MIAMI UNIVERSITY

The Graduate School

Certificate for Approving the Dissertation

We hereby approve the Dissertation of Jason B. Williams

Candidate for the Degree:

Doctor of Philosophy

______Director R.E. Lee Jr.

______Reader Alan B. Cady

______Reader James T. Oris

______Reader Jack C. Vaughn

______Graduate School Representative Robert L. Schaefer

ABSTRACT

LINKS BETWEEN DESICCATION RESISTANCE AND COLD-TOLERANCE IN AN OVERWINTERING INSECT: SEASONAL AND GEOGRAPHIC TRENDS

by Jason B. Williams In this dissertation, I have examined possible links between physiological parameters associated with survival at low temperature and water balance of an overwintering insect. The first study provided a seasonal characterization of cold-tolerance and desiccation resistance in overwintering larvae of the goldenrod gall fly, Eurosta solidaginis. From September 20 to October 30 larvae exhibited a gradual increase in cold-tolerance that was associated with increases in cryoprotectants. In contrast, larvae exhibited a two-phase increase in desiccation resistance. The first was a dramatic six- fold reduction in rate of water loss that occurred between October 3 and October 16 as the gall tissue senesced. The second, more subtle reduction occurred between October 16 and December 11 and was associated with cryoprotectant production. The second study examined cues for the rapid, seasonal increase in desiccation resistance of E. solidaginis larvae associated with senescing of the gall tissue. Desiccation resistance increased dramatically within three days of removal from the gall, and was primarily due to reductions in respiratory water loss as larvae entered dormancy. This study illustrated that dormancy in overwintering insects that was primarily thought to be an adaptation to conserve metabolic fuels, also may be essential for water conservation. I also examined cold-tolerance and desiccation resistance in three widely separated populations of overwintering E. solidaginis larvae from Michigan, Ohio and Alabama. Larvae from the most northern population had higher concentrations of the cryoprotectant glycerol, were more cold-tolerant, and had lower rates of overall water loss after acclimation to 5 °C. In contrast, southern larvae had lower rates of metabolism and transpiratory rates of water loss after acclimation to 20 °C. Lastly, I examined possible links between extracellular solute regulation and cell volume maintenance in larvae subjected to dehydration and freezing. After dehydration, low temperature, and freezing exposures, larvae had lower relative hemolymph volumes and lower than expected hemolymph osmolalities compared to controls, suggesting that hemolymph solutes are regulated and extracellular water was removed during the treatments. There was no substantial movement of ions between fluid compartments in dehydrated or frozen larvae, but cryoprotectants may have accumulated in intracellular fluids during these stresses.

LINKS BETWEEN DESICCATION RESISTANCE AND COLD-TOLERANCE IN AN

OVERWINTERING INSECT: SEASONAL AND GEOGRAPHIC TRENDS

A DISSERTATION

Submitted to the

Faculty of Miami University

in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

Department of Zoology

By

Jason B. Williams

Miami University

Oxford, Ohio

2005

Dissertation Director: Richard E. Lee, Jr Table of Contents

Item Page

Table of contents ii List of figures iv List of tables x

Chapter 1: General Introduction 1 Literarature Cited 5

Chapter 2: Partial link between the seasonal acquisition of cold-tolerance and 9 desiccation resistance in the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae).

Introduction 10 Materials and Methods 11 Results 14 Discussion 24 Acknowledgements 29 Literature Cited 29

Chapter 3: Plant senescence cues entry into diapause in the gall fly, Eurosta 34 solidaginis: resulting metabolic depression is critical for water conservation.

Introduction 35 Materials and Methods 37 Results 40 Discussion 47 Acknowledgements 53

ii Literature Cited 54

Chapter 4: Latitudinal variation in cold-tolerance and desiccation resistance in 58 the goldenrod gall fly, Eurosta solidaginis.

Introduction 59 Materials and Methods 61 Results 64 Discussion 73 Acknowledgments 81 Literature Cited 81

Chapter 5: Effect of freezing and dehydration on hemolymph volume and the 86 distribution of ions and cryoprotectants in the goldenrod gall fly, Eurosta solidaginis.

Introduction 87 Materials and Methods 89 Results 92 Discussion 98 Acknowledgments 103 Literature Cited 103

Chapter 6: Concluding Remarks 107

iii

List of Figures Figure Page

Chapter 2:

Figure 1. Daily minimum and maximum air temperatures taken from Sept. 1, 15 2001 to Dec. 31, 2001 at the weather station located at the Miami University Ecology Research Center, Oxford Ohio, USA.

Figure 2. Mean gall water contents (A), body water contents (B), hemolymph 16 osmolalities (C) and rates of water loss (D) for Eurosta solidaginis larvae collected from September 20, 2001 to January 15, 2002. Data points not sharing a letter are significantly different. Values are mean ±

S.E.M., n = 10.

Figure 3. Seasonal changes in cold-tolerance of Eurosta solidaginis larvae (n = 18 10), as indicated by survival after 24 h exposure to -2, -4, -8, -12, or -20 ºC from September 20 to October 30, 2001.

Figure 4. Mean rates of water loss versus hemolymph osmolality in Eurosta 19 solidaginis larvae collected from October 16, 2001 to December 11, 2001. To ensure all larvae were in the state of diapause for this comparison, data collected on January 15, 2001 were not used.

Figure 5. Mean water potential (bars) for goldenrod gall tissue and Eurosta 21 solidaginis hemolymph from September 20 to October 30, 2001. An asterisk indicates a significant difference between gall and larval values

for the same date of collection (p < 0.05). Values are means ± S.E.M., n = 10 for all values except gall tissue measurements on Oct 30, where n = 5.

iv Figure 6. The effects of moderate desiccation stress (95 or 76% RH) at 15 ºC for 22 10 days on cold-tolerance of Eurosta solidaginis larvae (n=20) collected on October 5, 2001. Field group data were taken on larvae collected and analyzed on October 3, 2001.

Figure 7. The effects of moderate desiccation stress (95 or 76% RH) at 15 ºC for 23 10 days on mean body water content (A), and mean rate of water loss (B) on Eurosta solidaginis larvae collected on October 5 and November 2, 2001. Field group data were taken on larvae collected and analyzed on October 3 and October 30, 2001 respectively. Values not sharing a letter are significantly different. Values are mean ± S.E.M.

Chapter 3:

Figure 1. Mean (± SEM) body water content (n = 10) for Eurosta solidaginis 42 larvae analyzed immediately after collection from the field or after 3, 6, or 10 days exposure to various relative humidities in the laboratory. On a given day, laboratory group data with a + are significantly different from the 100% RH treatment using a one-way ANOVA and Bonferroni multiple comparisons test.

Figure 2. (A) Mean total rates of water loss (n =10), (B) mean rates of cuticular 43 water loss (n = 10), and (C) mean rates of metabolism (n = 7) for Eurosta solidaginis larvae analyzed immediately after collection from the field or after 3, 6, or 10 days exposure to various relative humidities in the laboratory. Rates of cuticular water loss were measured on larvae after their spiracles were topically blocked with stopcock grease. Means (± SEM) with an * are significantly different than the Oct. 1 data points using a one-way ANOVA and Bonferroni multiple comparisons test. On a given day, laboratory group data with a + are significantly

v different from the 100% RH treatment using a one-way ANOVA and Bonferroni multiple comparisons test.

Figure 3. (A) Mean hemolymph osmolality (n = 10), and (B) mean glycerol 46 concentration (n = 7) for Eurosta solidaginis larvae analyzed immediately after collection from the field or after 3, 6, or 10 days exposure to various relative humidities. Means (± SEM) with an * are significantly different from the Oct. 1 data points using a one-way ANOVA and Bonferroni multiple comparisons test. On a given day, laboratory group data with a + are significantly different from the 100% RH treatment using a one-way ANOVA and Bonferroni multiple comparisons test.

Figure 4. Mean reductions in the total rate and cuticular rate of larval water loss 49 for Eurosta solidaginis between the non-diapausing Oct. 1 control and diapausing Oct. 20, 75% RH day 3, and 75% RH day 10 experimental groups. These three experimental groups were chosen for this comparison because they had significantly reduced both rate of total water loss and rate of cuticular water loss compared to the Oct. 1 control.

Chapter 4:

Figure 1. (A) Responsive and (B) developed larvae of Eurosta solidaginis (n = 65 50 per collection site) collected from Michigan, Ohio, and Alabama after exposure to -40 °C for 96 h. Larvae were held for 16 weeks at 5 °C and judged to be responsive if they moved after tactile stimulation. After that period, larvae were transferred to 23 °C and larvae were determined to be alive if they pupated, eclosed or became fully formed adults. At a given testing period or developmental stage, values not sharing the same letter were significantly different using a one-way

vi ANOVA followed by Tukey’s multiple comparison procedure after the proportions were angularly transformed.

Figure 2. (A) Mean glycerol concentration (n =10) and (B) mean sorbitol 67 concentrations (n = 10 per data point), for larvae of Eurosta solidaginis collected from Michigan, Ohio, and Alabama and acclimated to either 5 °C or 20 °C. Means (± SEM) of larvae acclimated at 5 °C not sharing the same letter or means of larvae acclimated at 20 °C not sharing the same number were significantly different when analyzed with a one- way ANOVA followed with a Bonferroni multiple comparisons test. Values in the 5 °C temperature treatment with an asterisk indicate a significant difference between 5 °C and 20 °C data from larvae collected at the same site when using a t-test.

Figure 3. Mean hemolymph osmolality (n =10) for larvae of Eurosta solidaginis 68 collected from Michigan, Ohio, and Alabama and acclimated to either 5 °C or 20 °C. Means (± SEM) of larvae acclimated at 5 °C not sharing the same letter or means of larvae acclimated at 20 °C not sharing the same number were significantly different when analyzed with a one- way ANOVA followed with a Bonferroni multiple comparisons test. Values in the 5 °C temperature treatment with an asterisk indicate a significant difference between 5 °C and 20 °C data from larvae collected at the same site when using a t-test.

Figure 4. (A) Mean hemolymph volume (n =10), and (B) mean body water 69 content (n = 10) for larvae of Eurosta solidaginis collected from Michigan, Ohio, and Alabama and acclimated to either 5 °C or 20 °C. Means (± SEM) of larvae acclimated at 5 °C not sharing the same letter or means of larvae acclimated at 20 °C not sharing the same number were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test.

vii Figure 5. (A) Mean total rate of water loss (n =10), and (B) mean blocked 72

spiracles rate of water loss (n = 10), and (C) mean CO2 production (n = 8) for larvae of Eurosta solidaginis collected from Michigan, Ohio, and Alabama and acclimated to either 5 °C or 20 °C. Means (± SEM) of larvae acclimated at 5 °C not sharing the same letter or means of larvae acclimated at 20 °C not sharing the same number were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test. Values in the 5 °C temperature treatment with an asterisk indicate a significant difference between 5 °C and 20 °C data from larvae collected at the same site when using a t- test.

Figure 6. (A) October to March weekly mean air temperature from 1971 to 2000 74 from weather stations located within 15 km of the larval collection sites. (B) Average daily minimum temperatures from December to mid-February, the coldest part of the winter, from 1971 to 2000 from weather stations located within 15 km of the larval collection sites. Michigan temperature data was recorded from the weather station located at the Grand Rapids airport, Ohio temperature data was recorded at the Fairfield weather station, and Alabama temperature data was recorded at the Auburn Agronomy weather station. All weather station data can be accessed on the National Oceanic and Atmospheric website: http://www.ncdc.noaa.gov/oa/climate/climateresources.html

Chapter 5:

Figure 1. Whole body and extracellular concentrations of (A) mean glycerol (n = 94 10), (B) mean sorbitol (n = 10), and (C) relative of proportion of total glycerol and sorbitol located in the hemolymph for mid-winter- collected larvae of Eurosta solidaginis larvae exposed to various

viii treatment conditions. I estimated the precentage of total cryoprotectants in the hemolymph by combining sorbitol and glycerol data in parts A and B of this figure and deviding the extracellular componenet from the whole body cocentrations. Whole body means (± SEM) not sharing a letter and extracellular means not sharing a number are significantly different when analyzed using a one-way ANOVA followed with a Bonferroni multiple comparisons test.

Figure 2. Mean whole body and extracellular ion concentrations (n = 15) of (A) 96 sodium, (B) potassium, and (C) magnesium for mid-winter collected larvae of Eurosta solidaginis larvae exposed to various treatment conditions. Whole body means (± SEM) not sharing a letter and extracellular means not sharing a number are significantly different when analyzed using a one-way ANOVA followed with a Bonferroni multiple comparisons test.

Figure 3. Mean carcass ion concentrations (n = 15) of (A) sodium, (B) 97 potassium, and (C) magnesium for mid-winter collected larvae of Eurosta solidaginis larvae exposed to various treatment conditions. Whole body and extracellular means (± SEM) not sharing a letter are significantly different when analyzed using a one-way ANOVA followed with a Bonferroni multiple comparisons test.

