Joshua B. Benoit

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.

Suppression of water loss during adult diapause in the northern house mosquito, Culex pipiens.

SUMMARY One of the major challenges of overwintering in the mosquito, Culex pipiens, is prevention of dehydration. In this study, we compare the water balance requirements of nondiapausing and diapausing adult females of C. pipiens. Although their percentage water content is lower, diapausing females contain both higher initial and dry masses than nondiapausing individuals. Both nondiapausing and diapausing females tolerate a loss of up to 40% of their water mass before dying, but diapausing female C. pipiens reach this point after a longer period due to their lower rate of water loss. Males, which do not overwinter in diapause, showed no differences in their water balance characteristics when reared under diapausing or nondiapausing conditions. Likewise, no changes were noted in the water balance of pupae, indicating that diapause-related changes do not occur prior to adult eclosion. This mosquito does not replenish internal water stores by generating metabolic water or by absorbing vapor from the atmosphere, but instead relies on drinking liquid water (or blood feeding in the case of nondiapausing females). The critical transition temperature, a point where water loss increases rapidly with temperature, was the highest for females, then males, then pupae, but was not influenced by the diapause program. Females in diapause did not utilize common polyols (glycerol, trehalose and sorbitol) to retain water, but instead the presence of twice the amount of cuticular hydrocarbons in diapausing compared with nondiapausing females suggests that the deposition of hydrocarbons contribute to the reduced rates of water loss. The laboratory results were also verified in field-collected specimens: mosquitoes in the late fall and winter had a lower percentage water content and water loss rate, higher initial mass, dry mass and more cuticular hydrocarbons than individuals collected during the summer. Thus, the major features of diapause that contribute to the suppression of water loss are the large size of diapausing females (reduction of surface area to volume ratio lowers cuticular water loss), their low metabolic rate and the deposition of extra cuticular hydrocarbons.

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.

Meeting the challenges of on-host and off-host water balance in blood-feeding arthropods

In this review, we describe water balance requirements of blood-feeding arthropods, particularly contrasting dehydration tolerance during the unfed, off-host state and the challenges of excess water that accompany receipt of the bloodmeal. Most basic water balance characteristics during the off-host stage are applicable to other terrestrial arthropods, as well. A well-coordinated suite of responses enable arthropods to conserve water resources, enhance their desiccation tolerance, and increase their water supplies by employing a diverse array of molecular, structural and behavioral responses. Water loss rates during the off-host phase are particularly useful for generating a scheme to classify vectors according to their habitat requirements for water, thus providing a convenient tool with potential predictive power for defining suitable current and future vector habitats. Blood-feeding elicits an entirely different set of challenges as the vector responds to overhydration by quickly increasing its rate of cuticular water loss and elevating the rate of diuresis to void excess water and condense the bloodmeal. Immature stages that feed on blood normally have a net increase in water content at the end of a blood-feeding cycle, but in adults the water content reverts to the pre-feeding level when the cycle is completed. Common themes are evident in diverse arthropods that feed on blood, particularly the physiological mechanisms used to respond to the sudden influx of water as well as the mechanisms used to counter water shortfalls that are encountered during the non-feeding, off-host state.

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.

Addition of alarm pheromone components improves the effectiveness of desiccant dusts against Cimex lectularius.

ABSTRACT We demonstrate that the addition of bed bug, Cimex lectularius,, alarm pheromone to desiccant formulations greatly enhances their effectiveness during short-term exposure. Two desiccant formulations, diatomaceous earth (DE) and Dri-die (silica gel), were applied at the label rate with and without bed bug alarm pheromone components, (E)-2-hexenal, (E)-2-octenal, and a (E)-2-hexenal: (E)-2-octenal blend. First-instar nymphs and adult females were subjected to 10-min exposures, and water loss rates were used to evaluate the response. Optimal effectiveness was achieved with a pheromone concentration of 0.01 M. With Dri-die alone, the water loss was 21% higher than in untreated controls, and water loss increased nearly two times with (E)-2-hexenal and (E)-2-octenal and three times with the (E)-2-hexenal: (E)-2-octenal blend. This shortened survival of first-instar nymphs from 4 to 1 d, with a similar reduction noted in adult females. DE was effective only if supplemented with pheromone, resulting in a 50% increase in water loss over controls with the (E)-2-hexenal: (E)-2-octenal blend, and a survival decrease from 4 to 2 d in first-instar nymphs. Consistently, the addition of the pheromone blend to desiccant dust was more effective than adding either component by itself or by using Dri-die or DE alone. Based on observations in a small microhabitat, the addition of alarm pheromone components prompted bed bugs to leave their protective harborages and to move through the desiccant, improving the use of desiccants for control. We concluded that short exposure to Dri-die is a more effective treatment against bed bugs than DE and that the effectiveness of the desiccants can be further enhanced by incorporation of alarm pheromone. Presumably, the addition of alarm pheromone elevates excited crawling activity, thereby promoting cuticular changes that increase water loss.