sterility can improve the feasibility and economy of SIT programs. Exposure to ionizing radiation induces the formation of damaging free radicals in biological systems that may reduce sterile male performance. Here, we test whether exposure to an anoxic environment for 1 h before and during irradiation improves male performance, while maintaining sterility in males of the cactus moth, Cactoblastis cactorum (Berg). We show that exposure to 1 h of anoxia increases the moth's antioxidant capacity and that irradiation in anoxia after 1 h of anoxic conditioning decreases irradiation-induced oxidative damage to the moth's lipids and proteins. Anoxia treatment that reduced oxidative damage after irradiation also produced moths with greater flight performance, mating success, and longevity, while maintaining F1 male sterility at acceptable levels for SIT. We conclude that anoxia pretreatment followed by irradiation in anoxia is an efficient way to improve the quality of irradiated moths and perhaps lower the number of moths needed for release SIT moth operations.

Early life hormetic treatments decrease irradiation-induced oxidative damage, increase longevity, and enhance sexual performance during old age in the Caribbean fruit fly.

Early life events can have dramatic consequences on performance later in life. Exposure to stressors at a young age affects development, the rate of aging, risk of disease, and overall lifespan. In spite of this, mild stress exposure early in life can have beneficial effects on performance later in life. These positive effects of mild stress are referred to as physiological conditioning hormesis. In our current study we used anoxia conditioning hormesis as a pretreatment to reduce oxidative stress and improve organismal performance, lifespan, and healthspan of Caribbean fruit flies. We used gamma irradiation to induce mild oxidative damage in a low-dose experiment, and massive oxidative damage in a separate high-dose experiment, in pharate adult fruit flies just prior to adult emergence. Irradiation-induced oxidative stress leads to reduced adult emergence, flight ability, mating performance, and lifespan. We used a hormetic approach, one hour of exposure to anoxia plus irradiation in anoxia, to lower post-irradiation oxidative damage. We have previously shown that this anoxic-conditioning treatment elevates total antioxidant capacity and lowers post-irradiation oxidative damage to lipids and proteins. In this study, conditioned flies had lower mortality rates and longer lifespan compared to those irradiated without hormetic conditioning. As a metric of healthspan, we tracked mating both at a young age (10 d) and old age (30 d). We found that anoxia-conditioned male flies were more competitive at young ages when compared to unconditioned irradiation stressed male flies, and that the positive effects of anoxic conditioning hormesis on mating success were even more pronounced in older males. Our data shows that physiological conditioning hormesis at a young age, not only improves immediate metrics of organismal performance (emergence, flight, mating), but the beneficial effects also carry into old age by reducing late life oxidative damage and improving lifespan and healthspan.

Short-term anoxic conditioning hormesis boosts antioxidant defenses, lowers oxidative damage following irradiation and enhances male sexual performance in the Caribbean fruit fly, Anastrepha suspensa.

Most organisms are repeatedly exposed to oxidative stress from multiple sources throughout their lifetimes, potentially affecting all aspects of organismal performance. Here we test whether exposure to a conditioning bout of anoxia early in adulthood induces a hormetic response that confers resistance to oxidative stress and enhances male sexual performance later in life in the Caribbean fruit fly, Anastrepha suspensa. Anoxic conditioning of adults prior to emergence led to an increase in antioxidant capacity driven by mitochondrial superoxide dismutase and glutathione peroxidase. When exposed to gamma irradiation, a strong oxidative stressor, males that received anoxic conditioning had lower lipid and protein oxidative damage at sexual maturity. Anoxia conditioning led to greater male sexual competitiveness compared with unconditioned males when both were irradiated, although there was no effect of anoxia conditioning on mating competitiveness in unirradiated males. Anoxia also led to higher adult emergence rates and greater flight ability in irradiation-stressed flies while preserving sterility. Thus, hormetic treatments that increased antioxidant enzyme activity also improved male performance after irradiation, suggesting that antioxidant enzymes play an important role in mediating the relationship between oxidative stress and sexual selection. Furthermore, our work has important applied implications for the sterile insect technique (SIT), an environmentally friendly method of insect pest control where males are sterilized by irradiation and deployed in the field to disrupt pest populations via mating. We suggest that hormetic treatments specifically designed to enhance antioxidant activity may produce more sexually competitive sterile males, thus improving the efficacy and economy of SIT programs.

Rehydration driven RNAi: a novel approach for effectively delivering dsRNA to mosquito larvae.