Figure 4. Mean fat body ion concentrations (n = 15) of (A) sodium, (B) 99 potassium, and (C) magnesium for mid-winter collected larvae of Eurosta solidaginis larvae exposed to various treatment conditions. Whole body and extracellular means (± SEM) not sharing a letter are significantly different when analyzed using a one-way ANOVA followed with a Bonferroni multiple comparisons test.

ix List of Tables Table

Chapter 3: Page

Table 1. Mean gall water content and water activity of goldenrod galls (Salidago 41 altissima) containing Eurosta solidaginis larvae (n=10) collected during October, 2003. In the stem cutting group, plants had their stems cut at ground level two weeks prior to the Oct. 1 collection. Galls were too dry in the stem cutting and Oct. 20 collections to accurately measure gall water activity. Means (± SEM) for gall water content not sharing a letter are significantly different.

Chapter 4:

Table 1. Mean lipid content (n =10 per) and glycogen content (n = 10), for 71 larvae of Eurosta solidaginis collected from Michigan, Ohio, and Alabama. Means (± SEM) within a column not sharing the same letter were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test.

Chapter 5:

Table 1. Mean body water content (n = 15), mean dry mass (n = 15), hemolymph 93 content (n = 25) and mean hemolymph osmolality (n = 10), for mid- winter-collected larvae of Eurosta solidaginis subjected to five different treatment conditions. Means (± SEM) within a column not sharing the same letter were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test.

x Chapter 1

General Introduction

How insects survive low temperature The majority of the planet experiences seasonally low temperatures. Consequently, both animals and plants have evolved strategies to survive low temperatures in addition to other restrictions associated with winter, such as reduced or no feeding, drinking, growth or reproduction (Marchand, 1996). To survive winter, most organisms behaviorally avoid severely low temperatures. Certain organisms, such as the Monarch butterfly, migrate to warmer climates prior to winter. Overwintering underwater or deep underground typically eliminates exposure to subzero temperatures (Pinder et al., 1992). Still other organisms avoid low temperatures by overwintering in the subnivean space (or under the snow pack) which is maintained at a relatively mild temperature of ~ 0 °C (Marchand, 1996). However, certain organisms must endure the extremely low temperatures of winter as they overwinter exposed above the snow pack. Investigators of cold hardiness typically place insects into one of two categories: Freeze susceptible or freeze tolerant (Lee, 1991; Denlinger and Lee, 1998). Most insects are freeze susceptible and only survive exposure to temperatures below the melting point of their body fluids by remaining unfrozen, termed supercooled (Lee, 1991). In contrast, freeze tolerant insects can survive extensive freezing of their body fluids as long as the ice remains confined to the extracellular space (Lee, 1991). During freezing, organisms that are freeze tolerant must survive a variety of unique stresses. Such possible stresses include hypoxia as extracellular ice formation halts hemolymph circulation (Storey and Storey, 1996) and membrane damage due to free-radical formation as oxygen is reintroduced upon thawing (Rojas and Leopold, 1996). Freezing may also stop vital functions such as the ability of membrane transporters to function (Kristiansen and Zachariassen, 2001) and skeletal and smooth muscle contraction. However, the primary mode of damage due to extracellular ice formation occurs as cells shrink below a critical volume as they osmotically lose water to the freeze concentrated extracellular fluids (Mazur, 1984; Lee, 1991, Storey and Storey, 1996). Potential benefits of the freeze

1 tolerant strategy include increased chances of survival when exposed to temperatures below the melting point of their body fluids for extended periods (se references in Sinclair et al., 2003) and reduced water loss as the water potential gradients are lowered between the frozen insect’s hemolymph and the frozen environment (Zachariassen, 1991). Both freeze tolerance and freeze susceptible strategies include unique behavioral, physiological, and biochemical adaptations (Bale, 1987; Zachariassen, 1985; Duman et al., 1991; Lee, 1991). However, certain adaptations are shared between the two overwintering strategies. For instance, most overwintering insects produce high concentrations of low-molecular-mass polyols and sugars termed cryoprotectants. Freeze susceptible insects produce cryoprotectants to colligatively lower the supercooling point of their body fluids (Lee, 1991). Freeze tolerant insects produce cryoprotectants to colligatively lower the amount of ice formed at a given temperature. Membrane penetrating cryoprotectants may also move from the freeze concentrated hemolymph into the cell during freezing to reduce cellular dehydration and maintain a critical cell volume (Storey and Storey, 1996).

Other stresses of winter In the past decade, studies of overwintering insects have broadened beyond focusing only on adaptations that promote survival to low temperatures. For instance, unseasonably warm winter temperatures can be detrimental by increasing an insect’s metabolism, reducing spring metabolic reserves, resulting in a lowered potential fecundity (Irwin and Lee, 2003; Williams et al., 2003). In addition, insects that must endure long periods of low temperature are at extreme risk of desiccation because cold air contains very little water and environmental water is typically in the form of ice or snow and unusable (Danks, 2000). Therefore, overwintering insects must not only survive low temperature, but also long periods of severe water stress. Recently, several authors have suggested that certain adaptations primarily associated with survival to low temperature may also effect, or were original adaptations for, water conservation (Ring and Danks, 1994; Block 1996; Danks, 2000). For instance, insects behaviorally avoid low temperature and also severely desiccating conditions by

2 overwintering below the snow pack, where conditions are stable and relatively mild, ~0 ˚C and 100% RH (Marchand, 1996). In addition, seasonal production of cryoprotectants may also lower rates of water loss by colligatively reducing the vapor pressure gradient between the insect’s hemolymph and environmental water vapor (Ring and Danks, 1994; Block, 1996; Bayley and Holmstrup, 1999; Sjurnsen et al., 2001). The evidence that cryoprotectants may affect both cold hardiness and desiccation resistance is indirect or circumstantial. For example, the Bruce spanworm Operophtera brumata produces cryoprotectants during desiccation stress in the summer (Ring and Danks, 1994). Also, certain tropical insects which never experience subzero temperatures produce cryoprotectants during seasonal drought stress (Pullin and Wolda, 1991). Taken together, these lines of evidence suggest that cryoprotectants are important for water retention, however, the hypothesis that cryoprotectants lower rates of water loss in overwintering insects has never been directly investigates.

Overall aim of the dissertation This dissertation is a collection of four studies, each with the underlying hypothesis: Cryoprotectants not only increase cold tolerance but also water retention in overwintering insects.

Study animal I chose the goldenrod gall fly Eurosta solidaginis as the model organism for these studies. The gall fly ranges throughout much of the United States, from southern Texas to southern Canada, and produce stem galls on goldenrod plants, Solidago spp. (Uhler, 1951). In late spring (beginning of June for populations near Ithaca, New York), adult flies emerge from their galls and search for suitable mates (Uhler, 1951). After mating, female flies lay eggs on the unfolded leaves of the terminal bud of the goldenrod plant. Eggs incubate for 5-7 days, depending on temperature, prior to hatching (Uhler, 1951). After the larva emerges from the egg, it bores through the unfolded leaf into the meristematic tissue and begins to chew out a chamber where it will reside. The plant forms a gall around the insect as the larva feeds and grows within the plant tissue. Growth continues and larval masses peak, at ~55 mg, just prior to plant senescence in

3 early October for larvae in southwest Ohio (Bennett and Lee, 1997). Larvae enter diapause at this time and remain in the gall throughout the winter as freeze-tolerant third instars. Larvae begin post-diapause development after exposure to the appropriate thermal cues in early spring (Irwin et al., 2001). In early fall, larvae of E. solidaginis can only survive relatively mild subzero temperatures, ~-15 °C for 24 h (Lee and Hankinson, 2003). During the fall and winter, however, larvae produce high quantities of the cryoprotectants glycerol and sorbitol to increase cold-hardiness. Production of these cryoprotectants are highly temperature dependent as glycerol is produced at temperatures above 15 °C and sorbitol is produced at temperatures below 5 °C (Storey et al., 1981). In mid-winter, concentrations of these cryoprotectants may reach a combined 1 molar which, at least in part, allows these larvae to survive exposures to temperatures below -40 °C (Lee, 1991). Mid-winter larvae of E. solidaginis are extremely resistant to desiccation; in fact they have rates of water loss similar to adult, heavily sclerotized desert beetles (Ramløv and Lee, 2000). However the mechanisms by which E. solidaginis larvae attain such low rates of water loss is unknown, also, it has yet to be determined if rates of water loss change seasonally similar to changes in cold-tolerance.

Dissertation layout and specific hypothesis My dissertation consists of six chapters. This initial introduction chapter, chapters two to five consist of individual studies, and a final concluding chapter. In chapter two, I specifically test the hypothesis: cold-tolerance and desiccation resistance increase with seasonal production of cryoprotectants. In this study I tested parameters associated with desiccation resistance and cold tolerance on larvae collected bi-weekly from late summer to mid-winter. Data from this study illustrated that larvae undergo two phases of reduced rates of water loss. An initial 6-fold reduction in rate of water loss occurs in a two-week period, associated with plant senescence, followed by a more subtle 4-fold reduction over an 8-week period. In chapter three I closely examined the initial 6- fold reduction in rates of water loss illustrated in chapter two. I specifically tested the hypothesis that the reduced rate of water loss during this period is triggered by senescing

4 of the gall tissue and is due primarily to lowered respiratory transpiration as larvae enter diapause? In chapter four I exploited the large range of E. solidaginis and examined parameters of cold-hardiness and desiccation tolerance in three widely separated larval populations (from Michigan, Ohio and Alabama). I specifically asked the question: are northern larvae, which experiences lower overwintering temperatures and longer periods of desiccation stress, more cold-tolerant, more desiccation resistance and produce higher concentrations of cryoprotectants than southern-collected larvae. Lastly, I examined the role of cryoprotectants in regulating cell volume during both dehydration and freezing in E. solidaginis. I hypothesized that mid-winter collected larvae would regulate cell volume during both freezing and dehydration by moving solutes (i.e. cryoprotectants and/or ions) from the hemolymph into the cells.

Broader impacts of the dissertation Traits associated with water retention, such as cryoprotectant production, may represent a pre-adaptation that facilitated the evolution of increased cold-tolerance in insects. This, in turn, could have allowed for dispersal of these species from southern to northern climates. For example, the biochemical pathways for synthesizing and accumulating cryoprotectants in warmer climates may have been selected for because quantitative increases in production of these solutes would promote supercooling in freeze susceptible insects and lower ice content for freeze tolerant organisms. Thus, increased hemolymph solute levels together with other physiological and morphological parameters associated with cold-hardiness, such as small body size (Lee and Costanzo, 1998) and low temperature adapted transporters (Storey and Storey, 1996), may have facilitated the radiation of insects northward and or the evolution of freeze-tolerance.

Literature Cited

Bale, J.S. (1987). Insect cold hardiness: Freezing and supercooling – an ecophysiological prespective. J. Insect Physiol. 33, 899-908.

5 Bayley, M. and Holmstrup, M. (1999). Water vapor absorption in arthropods by accumulation of myoinositol and glucose. Science 285, 1909-1911. Bennett, V. A. and Lee, R. E. (1997). Modeling seasonal changes in intracellular freeze- tolerance of fat body cells of the gall fly Eurosta solidaginis (Diptera, Tephritidae). J. Exp. Biol. 200, 185-192. Block, W. (1996). Cold or drought - The lesser of two evils for terrestrial arthropods? Eur. J. Entomol.93, 325-339. Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46, 837-852. Denlinger, D. L. and Lee, R. E. (1998). Physiology of cold sensitivity. In Temperature Sensitivity in Insects and Application in Integrated Pest Management, (eds. G. J. Hallman and D. L. Denlinger), pp. 55-95. Boulder: Westview Press. Duman, J. G., Xu, L., Neven, L. G., Tursman, D. and WU, D. W. (1991). Hemolymph proteins involved in insects subzero-temperature tolerance: ice nucleators and anti- freeze protiens. In Insects at Low Temperature, (ed. R. E. Lee and D. L. Denlinger), pp. 94-127. New York: Chapman and Hall. Irwin, J. T., Bennett, V. A. and Lee, R. E. (2001). Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera: Tephritidae). J. Comp. Phys. B 171, 181-188. Irwin, J. T. and Lee, R. E. (2003). Cold winter microenvironments conserve energy and improve overwintering survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis. Oikos 100, 71-78. Kristiansen, E. and Zachariassen, K. E. (2001). Effect of freezing on the transmembrane distribution of ions in freeze-tolerant larvae of the wood fly Xylophagus cinctus (Diptera, Xylophagidae). J. Insect Physiol. 47, 585-592. Lee, R. E. (1991). Principles of insect low temperature tolerance. In Insects at Low Temperature, eds. R. E. Lee and D. L. Denlinger), pp. 17-46. New York and London: Chapman and Hall. Lee, R. E. and Costanzo, J. P. (1998). Biological ice nucleation and ice distribution in cold-hardy ectothermic animals. An. Rev. Physiol. 60, 55-72.

