Giancarlo Lopez-Martinez

Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing and desiccation in the Antarctic midge, Belgica antarctica

The midge, Belgica antarctica Jacobs, is subjected to numerous environmental stressors during its 2-year life cycle on the Antarctic Peninsula, and in response it has evolved a suite of behavioral, physiological, and life-cycle modifications to counter these stressors, but thus far only a limited number of biochemical adaptations have been identified. In this study, we use a metabolomics approach to obtain a broad overview of changes in energy metabolism, amino acids, and polyols in response to three of the midges major stresses: heat, freezing, and desiccation. Using GC-MS analysis, a total of 75 compounds were identified. Desiccation (50% water loss) elicited the greatest physiological response (as determined by principal components analysis) when compared to untreated controls, with many elevated metabolites from pathways of central carbohydrate metabolism and a decrease in free amino acids. When larvae were frozen (6h at -10 degrees C), alanine and aspartate increased as well as urea. Freezing also increased three polyols (glycerol, mannitol, erythritol), while desiccation increased only two polyols (glycerol, erythritol). Heating the midges for 1h at 30 degrees C elevated alpha-ketoglutarate and putrescine while suppressing glycerol, glucose, and serine levels. Freezing and desiccation elicited elevation of four shared metabolites, whereas no shared metabolites were elevated by heat. All three treatments resulted in a reduction in serine, potentially identifying this amino acid as a marker for stress in this species. A number of metabolic changes, especially those in the sugar and polyol pools, are adaptations that have potential to enhance survival during both cold and desiccation.

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.

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.

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.

Responses of the bed bug, Cimex lectularius, to temperature extremes and dehydration: levels of tolerance, rapid cold hardening and expression of heat shock proteins

This study of the bed bug, Cimex lectularius, examines tolerance of adult females to extremes in temperature and loss of body water. Although the supercooling point (SCP) of the bed bugs was approximately −20°C, all were killed by a direct 1 h exposure to −16°C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4°C for 2 weeks nor dehydration (15% loss of water content) enhanced cold tolerance. However, bed bugs have the capacity for rapid cold hardening, i.e. a 1‐h exposure to 0°C improved their subsequent tolerance of −14 and −16°C. In response to heat stress, fewer than 20% of the bugs survived a 1‐h exposure to 46°C, and nearly all were killed at 48°C. Dehydration, heat acclimation at 30°C for 2 weeks and rapid heat hardening at 37°C for 1 h all failed to improve heat tolerance. Expression of the mRNAs encoding two heat shock proteins (Hsps), Hsp70 and Hsp90, was elevated in response to heat stress, cold stress and during dehydration and rehydration. The response of Hsp90 was more pronounced than that of Hsp70 during dehydration and rehydration. Our results define the tolerance limits for bed bugs to these commonly encountered stresses of temperature and low humidity and indicate a role for Hsps in responding to these stresses.

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

The mosquitos 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 mosquitos 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.

Rapid cold-hardening in larvae of the Antarctic midge Belgica antarctica: cellular cold-sensing and a role for calcium

In many insects, the rapid cold-hardening (RCH) response significantly enhances cold tolerance in minutes to hours. Larvae of the Antarctic midge, Belgica antarctica, exhibit a novel form of RCH, by which they increase their freezing tolerance. In this study, we examined whether cold-sensing and RCH in B. antarctica occur in vitro and whether calcium is required to generate RCH. As demonstrated previously, 1 h at -5 degrees C significantly increased organismal freezing tolerance at both -15 degrees C and -20 degrees C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at -20 degrees C. Furthermore, isolated tissues retained the capacity for RCH in vitro, as demonstrated with both a dye exclusion assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based viability assay, thus indicating that cold-sensing and RCH in B. antarctica occur at the cellular level. Interestingly, there was no difference in survival between tissues that were supercooled at -5 degrees C and those frozen at -5 degrees C, suggesting that temperature mediates the RCH response independent of the freezing of body fluids. Finally, we demonstrated that calcium is required for RCH to occur. Removing calcium from the incubating solution slightly decreased cell survival after RCH treatments, while blocking calcium with the intracellular chelator BAPTA-AM significantly reduced survival in the RCH treatments. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) also significantly reduced cell survival in the RCH treatments, thus supporting a role for calcium in RCH. This is the first report implicating calcium as an important second messenger in the RCH response.

Habitat requirements of the seabird tick, Ixodes uriae (Acari: Ixodidae), from the Antarctic Peninsula in relation to water balance characteristics of eggs, nonfed and engorged stages.

The seabird tick Ixodes uriae is exposed to extreme environmental conditions during the off-host phase of its life cycle on the Antarctic Peninsula. To investigate how this tick resists desiccation, water requirements of each developmental stage were determined. Features of I. uriae water balance include a high percentage body water content, low dehydration tolerance limit, and a high water loss rate, which are characteristics that classify this tick as hydrophilic. Like other ticks, I. uriae relies on water vapor uptake as an unfed larva and enhanced water retention in the adult, while nymphs are intermediate and exploit both strategies. Stages that do not absorb water vapor, eggs, fed larvae and fed nymphs, rely on water conservation. Other noteworthy features include heat sensitivity that promotes water loss in eggs and unfed larvae, an inability to drink free water from droplets, and behavioral regulation of water loss by formation of clusters. We conclude that I. uriae is adapted for life in a moisture-rich environment, and this requirement is met by clustering in moist, hydrating, microhabitats under rocks and debris that contain moisture levels that are higher than the tick’s critical equilibrium activity.

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

SUMMARY 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.

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

SUMMARY 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 ∼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.