The soft bodies and aquatic habitats of mosquito larvae pose a challenge for applying standard RNA-interference techniques to silence expression of target genes. Here we describe a novel technique for delivering double-stranded RNA into mosquito larvae by exploiting the larva's dehydration tolerance. Larvae were dehydrated in a NaCl solution and then rehydrated in water containing double-stranded RNA. Using larvae of Culex pipiens (L.) we demonstrated the principle by knocking down expression of the gene encoding heat shock protein 90. The knockdown persisted through the pupal stage and into adulthood, with a knockdown of approximately 77% still evident on the third day of adult life. We anticipate that this relatively simple procedure will prove useful for knocking down expression of other genes as well, in larvae of this mosquito and in others.

Drinking a hot blood meal elicits a protective heat shock response in mosquitoes.

The mosquito's body temperature increases dramatically when it takes a blood meal from a warm-blooded, vertebrate host. By using the yellow fever mosquito, Aedes aegypti, we demonstrate that this boost in temperature following a blood meal prompts the synthesis of heat shock protein 70 (Hsp70). This response, elicited by the temperature of the blood meal, is most robust in the mosquito's midgut. When RNA interference is used to suppress expression of hsp70, protein digestion of the blood meal is impaired, leading to production of fewer eggs. We propose that Hsp70 protects the mosquito midgut from the temperature stress incurred by drinking a hot blood meal. Similar increases in hsp70 were documented immediately after blood feeding in two other mosquitoes (Culex pipiens and Anopheles gambiae) and the bed bug, Cimex lectularius, suggesting that this is a common protective response in blood-feeding arthropods.

Heat shock proteins contribute to mosquito dehydration tolerance.

This study examines the responses of heat shock protein transcripts, Hsp70 and Hsp90, to dehydration stress in three mosquito species, Aedes aegypti, Anopheles gambiae and Culex pipiens. We first defined the water balance attributes of adult females of each species, monitored expression of the hsp transcripts in response to dehydration, and then knocked down expression of the transcripts using RNA interference (RNAi) to evaluate potential functions of the Hsps in maintenance of water balance. Fully hydrated females of all three species contained nearly the same amount of water (66-68%), but water loss rates differed among the species, with A. aegypti having the lowest water loss rate (2.6%/h), followed by C. pipiens (3.3%/h), and A. gambiae (5.1%/h). In all three species water could be replaced only by drinking water (or blood). Both A. aegypti and C. pipiens tolerated a loss of 36% of their body water, but A. gambiae was more vulnerable to water loss, tolerating a loss of only 29% of its body water. Dehydration elicited expression of hsp70 in all three species, but only C. pipiens continued to express this transcript during rehydration. Hsp90 was constitutively expressed and expression levels remained fairly constant during dehydration and rehydration, except expression was not noted during rehydration of C. pipiens. Injection of dsRNA to knock down expression of hsp70 (83% reduction) and hsp90 (46% reduction) in A. aegypti did not alter water content or water loss rates, but the dehydration tolerance was lower. Instead of surviving a 36% water loss, females were able to survive only a 28% water loss in response to RNAi directed against hsp70 and a 26% water loss when RNAi was directed against hsp90. These results indicate a critical function for these Hsps in mosquito dehydration tolerance.

Osmoregulation and salinity tolerance in the Antarctic midge, Belgica antarctica: seawater exposure confers enhanced tolerance to freezing and dehydration.

Summer storms along the Antarctic Peninsula can cause microhabitats of the terrestrial midge Belgica antarctica to become periodically inundated with seawater from tidal spray. As microhabitats dry, larvae may be exposed to increasing concentrations of seawater. Alternatively, as a result of melting snow or following rain, larvae may be immersed in freshwater for extended periods. The present study assessed the tolerance and physiological response of B. antarctica larvae to salinity exposure, and examined the effect of seawater acclimation on their subsequent tolerance of freezing, dehydration and heat shock. Midge larvae tolerated extended exposure to hyperosmotic seawater; nearly 50% of larvae survived a 10-day exposure to 1000 mOsm kg(-1) seawater and approximately 25% of larvae survived 6 days in 2000 mOsm kg(-1) seawater. Exposure to seawater drastically reduced larval body water content and increased hemolymph osmolality. By contrast, immersion in freshwater did not affect water content or hemolymph osmolality. Hyperosmotic seawater exposure, and the accompanying osmotic dehydration, resulted in a significant correlation between the rate of oxygen consumption and larval water content and induced the de novo synthesis and accumulation of several organic osmolytes. A 3-day exposure of larvae to hyperosmotic seawater increased freezing tolerance relative to freshwater-acclimated larvae. Even after rehydration, the freezing survival of larvae acclimated to seawater was greater than freshwater-acclimated larvae. Additionally, seawater exposure increased the subsequent tolerance of larvae to dehydration. Our results further illustrate the similarities between these related, yet distinct, forms of osmotic stress and add to the suite of physiological responses used by larvae to enhance survival in the harsh and unpredictable Antarctic environment.

Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration.

Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2-DE and nanoscale capillary LC/MS/MS. Twenty-four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration-regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.

Dehydration-induced cross tolerance of Belgica antarctica larvae to cold and heat is facilitated by trehalose accumulation.

Larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae), are frequently exposed to dehydrating conditions on the Antarctic Peninsula. In this study, we examined how rates and levels of dehydration alter heat and cold tolerance and how these relate to levels of trehalose within the insect. When dehydrated, larvae tolerated cold and heat stress more effectively, although resistance to cold was more pronounced than heat resistance. Slow dehydration was more effective than rapid dehydration in increasing temperature tolerance. Severe dehydration (50% reduction in water content) caused a much greater increase in temperature tolerance than did mild dehydration (e.g. 10% water loss). Larvae severely dehydrated at a slow rate (98% RH) were more temperature tolerant than those dehydrated quickly (0 or 75% RH). These results indicate that the slower dehydration rate allows the larvae to more effectively respond to reduced water levels and that physiological adjustments to desiccation provide cross tolerance to cold and heat. Levels of trehalose increased during dehydration and are likely a major factor increasing subsequent cold and heat resistance. This hypothesis was also supported by experimental results showing that injection of trehalose enhanced resistance to temperature stress and dehydration. We conclude that changes in temperature tolerance in B. antarctica are linked to the rate and severity of dehydration and that trehalose elevation is a probable mechanism enhancing this form of cross tolerance.

Dehydration, rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, Belgica antarctica.

We investigated molecular responses elicited by three types of dehydration (fast, slow and cryoprotective), rehydration and overhydration in larvae of the Antarctic midge, Belgica antarctica. The larvae spend most the year encased in ice but during the austral summer are vulnerable to summer storms, osmotic stress from ocean spray and drying conditions due to wind and intense sunlight. Using suppressive subtractive hybridization (SSH), we obtained clones that were potentially responsive to dehydration and then used northern blots to evaluate the gene's responsiveness to different dehydration rates and hydration states. Among the genes most responsive to changes in the hydration state were those encoding heat shock proteins (smHsp, Hsp70, Hsp90), antioxidants (superoxide dismutase, catalase), detoxification (metallothionein, cytochrome p450), genes involved in altering cell membranes (fatty acid desaturase, phospholipase A2 activating protein, fatty acyl CoA desaturase) and the cytoskeleton (actin, muscle-specific actin), and several additional genes including a zinc-finger protein, pacifastin and VATPase. Among the three types of dehydration evaluated, fast dehydration elicited the strongest response (more genes, higher expression), followed by cryoprotective dehydration and slow dehydration. During rehydration most, but not all, genes that were expressed during dehydration continued to be expressed; fatty acid desaturase was the only gene to be uniquely upregulated in response to rehydration. All genes examined, except VATPase, were upregulated in response to overhydration. The midge larvae are thus responding quickly to water loss and gain by expressing genes that encode proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in this Antarctic environment.

High resistance to oxidative damage in the Antarctic midge Belgica antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins.

Intense ultraviolet radiation, coupled with frequent bouts of freezing-thawing and anoxia, have the potential to generate high levels of oxidative stress in Antarctic organisms. In this study, we examined mechanisms used by the Antarctic midge, Belgica antarctica, to counter oxidative stress. We cloned genes encoding two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), and showed that SOD mRNA was expressed continuously and at very high levels in larvae, but not in adults, while Cat mRNA was expressed in both larvae and adults but at a somewhat reduced level. SOD mRNA was expressed at even higher levels in larvae that were exposed to direct sunlight. Catalase, a small heat shock protein, Hsp70 and Hsp90 mRNAs were also strongly upregulated in response to sunlight. Total antioxidant capacity of the adults was higher than that of the larvae, but levels in both stages of the midge were much higher than observed in a freeze-tolerant, temperate zone insect, the gall fly Eurosta solidaginis. Assays to measure oxidative damage (lipid peroxidation TBARS and carbonyl proteins) demonstrated that the Antarctic midge is highly resistant to oxidative stress.