6 Lee, R. E. and Hankison, S. J. (2003). Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae, Eurosta solidaginis (Diptera, Tephritidae). J. Insect Physiol. 49, 385-393. Marchand, P. J. (1996). Life in the cold: an introduction to winter ecology 3rd ed. Hanover, NH: University Press of New England. Mazur, P. (1984). Freezing of living cells. Am. J. Physiol. 16, C125-C142. Pinder, A. W., Storey, K.B. and Ultsch, G.R. (1992) Estivation and hibernation. In Environmental Biology of the Amphibia, (eds. M.E. Feder and W.W. Burggren), pp. 250-274. Chicago: University of Chicago Press. Pullin, A.S. and Wolda, A. (1993). Physiol. Ent. 18, 75-78. Ramløv, H. and Lee, R. E. (2000). Extreme resistance to desiccation in overwintering larvae of the gall fly Eurosta solidaginis (Diptera, Tephritidae). J. Exp. Biol. 203, 783-789. Ring, R. A. and Danks, H. V. (1994). Desiccation and cryoprotection - overlapping adaptations. Cryo-Lett. 15, 181-190. Rojas, R.R. and Leopold, R.A. (1996). Chilling injury in the house fly: Evidence for the role of oxidative stress between pupariation and emergence. Cryobiol. 33, 447-458. Sinclair, B.J., Addo-Bediako, A. and Chown, S.L. (2003). Climatic variability and the evolution of insect freeze tolerance. Biol. Rev. 28, 181-195. Sjursen, H., Bayley, M. and Holmstrup, M. (2001). Enhanced drought tolerance of a soil-dwelling springtail by pre-acclimation to a mild drought stress. J. Insect Physiol. 47, 1021-1027. Storey, K. B., Baust, J. G. and Storey, J. M. (1981). Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Eurosta solidaginis. J. Comp. Physiol. 144, 183-190. Storey, K. B. and Storey, J. M. (1996). Natural freezing survival in animals. Annu. Rev. Ecol. Syst. 27, 365-386. Uhler, L. D. (1951). Biology and Ecology of the Goldenrod Gall Fly, Eurosta solidaginis (Fitch). In Cornell Experiment Station Memoir 300, pp. 1-51. New York: New York State College of Agriculture.

7 Williams, J. B., Shorthouse, J. D. and Lee, R. E. (2003). Deleterious effects of mild simulated overwintering temperatures on survival and potential fecundity of rose- galling Diplolepis wasps (Hymenoptera : Cynipidae). J. Exp. Zool. 298A, 23-31. Zachariassen, K. E. (1985). Physiology of cold tolerance in insects. Physiol. Rev. 65, 799-832. Zachariassen, K. E. (1991). The water relations of overwintering insects. In Insects at Low Temperature, (ed. R. E. Lee and D. L. Denlinger), pp. 47-63. New York: Chapman and Hall.

8 Chapter 2

Partial link between the seasonal acquisition of cold-tolerance and desiccation resistance in the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae)

Published in The Journal of Experimental Biology (2004) 207:4407-4414

9

Introduction

Many insects that overwinter in temperate and polar regions must tolerate not only extreme cold but also desiccation stress. Recently, several reviews suggested that certain behavioral and physiological adaptations promoting cold-tolerance may also influence, or were originally adaptations for, desiccation resistance (Ring and Danks, 1994; Block, 1996; Danks, 2000). For instance, freeze-tolerant insects use glycerol and other low-molecular-mass polyols and sugars, termed cryoprotectants, to decrease the amount of body water that freezes at a given temperature, thereby preventing excessive cellular dehydration (Baust and Lee, 1981; Storey and Storey, 1992; Zachariassen, 1991). Increased cryoprotectant concentrations may also lower water loss rates by colligatively reducing the vapor pressure deficit between the insect’s hemolymph and environmental water vapor (Ring and Danks, 1994; Bayley and Holmstrup, 1999; Sjursen et al., 2001). However, most studies of overwintering insects have focused on how adaptations promote low temperature survival with little attention to the possible effects these adaptations may have on water conservation. Larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera, Tephritidae), have been used extensively as an insect model for studying freeze tolerance. The gall fly ranges throughout much of the United States and southern Canada where they induce stem galls on goldenrod plants (Solidago spp.; Uhler, 1951). Larvae feed on the mature, moist gall tissue throughout the summer. In early autumn, larvae cease feeding as the goldenrod plant senesces and overwinter as freeze-tolerant third instar larvae within their dried galls. Gall tissue offers little protection against winter extremes (Layne, 1993). Overwintering larvae experience ambient air temperatures and extremely desiccating conditions above the snow pack, although hydric parameters of the gall may change depending on precipitation (Layne, 1993). To survive the low temperatures and desiccating conditions of winter, larvae of E. solidaginis increase their cold-tolerance during the autumn and have extremely low rates of water loss. In early autumn, few larvae can survive -6 °C for 24 h, but as the season progresses larvae readily survive freezing at -20 °C (Lee and Hankinson, 2003). The

10 seasonal increase in cold-tolerance is correlated with the accumulation of the cryoprotectants glycerol and sorbitol, whose synthesis is triggered, respectively, by drying of the gall tissue and low temperature (Baust and Lee, 1982; Rojas et al., 1986; Storey and Storey, 1992). Recently, mid-winter E. solidaginis larvae were shown to have extremely low rates of water loss, rates comparable to heavily sclerotized desert beetles (Ramløv and Lee, 2000). However, it is unknown if resistance to water loss changes in this species from early autumn, when gall tissue is fully hydrated, to mid-winter, when their galls can be extremely dry. Also, if seasonal changes in the rate of water loss exist, are these reductions in rate of water loss linked to physiological process of increasing cold-tolerance? Previous studies examined several parameters of cold-tolerance and their possible link to desiccation resistance in cold-hardy insects collected in mid-winter (c.f. Williams et al., 2002). In contrast, the purpose of the present study was to characterize seasonal changes in cold-tolerance and resistance to water loss in E. solidaginis larvae to determine if the acquisition of desiccation resistance is linked to increases in cold- tolerance. To investigate this question we measured survival after exposure to subzero temperatures, hemolymph osmolality as a measure of cryoprotectant production, resistance to water loss, and body water content of field collected larvae from early autumn to mid-winter. To identify possible environmental cues for seasonal increases in cold-tolerance and enhanced desiccation resistance we monitored ambient temperature, gall water content and gall water activity. In conjunction with the field study, we also examined the effect of mild desiccation stress on rates of water loss and cold-tolerance prior to and after plant senescence and gall drying in the autumn.

Materials and Methods

Insect collection Galls containing third instar larvae of E. solidaginis were collected biweekly from Sept. 20 to Nov. 14, 2001 and then again on Dec. 11, 2001 and Jan. 15, 2002 from the Miami University Ecology Research Center in Oxford, Ohio. All tests were initiated

11 within 24 h of gall collection. To standardize for body size, only larvae weighing between 45-55 mg were used in this study.

Environmental and gall measurements Beginning September 1, 2001 air temperature was monitored by the Miami University weather station located at the Miami University Ecology Research Center approximately 0.2-0.4 km from the collection sites. Because the dried gall tissue offers little insulative value, larval body temperature should closely track ambient air temperatures, particularly on cloudy days or at night (Layne, 1993). Water activity of the galls was assessed by measuring the total water content of each gall and the water vapor potential of the gall tissue immediately surrounding the larvae. Gall water content was determined by weighing 10 galls that had contained larvae to ± 0.1 mg using a Mettler Toledo AG245 balance (Mettler-Toledo inc., Highstown, NJ USA), before and after drying in an oven at 65 ˚C until they reached a constant mass. Water vapor potential of the gall tissue was determined by the psychrometric vapor pressure depression technique described by Hølmstrup and Westh (1994). Immediately after opening an occupied gall, 10-20 mg of gall tissue directly surrounding the larval chamber was transferred to a Wescor C-52 sample chamber (sensitivity range 0 to -70 Bars; Wescor, Logan, UT, USA) and allowed to equilibrate for 30 min. Water potential was then determined with a Wescor HR 33T Dewpoint Microvoltmeter operated in the dewpoint mode. Measurements were taken on 10 selected galls for the first three testing dates. However, only five of the 10 selected galls on Oct. 30 were moist enough to obtain a reading. No vapor pressures were measured after Oct. 30 because gall tissues were too dry to measure.

Measurement of cold-tolerance Larval cold-tolerance was assessed by measuring survival rates after exposure to various subzero temperatures. Ten larvae were placed in temperature controlled baths and cooled at 1˚C•min-1 to either -2, -4, -8, or -12 ˚C. A fifth group was placed in an insulated container which provided a cooling rate of approximately 1 ˚C•min-1 until it reached equilibrium in a -20˚C freezer. After 24 h exposure to a treatment temperature,

12 larvae were warmed to room temperature (~23 °C) at 1 ˚C•min-1. Larvae were then held for 24 h at room temperature and considered alive if they moved after being gently touched with a blunt probe. The -12 ˚C experimental group was added on Oct. 3, 2001 to increase sensitivity for detecting changes in cold-hardiness. Cold-tolerance tests were not done after Oct. 30, when all larvae survived -20 ˚C for 24 h and were considered to be highly cold-tolerant. Hemolymph osmolality provided a measure of the seasonal accumulation of cryoprotectants. Hemolymph osmolality (n=10) was determined by drawing 7-10 µl of hemolymph into a capillary tube through a small incision in the larva’s cuticle. The hemolymph was then analyzed in a Wescor Vapro 550 Hemolymph Osmometer.

Measurement of desiccation resistance Resistance to desiccation was examined using measures of water loss rate in units of µg•mm-2•h-1, and body water content as a ratio of wet mass to dry mass. To determine water loss rates, 10 individuals per test date were weighed to ± 0.01 mg to obtain a fresh mass. Larvae were then re-weighed after being desiccated at 5˚C over Drierite (W.A. Hammond Drierite Co., Ohio, USA), providing a 4% RH, until they lost 5-10% of their fresh mass. Body water content was determined by placing the desiccated larvae in an oven at 65˚C until a constant dry mass was obtained. Cuticular surface area was estimated from initial wet mass using an equation derived from the best fit line for larvae of known mass and surface area. Surface area was calculated for 10 individuals of varying mass by puncturing the cuticle, expelling the internal contents by gently flattening the cuticle on millimeter-squared paper, and estimating the surface area (Williams et al., 2002; Ramløv and Lee, 2000). The derived equation was y = 0.912x + 4.204, r2 = 0.804, where y = surface area in mm2 and x = mass in mg.

Effect of moderate desiccation stress on cold-tolerance and desiccation resistance Larvae collected on Oct. 5 and Nov. 2 were used to determine the effects of mild water stress on cold-tolerance and desiccation resistance. Larvae were either held over a saturated solution of sodium sulfate producing a RH of 95% or over a saturated solution

13 of sodium chloride producing a RH of 76% at 15 ˚C. After 10 days of exposure to these conditions, larval cold-tolerance, water loss rate and body water content were measured using the techniques described previously. In contrast to the previous techniques, 15 larvae per treatment were used in these experiments as opposed to 10, and cold-tolerance was determined using only the -8 and -12 ˚C treatment conditions. Larvae collected on Oct. 3 and Oct. 30, as described in the previous sections, were compared to the 95% and 76% RH experimental groups and referred to as field groups.

Statistical analyses Seasonal data were analyzed using a one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test. To identify differences between gall and larval water activity for a given date, unpaired t-tests were used. When determining the effect of moderate desiccation on cold-tolerance and desiccation resistance, a one-way ANOVA followed by Student-Newman-Keuls test were used to indicate significant differences between treatment groups for a given date. A significance level of α = 0.05 was used for all tests. Linear regression analyses were used to estimate surface area of the larvae, as well as the relationships between events of cold hardening and acquisition of desiccation resistance.

Results

Seasonal acquisition of cold-tolerance and desiccation resistance Daily minimum air temperatures gradually decreased from early September to late December (Fig. 1). Temperatures decreased to below 5 °C for the first time on Oct. 6, and to below 0 °C on Oct. 7. However, minimum daily temperatures were not consistently below 0 °C until mid-December. In contrast to the gradual decrease in air temperature, gall water content decreased dramatically during two weeks in October as the goldenrod plants senesced. Galls were well hydrated from Sept. 20 to Oct. 16, ranging from 62 to 68% water (Fig. 2A). However, between Oct. 16 and Oct. 30, gall water content decreased significantly (p < 0.05) to 25%. Gall water content reached a minimum value of 15% on Jan. 15.

14 40

Daily Minimum Air Temperature 30 Daily Maximum Air Temperature

20

10

0 Temperature (°C) -10

-20 Sept-20 Oct-3 Oct-16 Oct-30 Nov-14 Dec-11 Jan-15

Collecting Dates

Figure 1. Daily minimum and maximum air temperatures taken from Sept. 1, 2001 to Dec. 31, 2001 at the weather station located at the Miami University Ecology Research Center, Oxford Ohio, USA.

15 different. Valuesarem Septem osm Figure 2.Meangallwatercontents(A

Mean Rate of Water Loss Mean Hemolymph Osmolality Mean Body Water Content Mean Gall Water Content o

lalities (C)andra -2 -1 -1 -1 -1 (µg mm h ) (mosm kg ) (mg H2O mg dry mass ) (mg H2O mg dry mass ) ber 20,2001toJanuary15, 2002.Datapoi 1000 1100 400 500 600 700 800 900 0. 0. 1. 1. 2. 2. 1. 1. 1. 1. 1. 2. 0 5 0 5 0 5 0 2 4 6 8 0 2 3 4 5 0 1 Se pt a a a a - te 2 e 0 s of an ± O c waterlo a, t a a a D S. - b Fi 0 E r 3 a s . t M Ph te . O a , n=10. s o e c ss (D)f a b t b a - f 1 ), bodywatercontents(B),hem I 6 n s O 16 e c S o A c b b a t eco c D r - B C 3 t n Eurosta solidaginis 0 C d P h N as o nts notsharingalette e ov- lle a b d bb 14 c t io D e n e c b a - 11 larvaecollectedf J a n- a b b f o 15 lymph r aresignificantly

r om

Even though gall water content decreased markedly during the study period, larval body water content remained statistically unchanged (Fig. 2B). No trends were evident in values for body water content, which ranged between 1.44 and 1.71 mg water • mg dry mass-1. As air temperatures decreased through the autumn and winter, larval cold- hardiness gradually increased (Fig. 3). Larvae collected in September already had a modest level of cold-tolerance, as all larvae survived a 24-h exposure to -2 °C and 90% survived -4 °C; however, no larvae survived -20 °C. Larvae were judged to be extremely cold-tolerant on Oct. 30, as all individuals survived -20 °C for 24 h. Notably, throughout the study larvae tolerated temperatures that were 10-20 °C lower than were measured in the field. The gradual increase in cold-tolerance of E. solidaginis larvae was mirrored by steady increases in hemolymph osmolality (Fig. 2C). Values for hemolymph osmolality increased significantly (p < 0.05) at each successive testing date after Oct. 16 and ranged from an initial value of 488 mosmol•kg-1 to the final measure of 967 mosmol•kg-1. In contrast to the gradual increase in larval cold-tolerance, there were two distinct periods in which water loss rates decreased during the autumn. The first phase of reduced rates of water loss was a substantial six-fold decrease that occurred between Oct. 3 (3.5 µg•mm-2•h-1) and Oct. 16 (Fig. 2D). This initial reduction in the rate of water loss was followed by a second phase in which rates of water loss decreased more slowly over an 8 week period (Fig. 2D). Even though the second phase of reduced rates of water loss was not as dramatic as the one that occurred in early October, the 3.9-fold decrease was significantly different when the Oct. 16, Oct. 30, Nov. 14 and Dec. 11 data were analyzed separately using an ANOVA followed by Student-Newman-Keuls test (Fig. 2D). Interestingly, the decrease in rates of water loss during this period correlated strongly with increases in hemolymph osmolality levels (Fig. 4). It is important to note that the data for the larvae collected on January 15 were excluded from this analysis because these individuals were probably no longer in the refractory phase of diapause (Irwin et al., 2001).

17 100

80

-2°C 60 -4°C -8°C -12°C 40 -20°C Percent Survival 20

0 Sept-20 Oct-3 Oct-16 Oct-30 Date of Insect Collection

Figure 3. Seasonal changes in cold-tolerance of Eurosta solidaginis larvae (n = 10), as indicated by survival after 24 h exposure to -2, -4, -8, -12, or -20 ºC from September 20 to October 30, 2001.

18 0.8

0.7

0.6 y = -0.001x + 1.355 r2 = 0.94 )

-1 0.5 h

-2 0.4 mm

g

µ 0.3 (

0.2 Mean Rate of Water Loss 0.1

0.0 500 600 700 800 900 -1 Mean Hemolymph Osmolality (mosm kg )

Figure 4. Mean rates of water loss versus hemolymph osmolality in Eurosta solidaginis larvae collected from October 16, 2001 to December 11, 2001. To ensure all larvae were in the state of diapause for this comparison, data collected on January 15, 2001 were not used.

19 The water potential of the gall tissue decreased from Sept. 20 to Oct. 16, ranging between -9.1 and -12.7 bars (Fig. 5). By Oct. 30, gall tissue was considerably drier, only 5 of the 10 randomly selected galls were moist enough to obtain a measure of water potential. Water potential of the gall tissue was significantly higher (p < 0.05) than the water potential of larval hemolymph on Sept. 20 and Oct. 3, suggesting larvae were not subjected to desiccation stress at this time. However, the water potential of the gall tissue and the insect’s hemolymph did not differ on Oct. 16, indicating the gall was transitioning between a non-desiccating and desiccating environment for the larvae.

Cold-tolerance and desiccation resistance after moderate desiccation stress To determine whether desiccation stress could induce changes in rates of water loss and cold-tolerance, larvae were collected on two different dates and were subjected to desiccating conditions in the laboratory. One group of larvae was collected on Oct. 5, when the goldenrod plant tissue was green and moist, and a second group on Nov. 2, after the plant had senesced and dried. Larvae collected on Oct. 5 were subjected to either 95% RH or 76% RH at 15 °C for 10 days prior to assessing their cold-tolerance and desiccation resistance. Even though there was an apparent trend toward increased survival at -8 °C and -12 °C for larvae in both the 76% and 95% RH treatment groups, these differences were not significant when compared to field samples taken on Oct. 3 (Fig. 6). Body water content for all larval groups for the Oct. 5 treatments were the same, averaging 1.49 mg water mass/mg dry mass (Fig. 7A). In contrast, moderate desiccation stress enhanced desiccation resistance as rates of water loss were significantly lower (p < 0.05) for larvae in the 95% RH and the 76% RH groups, 2.24 and 0.83 µg•mm-2•h-1 respectively, than the field group (Fig. 7B). These data suggests that mild desiccation stress induced an enhanced desiccation resistance in the larvae.

20 0

-5

-10 * * -15

-20

-25 Eurosta Hemolymph Mean Water Potential (Bars) -30 Gall Tissue

-35 Sept-20 Oct-3 Oct-16 Oct-30

Date of Insect Collection

Figure 5. Mean water potential (bars) for goldenrod gall tissue and Eurosta solidaginis hemolymph from September 20 to October 30, 2001. An asterisk indicates a significant difference between gall and larval values for the same date of collection (p < 0.05). Values are means ± S.E.M., n = 10 for all values except gall tissue measurements on Oct 30, where n = 5.

21 100 Control 95% RH 80 76% RH

60

40 Survival (%)

20

0 -8°C -12°C

Temperature

Figure 6. The effects of moderate desiccation stress (95 or 76% RH) at 15 ºC for 10 days on cold-tolerance of Eurosta solidaginis larvae (n=20) collected on October 5, 2001. Field group data were taken on larvae collected and analyzed on October 3, 2001.

22 not sharingaletterare larvae collectedandanalyzedonOctober3 larvae collectedonOctober5 on m Figure 7.T

Mean Rate of Water Loss Mean Body Water Content

ean bodywatercontent(A),and -1 -2 -1 (mg H O mg dry mass ) (µg mm h ) 2

0. 0. 1. 1. 2. 0 1 2 3 4 5 h 0 5 0 5 0 e effectsofm B A a significantly different.Va a oderate desiccation O c and Novem b D t - a 5 a te o m ean rateofwaterloss(B)on c f I b a er 2,2001.Field n 23 and October30,2001respectively.Values s stress (95or76%RH)at15ºCfor10days e c lues arem t C o lle a a c e group dataweretakenon an ±S.E.M. tio N n ov- a a 2 Eurosta solidaginis C 95% 76% ont a a R R r o H H l

A second group of larvae collected on Nov. 2 were tested for desiccation resistance after 10 days exposure to 95 or 76% RH at 15 °C. As with the Oct. 5 collection, the November-collected control group had the same body water content as the 95% and 76% RH experimental groups, ~ 1.68 mg water mass • mg dry mass-1 (Fig. 7A). In contrast to the Oct. 5 collection, water loss rates were very low and there were no differences in rates of water loss between larvae in the control and experimental groups (Fig. 7B). These results suggest that larvae were highly resistant to desiccation prior to being collected and subjected to these conditions on Nov. 2. Cold-tolerance was not examined for this collection date as the larvae were previously deemed to be extremely cold-tolerant on Oct. 30.

Discussion

During the autumn and winter, E. solidaginis larvae exhibited two phases of reduced rates of water loss. A rapid six-fold reduction in the rate of water loss occurred in a two-week period beginning on October 3, which did not appear to be linked to changes in cold-tolerance. A second, more subtle 3.9-fold decrease took place over a six- week period beginning on October 16, which may be linked to increasing hemolymph osmolality and cold-tolerance. The first phase of increased resistance to water loss is probably due to decreased respiratory transpiration as larvae entered diapause. Water loss through transpiration is positively linked to the activity level of a given insect. High levels of metabolic activity due to flight (Nicolson and Louw, 1982) or elevated temperatures (Ahearn, 1970), increase respiratory water loss. Irwin et al. (2001) showed that E. solidaginis larvae from southwest Ohio reduce their metabolic rate by more than 75% between October 1 and October 15, when they enter diapause. Diapause is defined as a genetically determined state of low metabolic activity, suppressed development and heightened resistance to environmental extremes that lasts longer than the adverse conditions (Danks, 1987; Tauber et al., 1986). A reduction of metabolic rate and consequent decrease in

24 respiratory transpiration, as larvae entered diapause, most likely contributed importantly to the rapid decrease in rates of water loss between October 3 and October 16. In addition to reduced transpiration, increased levels of cuticular lipids may have contributed to the first phase of reduced rates of water loss. Water loss for dormant insects primarily occurs as water diffuses across their cuticle and during respiratory transpiration (Edney, 1977; Hadley, 1994). Cuticular water loss is primarily regulated by the amount and type of epicuticular lipids on the integumental surface (see references in Hadley, 1994; Gibbs, 1998). Dormant stages of insects, which are at risk of dehydration, such as larvae of the flesh fly Sarcophaga crassipalpis (Yoder et al., 1992), the tobacco hornworm, Manduca sexta (Bell et al., 1975; Coudron and Nelson, 1981) and the moth Mamestra configurata (Hegdekar, 1979), increase the amount of their epicuticular lipids to diminish water loss. Epicuticular hydrocarbons increase 40-fold in E. solidaginis from late summer to mid-winter (D. R. Nelson and R. E. Lee, 2004) and may have contributed to the rapid phase one decrease rates of water loss (Fig. 2D). However, it is unknown if epicuticular lipids increased over the 13-day period that constituted phase one in the present study. Gall water content has been used as the primary indicator of desiccation stress in galling insects (Irwin et al., 2001; Layne and Medwith, 1997; Lee and Hankinson, 2003). However, this technique is unable to detect slight changes in water potential of the gall tissue immediately surrounding the insect that could profoundly impact its physiology and water balance. The springtail Folsomia candida increases its drought tolerance after being exposed to a water potential deficit between its environment and hemolymph of only 17 bars (Sjursen et al., 2001). Small changes in water potential between the body fluids of E. solidaginis larvae and its gall tissue may also influence its resistance to water loss. For instance, on September 20 and October 3 the water potential of larval hemolymph was significantly lower than the water potential of the surrounding gall tissue (Fig. 5), indicating the larvae were in a potentially hydrating environment. In contrast, between October 16 and 30 the water potential of the gall tissue decreased markedly, indicating a shift to a dehydrating environment. This small change in water potential deficit between the gall tissue and larval hemolymph correlates closely with the phase

25 one reduction in rates of water loss (Fig. 2D) and may be a cue that triggers larvae to increase their resistance to desiccation and to enter into diapause. We found no correlation between increased desiccation resistance and increased cold-tolerance early in the study. Between late September and October 30, larvae exhibited a gradual increase in cold-tolerance (Fig. 3) which is similar to other studies performed on this species in southwest Ohio (Lee and Hankinson, 2003) as well as in western Pennsylvania (Layne, 1991). This seasonal increase in cold-tolerance is due to the concomitant increase in cryoprotectants levels (Baust and Lee, 1981; Storey and Storey, 1992), as evidenced by hemolymph osmolality, which increased by 30% from September 20 to October 30 (Fig. 2C). However, cold-tolerance only gradually increased and hemolymph osmolality remained unchanged between October 3 and 16 when larval water loss rates decreased rapidly (Fig. 2D). In addition, larval rates of water loss were significantly lowered after being subjected to mild desiccation stress in early October (Fig. 7B), although larval cold-tolerance did not change (Fig. 6). Taken together, these data suggest that different mechanisms regulate desiccation resistance and cold-tolerance during this period. In contrast to phase one, the second phase of increased desiccation resistance correlates closely with increases in hemolymph osmolality and suggests a link between desiccation resistance and cold-tolerance in E. solidaginis (Fig. 4). It is unlikely that the decrease in the rate of water loss during the second phase was caused by changes in respiratory water loss because E. solidaginis larvae remain in diapause, with a depressed metabolic rate until mid-January (Irwin et al., 2001). As mentioned previously, epicuticular hydrocarbons increase 40-fold in E. solidaginis from late summer to mid- winter (D. R. Nelson and R. E. Lee, in press). Therefore, increased levels of epicuticular hydrocarbons may be partly responsible for the increased desiccation resistance between October 16 and December 11. However, it is likely that most cuticular hydrocarbons were added prior to experiencing desiccating conditions as the gall tissue senesced and dried in early October. The manner in which the elevated cryoprotectant concentrations could have affected water loss rates is unknown; however, it is unlikely that it is due to a colligative reduction in the water potential deficit between the insect’s hemolymph and

26 environmental water vapor (Edney, 1977; Williams et al., 2002). In response to desiccating conditions, the springtail F. candida rapidly synthesizes osmolytes, predominantly myoinositol and glucose, which colligatively lowers its hemolymph water activity and consequently reduces or even eliminates organismal water loss (Bayley and Holmstrup, 1999; Sjursen et al., 2001). The production of these solutes can reduce water loss colligatively only because the desiccating conditions the springtails experience are

quite mild (Av ~ 0.984) with a water potential deficit between the insect’s hemolymph and environmental water vapor of only ~17 bars (Bayley and Homlstrup, 1999). In contrast, gall fly larvae experience much drier conditions during winter. For example, the water potential deficit between larval hemolymph and their environment of 14,400 bars (simulated in the water loss trials of Fig. 2D) is commonly experienced by these insects in mid-winter. Between Oct. 16 and Dec. 11, larvae increased their hemolymph osmolality by 302 mosmol•kg-1 (Fig. 2C); this increase in solutes would reduce the water potential deficit between the hemolymph and the environment by only ~7 bars. Such a small reduction of the water potential deficit through colligative actions of increased solutes would have a negligible effect on rates of water loss over that period. Multiple lines of evidence indicate that carbohydrates influence arthropod water relations in a non-colligative manner. Trehalose, glycerol and sorbitol can protect cell membranes against severe desiccation stress and increase organismal tolerance to desiccation (Crow et al., 1984; Bryszewska and Epand, 1988; Crowe, 2002; Oliver et al., 2002). Certain cryoprotectants, like glycerol and sorbitol, are effective at binding water (Storey et al., 1981; Storey, 1983). Bound water differs markedly from bulk water as it is less likely to freeze than bulk water and is also highly resistant to removal when dried at biologically relevant temperatures (see references in Danks, 2000; Block, 1996). Intracellular bound water may increase post-freeze survival for freeze-tolerant organisms (Storey et al., 1981; Storey, 1983); however, little is known about the effect of extracellular bound water. The insect cuticle, a multi-layered structure with a single basal layer of epidermal cells, is the primary barrier to organismal water loss (Hadley, 1994). The cuticle also functions as the main barrier by which freeze susceptible insects resist inoculative freezing (Somme, 1982). Several insects seasonally increase their resistance to

27 inoculative freezing (Duman, 2001). Winter-acclimated larvae of the beetle, Dendroides canadensis, resist inoculative freezing better than summer larvae, in part because they produce antifreeze proteins that adhere to the epidermis (Olsen et al., 1998). Antifreeze proteins lower the non-equilibrium freezing point of a solution without affecting the melting point (Duman et al., 1991; Duman, 2001). Recently, Duman (2002) found that the cryoprotectant glycerol interacts synergistically with antifreeze proteins to increase their activity, apparently by stabilizing the protein. We speculate that cryoprotectants associate with proteins on the surface of the epidermal layer and, thereby, enhance resistance to desiccation. Although no antifreeze proteins are known to be produced by E. solidaginis, they do produce a novel, dehydrin- like protein during natural cold hardening (Pruitt and Shapiro, 2001). Dehydrins are a family of proteins produced in response to desiccation. Certain dehydrins are localized to leaf epidermal tissue in cold acclimated barley (Bravo et al., 2003) and may interact with low molecular mass cellular components (see references in Allagulova et al., 2003). Thus, it is possible that an epidermal proteins associate with cryoprotectants in E. solidaginis. Glycerol and sorbitol substantially increase the amount of bound water in E. solidaginis during natural cold-hardening; 10 to 20% of total body water is bound due to sorbitol and glycerol in mid-winter larvae (Storey et al., 1981; Storey, 1983). Taken together, bound water associated with epidermal cryoprotectants may collectively thicken the cuticular barrier, resulting in decreased rates of water loss (Fig. 4). In summary, hemolymph osmolality and cold-tolerance of E. solidaginis larvae steadily increased during the autumn. An initial rapid decrease in seasonal rates of water loss was correlated with drying of the gall tissue surrounding the larvae that was probably caused by decreased respiratory water loss as larval metabolism fell upon entering diapause. Later in the autumn, cryoprotectant accumulation may have affected water conservation through non-colligative actions.

28 Acknowledgements

I thank my collaborators on this project, R.E. Lee and N.C. Ruehl. Noah Gordan and Shuxia Yi helped with gall collection. I also thank Jim Oris, Alan Cady, and Jack Vaughn for help in the design of this project, and Robert Schaefer for aide with statistical analysis. Support for this project was provided by the National Science Foundation IBN# 0090204.

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Ahearn, G. A. (1970). The control of water loss in desert tenebrionid beetles. J. Exp. Zool. 53, 573-595. Allagulova, C. R., Gimalov, F. R., Shakirova, F. M. and Vakhitov, V. A. (2003). The plant dehydrins: Structure and putative functions. Biochem.-Moscow 68, 945-951. Baust, J. G. and Lee, R. E. (1981). Divergent mechanisms of frost-hardiness in two populations of the gall fly, Eurosta solidaginsis. J. Insect Physiol. 27, 485-490. Baust, J. G. and Lee, R. E. (1982). Environmental triggers to cryoprotectant modulation in separate populations of the gall fly, Eurosta solidaginis (Fitch). J. Insect Physiol. 28, 431-436. Bayley, M. and Holmstrup, M. (1999). Water vapor absorption in arthropods by accumulation of myoinositol and glucose. Science 285, 1909-1911. Bell, R. A., Nelson, D. R. and Borg, T. K. (1975). Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21, 1725-1729. Block, W. (1996). Cold or drought - The lesser of two evils for terrestrial arthropods? Eur. J. Entomol. 93, 325-339. Bravo, L. A., Gallardo, J., Navarrete, A., Olave, N., Martinez, J., Alberdi, M., Close, T. J. and Corcuera, L. J. (2003). Cryoprotective activity of a cold-induced dehydrin purified from barley. Physiol. Plant. 118, 262-269.

29 Bryszewska, M. and Epand, R. M. (1988). Effects of sugar alcohols and disaccharides in inducing the hexagonal phase and altering membrane-properties - implications for diabetes-mellitus. Biochim. Biophys. Acta 943, 485-492. Coudron, T. A. and Nelson, D. R. (1981). Characterization and distribution of the hydrocarbons found in diapausing pupae tissues of the tobacco hornworm, Manduca sexta (L). J. Lipid Res. 22, 103-112. Crowe, L. M. (2002). Lessons from nature: the role of sugars in anhydrobiosis. Comp. Biochem. Physiol. A 131, 505-513. Crowe, L. M., Mouradian, R., Crowe, J. H., Jackson, S. A. and Womersley, C. (1984). Effects of carbohydrates on membrane stability at low water activities. Biochim. Biophys. Acta 769, 141-150. Danks, H. V. (1987). Insect dormancy: an ecological perspective. Ottawa: Biological Survey of Canada (Terrestrial Artropods). Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46, 837-852. Duman, J. G. (2001). Antifreeze and ice nucleator proteins in terrestrial arthropods. Annu. Rev. of Physiol. 63, 327-357. Duman, J. G. (2002). The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate. J Comp Physiol B 172, 163-168. Duman, J. G., Xu, L., Neven, L. G., Tursman, D. and WU, D. W. (1991). Hemolymph proteins involved in insects subzero-temperature tolerance: ice nucleators and anti- freeze protiens. In Insects at Low Temperature, (ed. R. E. Lee and D. L. Denlinger), pp. 94-127. New York: Chapman and Hall. Edney, E. B. (1977). Water balance in land arthropods. New York: Springer-Verlag. Gibbs, A. G. (1998). Water-proofing properties of cuticular lipids. Amer. Zool. 38, 471- 482. Hadley, N. F. (1994). Water relations of terrestrial arthropods. San Diego: Academic Press. Hegdekar, B. M. (1979). Epicuticular wax secretion in diapause and non-diapause pupae of the Bertha armyworm Mamestra configurata. Ann. Entomol. Soc. Amer. 72, 13-15. Holmstrup, M. and Westh, P. (1994). Dehydration of earthworm cocoons exposed to cold - a novel cold-hardiness mechanism. J. Comp. Physiol. B 164, 312-315.

30 Irwin, J. T., Bennett, V. A. and Lee, R. E., Jr. (2001). Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera: Tephritidae). J. Comp. Physiol. B 171, 181-188. Layne, J. R. (1991). Microclimate variability and the eurythermic nature of goldenrod gall fly (Eurosta solidaginis) larvae (Diptera, Tephritidae). Can. J. Zool. 69, 614-617. Layne, J. R. (1993). Winter microclimate of goldenrod spherical galls and its effects on the gall inhabitant Eurosta solidaginis (Diptera, Tephritidae). J. Therm. Biol. 18, 125- 130. Layne, J. R. and Medwith, R. E. (1997). Winter conditioning of third instars of the gall fly Eurosta solidaginis (Diptera:Tephritidae) from western Pennsylvania. Physiol. Chem. Ecol. 26, 1378-1384. Lee, R. E. and Hankison, S. J. (2003). Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae, Eurosta solidaginis (Diptera, Tephritidae). J. Insect Physiol. 49, 385-393. Nelson, D.R. and Lee, R.E. (2004). Accumulation of cuticular lipids and increased desiccation resistance in overwintering larvae of the gall fly Eurosta solidaginis (Diptera: Tephritidae). Comp. Biochem. Physiol. B 138, 313-320. Nicolson, S. W. and Louw, G. N. (1982). Simultaneous measurement of evaporative water-loss, oxygen- consumption, and thoracic temperature during flight in a carpenter bee. J. Exp. Zool. 222, 287-296. Oliver, A. E., Hincha, D. K. and Crowe, J. H. (2002). Looking beyond sugars: the role of amphiphilic solutes in preventing adventitious reactions in anhydrobiotes at low water contents. Comp. Biochem. Physiol. A 131, 515-525. Olsen, T. M., Sass, S. J., Li, N. and Duman, J. G. (1998). Factors contributing to seasonal increases in inoculative freezing resistance in overwintering fire-colored beetle larvae Dendroides canadensis (Pyrochroidae). J. Exp. Biol. 201, 1585-1594. Pruitt, N. L. and Shapiro, C. (2001). Evidence for a cryoprotective protein in freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis. Am. Zool. 41, 1561- 1561.

31 Ramløv, H. and Lee, R. E. (2000). Extreme resistance to desiccation in overwintering larvae of the gall fly Eurosta solidaginis (Diptera, Tephritidae). J. Exp. Biol. 203, 783-789. Ring, R. A. and Danks, H. V. (1994). Desiccation and cryoprotection - overlapping adaptations. Cryo-Lett. 15, 181-190. Rojas, R. R., Lee, R. E. and Baust, J. G. (1986). Relationship of environmental water- content to glycerol accumulation in the freezing tolerant larvae of Eurosta solidaginis (Fitch). Cryobiol. 23, 564-564. Sjursen, H., Bayley, M. and Holmstrup, M. (2001). Enhanced drought tolerance of a soil-dwelling springtail by pre-acclimation to a mild drought stress. J. Insect Physiol. 47, 1021-1027. Somme, L. (1982). Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. A 73, 519-543. Storey, K. B. (1983). Metabolism and bound water in overwintering insects. Cryobiol. 20, 365-379. Storey, K. B., Baust, J. G. and Buescher, P. (1981). Determination of water "bound" by soluble subcellular components during low-temperature acclimation in the gall fly larva, Eurosta solidagensis. Cryobiol. 18, 315-321. Storey, K. B. and Storey, J. M. (1992). Biochemical adaptations for winter survival in insects. In Advances in Low-Temperature Biology, vol. 1 (ed. P. L. Steponkus), pp. 101-140. London: JAI Press. Tauber, M. J., Tauber, C. A. and Masaki, S. (1986). Seasonal Adaptations of Insects. New York: Oxford University Press. Uhler, L. D. (1951). Biology and ecology of the goldenrod gall fly, Eurosta solidaginis (Fitch). New York: New York State College of Agriculture. Williams, J. B., Shorthouse, J. D. and Lee, R. E. (2002). Extreme resistance to desiccation and microclimate-related differences in cold-hardiness of gall wasps (Hymenoptera : Cynipidae) overwintering on roses in southern Canada. J. Exp. Biol. 205, 2115-2124. Yoder, J. A., Denlinger, D. L., Dennis, M. W. and Kolattukudy, P. E. (1992). Enhancement of diapausing flesh fly puparia with additional hydrocarbons and

32 evidence for alkane biosynthesis by a decarbonylation mechanism. Insect Biochem. Mol. Biol. 22, 237-243. Zachariassen, K. E. (1991). The water relations of overwintering insects. In Insects at Low Temperature, (ed. R. E. Lee and D. L. Denlinger), pp. 47-63. New York: Chapman and Hall.

33

Chapter 3

Plant senescence cues entry into diapause in the gall fly, Eurosta solidaginis: Resulting metabolic depression is critical for water conservation.

Submitted to The Journal of Experimental Biology, May 2005

34

Introduction

Insects are intrinsically at risk of desiccation because of their small size and high surface area to volume ratio. Nevertheless, the risk of desiccation can vary greatly throughout an insect’s life cycle. For example, temperate and polar insects are potentially at a much higher risk of desiccation in the winter because cold air contains little water vapor and overwintering insects are typically dormant and unable to imbibe free water. The seasonal contrast in desiccation stress for galling insects can be even greater. Temperate galling insects likely do not experience desiccation stress in the summer while the active plant tissue they inhabit is fully hydrated (Williams et al., 2004). However, galls can quickly desiccate as the plants senesce and the gall tissues dry in the autumn. In addition, dried gall tissue provides little buffering against environmental extremes and insects remaining in their galls experience the full desiccating conditions of winter (Layne, 1993; Layne and Medwith, 1997). To survive the desiccating conditions of winter, galling insects such as cynipid wasps (Williams et al., 2002) and the goldenrod gall fly, Eurosta solidaginis (Ramløv and Lee, 2000), have extremely low rates of water loss. These soft-bodied, immature insects have rates of water loss that are similar to heavily sclerotized, adult desert beetles (see references in Edney, 1977; Hadley, 1994a). To attain such high levels of desiccation resistance, overwintering goldenrod gall fly larvae seasonally reduce their rates of water loss from late summer to mid-winter. In particular, E. solidaginis reduce their rate of water loss more than six-fold, within a two-week period in early autumn (Williams et al., 2004). However, the mechanisms by which E. solidaginis reduce their rate of water loss or the cues triggering the enhanced desiccation resistance during this period are unknown. The goldenrod gall fly (Diptera: Tephritidae) ranges throughout much of central and eastern North America, from Texas to southern Canada. In late spring or early summer, females oviposit in terminal buds (future stems) of goldenrod plants (Solidago spp.) (Uhler, 1951). Larvae feed and grow within the moist gall tissue throughout the summer and early autumn. In southwest Ohio goldenrod plants and gall tissues senesce

35 and rapidly dry in late September and early October (Irwin et al., 2001; Williams et al., 2004). In addition to reducing their rate of water loss, larvae undergo other physiological changes as their gall tissue senesces. For instance, larvae produce the cryoprotectant glycerol in response to plant senescence (Rojas et al., 1986; Baust and Lee, 1982), suggesting this low molecular weight polyol may be important in preventing dehydration. Larvae also enter diapause during this period (Irwin et al., 2001). Diapause is defined as a genetically determined state of low metabolic activity, suppressed development, and heightened resistance to environmental extremes, which generally begins before and lasts longer than the adverse conditions (Danks, 1987; Tauber et al., 1986). Overwintering and dormant insects lose water to the environment primarily through cuticular and respiratory transpiration (Edney, 1977; Hadley, 1994a; Danks, 2000). During the fall and winter, larvae of E. solidaginis reduce their rates of cuticular water loss by producing epicuticular lipids, which increase by 40-fold from summer to mid-winter (Nelson and Lee, 2004), and possibly by producing cryoprotectants (Williams et al., 2004). These larvae also may reduce their rates of water loss by lowering respiratory transpiration, as their metabolism decreases when entering diapause. However, reductions in metabolic rate for inactive insects frequently have a relatively minor effect on desiccation resistance (see reviews by Chown, 2002; Chown and Nicolson, 2004). Regardless, larvae of E. solidaginis likely reduce both avenues of water loss as they increase their desiccation resistance in early autumn as gall tissue senesces, yet the relative contributions of decreased cuticular and respiratory transpiration are unknown. The purpose of this study was to determine whether plant senescence and diapause induction were closely associated with the seasonal reduction in rates of larval water loss that occurs in early October as documented previously (Williams et al., 2004). Specifically, I measured the cuticular and estimated respiratory contributions to total organismal water loss as gall tissue naturally senesced. I also determined whether mild desiccation stress and premature plant senescence would trigger enhancement of larval desiccation resistance. To investigate these questions, I measured rates of total water loss, rates of cuticular water loss, carbon dioxide production (as an estimate of metabolic rate and diapause induction), body water content, hemolymph osmolality, and glycerol

36 content of larvae collected from the field just prior to, and after, natural plant senescence. To determine if the field-collected larvae were taken from desiccating and senescent plant tissue, I measured the water content and water activity of gall tissues.

Materials and Methods

Insect collection Fresh galls were collected before (October 1, 2003) and after (October 20, 2003) the rates of larval water loss decrease markedly (Williams et al., 2004). To induce premature drying of the gall tissue, goldenrod plants (Solidago altissima) were cut at ground level and tied upright to a stake on September 17, 2003. The cut plants remained in the field until being brought into the lab and analyzed on October 1, 2003 and were termed the “stem cutting” treatment. All galls were collected at the Ecology Research Center near Oxford, Ohio and tested within 12 h of being harvested. To standardize for size, only larvae weighing between 48 and 58 mg were used in all experimental groups except the stem cutting treatment. Because larvae grow rapidly prior to gall senescence (Bennett and Lee, 1997; Layne and Medwith, 1997) and goldenrod plants in the stem cutting treatment senesced prematurely, larval mass in this group were lower than normal, averaging 41.6 ± 1.5 mg (mean ± SEM).

Gall measurements Hydric conditions of the gall tissue were assessed by measuring the total water content and water vapor potential of the gall tissue immediately surrounding the larvae. Gall water content was determined by weighing 10 galls that had contained larvae to ± 0.1 mg using a Mettler Toledo AG245 balance, before and after drying in an oven at 65 ˚C until they reached a constant mass. Water vapor potential of the gall tissue was determined by the psychrometric vapor pressure depression technique described by Hølmstrup and Westh (1994). Immediately after opening an occupied gall, 10-20 mg of gall tissue surrounding the larval chamber was transferred to a Wescor C-52 sample chamber (range of sensitivity 0 to -70 Bars; Logan, Utah) and allowed to equilibrate for 30 min. Water potential was then determined with a Wescor HR 33T Dewpoint

37 Microvoltmeter (Logan, Utah) operated in the dewpoint mode. Measurements were taken on 10 galls collected on October 1. Galls in the stem cut treatment and those collected on October 20 were too dry to accurately measure water activity.

Measurements of desiccation resistance Resistance to desiccation was determined by measuring of rate of water loss, in units of µg•mm-2•h-1; body water content, as a ratio of wet mass to dry mass; and cuticular rate of water loss. To determine rate of organismal water loss, which includes both respiratory and cuticular components, larvae (n = 10) were weighed to ± 0.01 mg to obtain a fresh mass. Larvae were then re-weighed after being desiccated over Drierite (W.A. Hammond Drierite Co., Ohio, USA) at 4% RH and 20 ˚C until they lost 5-10% of their fresh mass (24-160 h). Cuticular surface area was estimated from initial wet mass using an equation determined by Williams et al. (2004): y = 0.912x + 4.204, r2 = 0.804, where y = surface area in mm2 and x = mass in mg. Body water content was determined by placing the desiccated larvae in an oven at 65 ˚C until a constant dry mass was obtained. Rate of cuticular water loss (µg•mm-2• h-1) was measured to determine the relative contributions of respiratory and cuticular components of overall organismal water loss. Cuticular water loss was assessed by weighing larvae (n = 10) before and after exposure to 4% RH at 20 ˚C as described above. However, prior to testing the spiracles of each larva were topically blocked with a small amount of Thomas Scientific Lubriseal stop cock grease (Swedesboro, New Jersey), to eliminate respiratory water loss.

Hemolymph osmolality and cryoprotectant concentration Hemolymph osmolality (n = 10) was determined by drawing 7-10 µl of hemolymph into a capillary tube through a small incision in the larval cuticle. The hemolymph was then analyzed in a Wescor Vapro 5520 Hemolymph Osmometer (Logan, Utah). To measure glycerol concentration, larvae (n = 7) were frozen at -80 °C until whole body measurements were performed by enzymatic assay (Sigma Chemical Co., no. 337) as described by Hølmstrup et al. (1999).

38 Measurement of metabolism

To determine larval diapause status, we assessed metabolic rate by measuring

CO2 emission. Larvae (n = 7 per treatment) were weighed and individually placed into small glass respirometry chambers kept within a temperature controlled bath held at 20 °C. Carbon dioxide was measured using a flow through (50 ml•l-1) respirometer (TR-3 model, Sable Systems, Las Vegas, Nevada) after the larvae had equilibrated to the chamber for one hour. Metabolic rate data was converted into the units of microliters of

CO2 emitted per milligram fresh mass per hour using DATACAN software (Sable Systems).

Effect of mild and moderate desiccation stress on larval desiccation resistance

A second group of larvae were collected between September 28 and October 3, prior to the dramatic seasonal decrease in rates of water loss, and were used to determine the effect of mild and moderate desiccation stress on desiccation resistance. Immediately after collection, larvae were removed from their galls and placed in desiccators over either double distilled water to produce a RH of 100%, a saturated solution of sodium sulfate producing a RH of 95% or a saturated solution of sodium chloride producing a RH of 75% at 20 °C. After 3, 6, or 10 days exposure to these conditions, larvae were removed and assessed for water loss rate, body water content, cuticular permeability, hemolymph osmolality, glycerol concentration, and metabolic rate using the methods described above.

Statistical analysis

A one-way analysis of variance followed by a Bonferroni multiple comparisons (Statview 5.0, SAS Institute Inc., Cary, NC) was used to determine differences in means between larvae collected on Oct. 1 and 20, the stem cutting treatment, and all relative humidity treatments. To increase statistical power when examining the larval treatments

39 exposed to various relatively humidities, a one-way ANOVA followed by a Bonferroni multiple comparisons was used to identify differences in RH treatments on a given day. A significance level of α = 0.05 was used for all tests.

Results

Gall measurements

Hydric characteristics of the gall tissue changed dramatically in post-senescent goldenrod plants. Gall water content was more than four-fold lower in the stem cutting group and galls collected on Oct. 20 than those examined on Oct. 1, which averaged 1.97 ± 0.01 (Mean ± SEM) mg water • mg dry mass-1 (Table 1). Hydric changes in the gall tissue were also evident as water potential of the tissue surrounding the larvae was high on Oct. 1 (-12.8 ± 0.7 bars) but was so low in the stem cutting group and those collected on Oct. 20 that no measurement could be made (Table 1). Taken together, and compared to previous reports (Williams et al., 2004), the high water content and water activity of the Oct. 1 control group indicated these galls were pre-senescent, while the low values for the stem cutting and Oct. 20 galls were fully senesced.

Measures of desiccation resistance

Although the larvae were exposed to desiccating conditions after gall senescence, they were able to maintain similar hydration levels as their body water content did not differ among the Oct. 1, stem cutting and Oct. 20 groups (Fig. 1). In addition, body water content did not differ among the laboratory RH treatments (Fig. 1). Values ranged between 1.62 ± 0.03, and 1.37 ± 0.05 mg water • mg dry mass-1 (58 to 62% of fresh mass). Rates of larval water loss were dramatically (p < 0.05) lower for larvae in the stem cutting group and those collected on Oct. 20 (averaging 0.7 ± 0.2 µg•mm-2•h-1)

40 Table 1. Mean gall water content and water activity of goldenrod galls (Salidago altissima) containing Eurosta solidaginis larvae (n=10) collected during October, 2003. In the stem cutting group, plants had their stems cut at ground level two weeks prior to the Oct. 1 collection. Galls were too dry in the stem cutting and Oct. 20 collections to accurately measure gall water activity. Means (± SEM) for gall water content not sharing a letter are significantly different.

Collection Date Gall Water Content Gall Water Activity (mg water • mg dry mass-1) (Bars) Oct. 1 1.97 ± 0.07 a -12.8 ± 0.7

Oct. 1 (stem cutting) 0.30 ± 0.04 b - Oct. 20 0.46 ± 0.12 b -

41

1.70 Field Groups Laboratory Groups

1.65

1.60 -1 s ent s

ma 1.55 y r d

mg 1.50 Water Cont

r e ody t

a 1.45 w an B e mg M 1.40 Field Groups + 100% RH 1.35 95% RH 75% RH

1.30 Oct. 1 Oct. 1 Oct. 20 Day 3 Day 6 Day 10 (Stem Cut) Treatment

Figure 1. Mean (± SEM) body water content (n = 10) for Eurosta solidaginis larvae analyzed immediately after collection from the field or after 3, 6, or 10 days exposure to various relative humidities in the laboratory. On a given day, laboratory group data with a + are significantly different from the 100% RH treatment using a one-way ANOVA and Bonferroni multiple comparisons test.

42

ANOVA andBonf data witha+aresignificantly differentfr ANOVA andBonf SEM) withan*aresignificantly different on larvaeaf various relativehum analyzed imm loss (n=10),and(C)m Figure 2.(A)Meantotalratesofwaterloss Mean Metabolic Rate Mean Cuticular Rate of Water Loss Mean Rate of Water Loss -1 -1 (µl CO g h ) -2 -1 -2 -1 2 (µg mm h ) (µg mm h ) 0. 0. 0. 0. 0. 0. 0. 0. 0. 1. 1. 1. 1. 1. 10 0 1 2 3 4 2 4 6 8 0 2 4 6 8 0 2 4 6 8 ter the e C B A diately aftercolle Oc t . i 1 r sp erroni m erroni m idities inthelaboratory. ira Fi (S e e t an ra c Oc e l les wereto m d G u u t C . 1 ltiple co ltiple co ∗ ∗ u tes ofm r t o ) ups ction from Oc mparisons te mparisons te T pically t . e 2 r tabolism ∗ ∗ eat ∗ 0 om than theOct.1datapoints usingaone-way m thefieldorafte 43 blocked withstopcockgrease.Means(± the (n =10),(B)meanratesofcuticularwater Da en Rates ofcuticularwate y (n=7)for 100%RHtreatm t 3 ∗ ∗ ∗ ∗ st. st. Onagiv L ∗ ∗ + + a + bor D at a or y 6 ∗ ∗ 75 95 10 Fi y ∗ r 3,6,or10daysexposureto ∗ E G el % % 0% u d e

rosta solidaginis G r n day,labo R R R o H H r ups ou Da H

e

ps nt usingaone-way y 1 ∗ ∗ r loss ∗ ∗ 0 + + ∗ + ratory g werem larvae r e oup asured

compared to larvae collected on Oct. 1 (6.0 ± 1.8 µg•mm-2•h-1) (Fig. 2A). After just 3 days of exposure to 75% RH, larvae had rates of water loss (1.3 ± .01 µg•mm-2•h-1) that were statistically similar to those in the stem cutting and Oct. 20 groups (Fig. 2A). Furthermore, humidity may have influenced E. solidaginis to increase desiccation resistance as 6-days exposure to 100% RH and 95% RH were required before rates of larval water loss were significantly lower that those collected and analyzed on Oct. 1 (Fig. 2A). In addition, larvae exposed to 75% RH for 10 days had significantly lower rates of water loss (0.8 ± 0.1 µg•mm-2•h-1; p < 0.05) than those in the 100% RH, 10 day treatment (2.1 ± 0.5 µg•mm-2•h-1).

To estimate the cuticular transpiration component of organismal water loss, I measured rates of larval water loss after topically blocking their spiracles. Rates of cuticular water loss were significantly lower for larvae analyzed on Oct. 20 (0.4 ± 0.1 µg•mm-2•h-1) compared to those in the Oct. 1 group (1.1 ± 0.4 µg•mm-2•h-1; p < 0.05) and there was a trend toward lowered cuticular rates of water loss for the stem cutting group (Fig. 2B). Larvae exposed to 75% and 95% RH for only 3 days had cuticular rates of water loss averaging 0.4 ± 0.1 µg•mm-2•h-1 that were significantly lower than for larvae analyzed on Oct. 1 (p < 0.05). In contrast, larvae in high humidity treatments did not lower their cuticular transpiration; larvae exposed to 100% RH for 3, 6, and 10 days had the same rates of cuticular water loss as those examined on Oct. 1 (Fig. 2B). In addition, rates of cuticular water loss were significantly higher in larvae exposed to 100% RH than those in the 95% and 75% RH treatments for day 3 and 10 of the analysis (Fig. 2B).

Measures of metabolism

Metabolic rate was significantly lower (p < 0.05) for larvae analyzed on Oct. 20 (post-plant senescence) and in the stem cutting group (premature plant senescence) -1 -1 averaging 0.05 ± 0.01 µl CO2•g •h compared to larvae from the Oct. 1 group (pre- -1 -1 senescent goldenrod; 0.31 ± 0.06 µl CO2•g •h ; Fig. 2C). Interestingly, metabolic rate lowered rapidly once the larvae are removed from their gall; all larvae in the 100%, 95%, -1 -1 and 75% RH treatments had similar metabolic rates (0.09 ± 0.05 µl CO2•g •h ) similar

44 to those in the stem cutting and Oct. 20 group after just three days after being removed from the gall. No larvae, in any experimental group, demonstrated discontinuous gas exchange.

Hemolymph osmolality and cryoprotectant production

Hemolymph osmolality increased by an average of 105 mOsm•kg-1 in the stem cutting and Oct. 20 treatments compared to the Oct. 1 collection, which averaged 534 ± 14 mOsm•kg-1 (Fig. 3A). Larvae exposed to 100%, 95%, and 75% RH for 6 and 10 days had significantly higher hemolymph osmolalities (averaging 673 ± 27 mOsm•kg-1) than those in the Oct. 1 collection (p < 0.05). Mildly desiccating conditions did not appear to influence hemolymph osmolality as solute concentrations did not differ between the 100% RH and 95 or 75% RH treatments during the 3, 6, or 10 days of exposure (Fig. 3A).

Changes in hemolymph osmolality were largely due to increases in glycerol content. Glycerol concentrations increased by over 3.4-fold in larvae collected on Oct. 20 compared to the Oct.1 group, which averaged 47 ± 5 mOsm•kg-1(Fig. 3B). The effect of relative humidity on glycerol concentration is less clear. Although there was no difference between RH treatments on day 3 and day 10, the larvae in the 75% RH treatment had higher glycerol levels than the Oct. 1 group on day 6 and day 10 of the study. Considering all field and laboratory groups together, glycerol production constituted approximately 61% of the overall increase in hemolymph osmolality compared to the Oct. 1 control group (Fig. 3A).

45

750 A Field Groups Laboratory Groups y

lit 700

la * o

) * 1 * - * * * kg h Osm

p 650 * * m m s ly O o * (m

Hem 600 n

Mea Field Groups 100% RH 550 95% RH 75% RH

500

240 B 220 200 * +

ion 180

160 * ) ncentrat

s * e

l 140 Co ol r 120 mmo ( yce 100 Gl n 80 Mea 60 40 20 Oct. 1 Oct. 1 Oct. 20 Day 3 Day 6 Day 10 (Stem Cut)

Treatment Figure 3. (A) Mean hemolymph osmolality (n = 10), and (B) mean glycerol concentration (n = 7) for Eurosta solidaginis larvae analyzed immediately after collection from the field or after 3, 6, or 10 days exposure to various relative humidities. Means (± SEM) with an * are significantly different from the Oct. 1 data points using a one-way ANOVA and Bonferroni multiple comparisons test. On a given day, laboratory group data with a + are significantly different from the 100% RH treatment using a one-way ANOVA and Bonferroni multiple comparisons test.

46 Discussion

Rapid increase in desiccation resistance

Since stem galls often project above the snowpack, larvae of E. solidaginis may experience severely desiccating conditions during winter. To survive six months or longer in their desiccated galls, in early autumn to mid-winter larvae must reduce their rates of water loss from relatively high levels (Ramløv and Lee, 2000; Williams et al., 2004). Larvae from prematurely and naturally senesced plants had significantly reduced rates of water loss compared to the Oct. 1 control group. The reduced rates of water loss for larvae from senesced plant tissue (stem cutting and Oct. 20 treatments) occurred quickly (within a few weeks) and were similar to rates measured from mid-winter acclimated larvae (Williams et al., 2004; Fig. 2A). In fact, mid-winter rates of water loss were probably attained within days after the plant tissue senesced because it only took three days for larvae in the 75% RH treatment and six days for larvae in the 95% and 100% RH treatments to reduce their rates of water loss to near mid-winter levels. Regardless of the speed at which these larvae reduced their rates of water loss, enhanced desiccation resistance for these larvae can be attributed to a combination of decreases in cuticular and respiratory transpiration.

Enhanced cuticular resistance plays a minor role in rapid increase in desiccation resistance

It is generally thought that increased desiccation resistance in arthropods is primarily due to a reduction in cuticular water loss (Hadley, 1994a; Hadley, 1994b). However, reduced cuticular transpiration contributed only a small component to lowering the rates of total water loss in this study (Fig. 4). Three groups of larvae significantly reduced both their rates of total water loss and rates of cuticular water loss compared to the Oct. 1 control (Oct. 20 field group, 75% RH day 3, and 75% RH day 10 groups; Figs. 2A and 2B). Larvae in these groups averaged a decrease in their total rate of water loss of 5.1 µg•mm-2•h-1, but reduced their cuticular rate of transpiration by only 0.8 µg•mm-

47 2•h-1 (Fig. 4). Thus, cuticular reductions in rate of water loss only accounted for a small portion (15%) of the overall decrease in total rate of water loss in these groups. Consequently, because the decrease in the rate of water loss for E. solidaginis larvae at this time in their life cycle is only due to changes in cuticular and respiratory transpiration, the majority (~85%) of the overall reduction in rate of water loss was due to a reduction in respiratory transpiration (Fig. 4).

Mechanisms of reduced cuticular water loss

Reductions in cuticular water loss were likely due to increases in cuticular lipids. The integument of an insect is the primary barrier to cuticular water loss (Hadley, 1994a; Gibbs, 1998) and many insects at risk of dehydration enhance their desiccation resistance by increasing epicuticular lipids when entering a dormant stage or diapause (s.f. Manduca sexta, Bell et al., 1975; Mamestra configurata, Hegdekar, 1979; Sarcophaga crassipalpis, Yoder et al., 1992). The goldenrod gall fly larvae, E. solidaginis, also increases the amount of its epicuticular lipids by 40-fold over several months from late summer to mid- winter (Nelson and Lee, 2004). Thus, even though the abrupt decrease in rates of water loss for E. solidaginis occurred within days, increased hydrocarbons likely reduced cuticular rates of water loss of the larvae exposed to desiccating conditions. Less clear is the effect that cryoprotectant accumulation may have had on reducing cuticular water loss. Cryoprotectants are so-called because they enhance insect cold tolerance. Recently, several authors have suggested that cryoprotectants may be beneficial for water conservation through colligative action (Ring and Danks, 1994; Block 1996; Bayley and Hølmstrup, 1999) or by binding water at the cuticular basement membrane (Williams et al., 2004). Synthesis of the cryoprotectant glycerol constituted most (~ 60%) of the overall increase in hemolymph osmolality for both field and laboratory groups (Figs. 3A and B). The observed increases in larval glycerol concentration confirmed earlier reports that E. solidaginis produces this cryoprotectant in response to gall tissue senescence (Baust and Lee 1982; Rojas et al., 1986), suggesting that glycerol may have a protective role when entering a dormant state or desiccating

48 Reduction in rate of cuticular water loss 6 Reduction in rate of total water loss

5 Loss r e t a W

) 4 f 1 - o h e t

a -2 R

n 3 mm

g on i i µ ( 2 duct Re

n a e 1 M

0 Oct. 20 Day 3 Day 10 75% RH 75% RH

Treatment

Figure 4. Mean reductions in the total rate and cuticular rate of larval water loss for Eurosta solidaginis between the non-diapausing Oct. 1 control and diapausing Oct. 20, 75% RH day 3, and 75% RH day 10 experimental groups. These three experimental groups were chosen for this comparison because they had significantly reduced both rate of total water loss and rate of cuticular water loss compared to the Oct. 1 control.

49 environment. However, it is unclear if glycerol production lowered rates of cuticular water loss. For instance, larvae in the 75% RH treatments had lower rates of cuticular water loss than larvae in the 100% RH treatments on day 3 and 10 of the study (Fig. 2B) and larvae in the 75% RH treatments were also the only laboratory groups to have an increased glycerol concentration when compared to the Oct. 1 control group (Fig. 3B). In contrast, larvae in the 95% RH treatment did not follow this trend. These larvae had lower rates of cuticular water loss than the 100 % RH treatment (Fig. 2B) but did not have increased glycerol levels compared to control values (Fig. 3B). Thus, it appears unlikely that glycerol production was a primary factor in reducing cuticular rates of water loss during this study.

Reductions in cuticular water loss were triggered by environmental moisture, or more specifically the presence of a water potential deficit between the larvae’s hemolymph and the environment. A water potential deficit of only 17 bars between the hemolymph of the collembolan Folsomia candida and its environment induce a marked increase in its drought tolerance (Sjurnsen et al., 2001). On Oct. 1, larvae were in a potentially hydrating environment as their gall tissues were quite moist and had a higher water activity than their hemolymph (plant tissue averaged = -12.8 bars, Table 1; larval hemolymph averaged -13.0 bars as calculated from osmolality measures). Larvae placed in the 100% RH treatment continued to experience a potentially hydrating environment and did not reduce their rate of cuticular water loss (Fig. 2B). In contrast, larvae placed in the 95% and 76% RH treatments were subjected to desiccating conditions with an average water potential deficit between their hemolymph and environmental water vapor of 58.2 and 392.1 bars respectively. In addition, larvae in the stem cut and Oct. 20 groups also had similarly reduced cuticular rates of water loss after being subjected to water potential deficits in their galls (Fig. 2B; Table 1). Consequently, mild desiccation stress cued larvae to reduce their rate of cuticular water loss and did so in as little as three days.

50 Plant senescence triggers entry into larval diapause

Low metabolic rates for various field and laboratory treated E. solidaginis larvae indicate that they had entered diapause. Larvae from southwest Ohio reduce their metabolic rate by more than 75% as they enter diapause and maintain a metabolic rate of -1 -1 ~ 0.1 µl CO2•g •h at 20 ˚C throughout diapause (Irwin et al., 2001). Carbon dioxide production of larvae taken from post-senescent galls or larvae exposed to various RH treatments decreased by an average of 75% (ranging between 61 and 84%) compared to -1 -1 the Oct. 1 control, and averaged 0.07 µl CO2•g •h . Taken together, the reduction and resulting level of CO2 production indicate that these larvae were in diapause. The induction of diapause occurred rapidly for larvae placed in the RH treatments as they entered the dormant state within three days of being removed from the gall.

Plant senescence triggered E. solidaginis larvae to enter diapause. A variety of cues induce insects to enter diapause, including food availability or quality, moisture, oxygen, and pH. However, the most common cues for temperate insects are related to temperature and/or photoperiod (Tauber et al., 1986; Danks, 1987). Irwin et al. (2001) suggested that a combination of low temperature, such as experiencing an initial frost, photoperiod, or host plant senescence induced larvae to enter diapause. Yet in our study, photoperiod and temperature did not appear to influence diapause induction. For example, all larvae in the various RH treatments entered diapause even though they were held at a relatively high constant temperature (20 ˚C) after collection (Fig. 2C). In addition, field groups experienced the same temperatures and photoperiods prior to testing; however, larvae in the stem cutting group had entered diapause by Oct. 1 while larvae from the control group had not (Fig. 2C). Yet, the fact that larvae in the stem cutting group (taken from dried gall tissue) entered diapause by Oct. 1 suggests that plant senescence induced these larvae to enter the dormant state. As gall tissues senesce the environment the larva inhabits changes in two distinct ways. The nutritive gall layer on which the larvae feed deteriorates, eliminating their only food source (Uhler, 1951). In addition, the larvae are subjected to a desiccating environment for the first time (Williams et al., 2004). Moisture is an important cue for many insects to enter diapause (see references in Tauber et al., 1998), yet moisture, or more specifically a desiccating

51 environment, does not appear to influence diapause induction for E. solidaginis. All larvae removed from their galls and placed in various relative humidities entered diapause regardless of whether they were in a non-desiccating environment (100% RH treatment) or a desiccating environment (95% and 75% RH treatments; Fig. 2C). However, larvae in all the RH treatments were removed from their food and water source, suggesting that food and water availability or quality is the primary cue that triggers E. solidaginis to enter diapause.

Importance of diapause in the water balance of dormant insects

Larvae of E. solidaginis likely reduced their respiratory transpiration by lowering their metabolic rate as they entered diapause. It is well established that elevated metabolic rate, due to activities such as flight, is directly related to increased respiratory water loss and, consequently, total water loss (Nicolson and Louw, 1982; Lehmann, 2001). However, for most inactive insects, respiratory water loss constitutes a minor portion of their total water loss, 20% or less (Chown, 2002). Therefore, most studies have found that reductions in basal metabolic rate have little effect on maintaining water balance for these insects (Quinlan and Hadley, 1993; Hadley, 1994b; Djawden and Bradley, 1997; Rourke, 2000). In spite of that, others contend that reductions in basal metabolic rate can be important in limiting insect water loss (Zachariassen, 1996; Davis et al., 2000; Addo-Bediako, et al., 2001). Reductions in metabolism may have a greater impact on water conservation for xeric-adapted insects in which respiratory transpiration constitutes the majority of their total water loss, such as the beetle Phrynocolus petrosus which loses 69% of its water through respiration (Zachariassen, 1991). For the non- diapausing Oct. 1 control larvae, respiratory water loss was 4.9 µg·mm-2·h-1 (estimated by subtracting cuticular rate of water loss, Fig. 2B, from total rate of water loss, Fig. 2A), or 80% of the total water loss. However, transpiratory water loss was dramatically reduced, to only 0.6 µg•mm-2•h-1, for larvae in diapause (Fig. 4). These larvae also reduced their metabolic rate by 4.2-fold over the same testing periods. Therefore, a substantially lowered metabolism due to diapause would reduce the need for gas exchange and would

52 allow the larvae to reduce the respiratory and total rate of water loss by regulating their spiracular openings (Gibbs et al., 2003).

Temperate insects benefit from diapause in a variety of ways. Insects in the state of diapause are often more resistant to adverse conditions. Diapause also synchronizes spring emergence and prevents premature development, which would be fatal, during unseasonably warm periods in late winter (Tauber et al., 1986; Danks, 1987). The reduced metabolic rate of diapausing insects also conserves stored energy needed for spring development and reproduction (Danks, 1987; Irwin and Lee, 2003; Williams et al., 2003). However, few authors ascribe desiccation resistance as an important function of diapause for temperate and polar insects. Our data suggest that a lowered metabolic rate, due to diapause induction, is extremely important in conserving body water. For instance, if the non-diapausing Oct. 1 control larvae maintained the respiratory transpiration rate of 4.9 µg•mm-2•h-1 (Fig.2A minus Fig. 2B), we estimate it would only take them 26 days to loose 10% of their body water to the desiccating conditions used in this experiment through respiration transpiration. In contrast, diapausing larvae had an average transpiration rate of 0.6 µg•mm-2•h-1 and it would take these larvae 214 days to loose 10% of their body water through respiratory transpiration. Since many temperate insects remain in diapause for six months or more, a dramatically reduced rate of respiratory water loss, through a lowered metabolic rate, would have a profound impact on their overwintering water balance.

In summary, the large seasonal reduction in rate of water loss for E. solidaginis larvae is cued by senescing of their gall tissue and is primarily due to reduced respiratory transpiration as the larvae entered diapause.

Acknowledgements

I thank Naomi Ruehl who aided in collecting preliminary data for this project. Patrick Baker, Shannon Pinkston, Shuxia Yi, and Noah Gordan helped collect galls. I

53 also thank Jim Oris, Alan Cady, and Jack Vaughn for help in the design of this project, and Robert Schaefer for aid with statistical analysis. Support for this project was provided by the National Science Foundation IOB# 0416720.

Literature Cited

Addo-Bediako, A., Chown, S. L. and Gaston, K. J. (2001). Revisiting water loss in insects: a large scale view. J. Insect Physiol. 47, 1377-1388. Baust, J. G. and Lee, R. E. (1982). Environmental triggers to cryoprotectant modulation in separate populations of the gall fly, Eurosta solidaginis (Fitch). J. Insect Physiol. 28, 431-436. Bayley, M. and Holmstrup, M. (1999). Water vapor absorption in arthropods by accumulation of myoinositol and glucose. Science 285, 1909-1911. Bell, R. A., Nelson, D.R., and Borg, T. K. (1975). Wax secretions in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21, 1725-1729. Bennett, V. A. and Lee, R. E. (1997). Modeling seasonal changes in intracellular freeze- tolerance of fat body cells of the gall fly Eurosta solidaginis (Diptera, Tephritidae). J. Exp. Biol. 200, 185-192. Block, W. (1996). Cold or drought - the lesser of two evils for terrestrial arthropods. Eur. J. Entomol. 93, 325-339. Chown, S. L. (2002). Respiratory water loss in insects. Comp. Biochem. Physiol. A 133, 791-804. Chown, S. L. and Nicolson, S. W. (2004). Insect Physiological Ecology. New York: Oxford University Press. Danks, H. V. (1987). Insect Dormancy: An Ecological Perspective. Ottawa: Biological Survey of Canada (Terrestrial Arthropods). Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46, 837-852.

54 Davis, A. L. V., Chown, S. L., McGeoch, M. A. and Scholtz, C. H. (2000). A comparative analysis of metabolic rate in six Scarabaeus species (Coleoptera: Scarabaeidae) from southern Africa: further caveats when inferring adaptation. J. Insect Physiol. 46, 553-562. Djawdan, M., Rose, M. R. and Bradley, T. J. (1997). Does selection for stress resistance lower metabolic rate? Ecology 78, 828-837. Edney, E. B. (1977). Water Balance in Land Arthropods. New York: Springer-Verlag. Gibbs, A. G. (1998). Water-proofing properties of cuticular lipids. Am. Zool. 38, 471- 482. Gibbs, A. G., Fukuzato, F. and Matzkin, L. M. (2003). Evolution of water conservation mechanisms in Drosophila. J. Exp. Biol. 206, 1183-1192. Hadley, N. F. (1994a). Ventilatory patterns and respiratory transpiration in adult terrestrial insects. Physiol. Zool. 67, 175-189. Hadley, N. F. (1994b). Water Relations of Terrestrial Arthropods. San Diego: Academic Press. Hegdekar, B. M. (1979). Epicuticular wax secretions in diapause and non-diapause pupae of the Bertha army worm Mamestra configurata. Annu. Entomol. Soc. Am. 72, 13-15. Irwin, J. T., Bennett, V. A. and Lee, R. E. (2001). Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera: Tephritidae). J. Comp. Physiol. B 171, 181-188. Layne, J. R. (1993). Winter microclimate of goldenrod spherical galls and its effects on the gall inhabitant Eurosta solidaginis (Diptera, Tephritidae). J. Therm. Biol. 18, 125- 130. Layne, J. R. and Medwith, R. E. (1997). Winter conditioning of third instars of the gall fly Eurosta solidaginis (Diptera: Tephritidae) from Western Pennsylvania. Physiol. Chem. Ecol. 26, 1378-1384. Lehmann, F.-O. (2001). Matching spiracular opening to metabolic need during flight in Drosophila. Science 294, 1926-1929.

55 Nelson, D. R. and Lee, R. E. (2004). Cuticular lipids and desiccation resistance in overwintering larvae of the goldenrod gall fly, Eurosta solidaginis (Diptera : Tephritidae). Comp. Biochem. Physiol. B 138, 313-320. Nicolson, S. W. and Louw, G. N. (1982). Simultaneous measurement of evaporative water-loss, oxygen-consumption, and thoracic temperature during flight in a carpenter bee. J. Exp. Zool. 222, 287-296.

Quinlan, M. C. and Hadley, N. F. (1993). Gas exchange, ventilatory patterns, and water loss in two lubber grasshoppers: quantifying cuticular and respiratory transpiration. Physiol. Zool. 66, 628-642. Ramløv, H. and Lee, R. E. (2000). Extreme resistance to desiccation in overwintering larvae of the gall fly Eurosta solidaginis (Diptera: Tephritidae). J. Exp. Biol. 203, 983-789. Ring, R. A. and Danks, H. V. (1994). Desiccation and cryoprotection: overlapping adaptations. Cryo-Lett. 15, 181-190. Rojas, R. R., Lee, R. E. and Baust, J. G. (1986). Relationship of environmental water content to glycerol accumulation in the freezing tolerant larvae of Eurosta solidaginis (Fitch). Cryo-Lett. 7, 234-245. Rourke, B. C. (2000). Geographic and altitudinal variation in water balance and metabolic rate in a California grasshopper, Melanoplus sanguinipes. J. Exp. Biol. 203, 2699-2712. Sjursen, H., Bayley, M. and Holmstrup, M. (2001). Enhanced drought tolerance of a soil-dwelling springtail by pre-acclimation to a mild drought stress. J. Insect Physiol. 47, 1201-1207. Tauber, M. J., Tauber, C. A. and Masaki, S. (1986). Seasonal Adaptations of Insects. New York: Oxford University Press. Tauber, M. J., Tauber, C. A., Nyrop, J. P. and Villani, M. G. (1998). Moisture, a vital but neglected factor in the seasonal ecology of insects: hypotheses and tests of mechanisms. Environ. Entomol. 27, 523-530. Uhler, L. D. (1951). Biology and ecology of the goldenrod gall fly, Eurosta solidaginis (Fitch). New York: New York State College of Agriculture.

56 Williams, J. B., Ruehl, N. C. and Lee, R. E. (2004). Partial link between the seasonal acquisition of cold-tolerance and desiccation resistance in the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae). J. Exp. Biol. 207, 4407-4414. Williams, J. B., Shorthhouse, J. D. and Lee, R. E. (2002). Extreme resistance to desiccation and microclimate-related differences in cold-hardiness of gall wasps (Hymenoptera: Cynipidae) overwintering on roses in southern Canada. J. Exp. Biol. 205, 2115-2124. Yoder, J. A., Denlinger, D. L., Dennis, M. W. and Kolattukudy, P. E. (1992). Enhancement of diapausing flesh fly puparia with additional hydrocarbons and evidence for alkane biosynthesis by a decarbonylation mechanism. Insect Biochem. Mol. Biol. 22, 237-243. Zachariassen, K. E. (1991). Routes of transpiratory water loss in a dry-habitat tenebrionid beetle. J. Exp. Biol. 157, 425-437. Zachariassen, K. E. (1996). The water conserving physiological compromise of desert insects. Eur. J. Entomol. 93, 359-367.

57 Chapter 4

Latitudinal variation in cold-tolerance and desiccation resistance in the goldenrod gall fly, Eurosta solidaginis.

58 Introduction

Many insects overwintering in temperate and polar regions must not only survive low temperature but also extremely desiccating conditions as well. To increase cold- tolerance, insects produce high concentrations of low-molecular mass polyols and sugars, termed cryoprotectants. Freeze tolerant insects use cryoprotectants to reduce the amount of body water frozen at a given temperature (Lee, 1991; Zachariassen, 1991; Storey and Storey, 1992), thereby reducing cellular dehydration, which is considered to be the primary mode of damage caused by extracellular ice formation (Mazur, 1981). Cryoprotectants may also lower rates of water loss by colligatively reducing the vapor pressure gradient between an insects' hemolymph and environmental water vapor (Ring and Danks, 1994; Bayley and Holmstrup, 1999; Sjursen et al., 2001) or possibly through non-colligative actions by binding water when desiccating conditions are more severe (Williams et al., 2004). The hypothesized link between cryoprotectant production and increased desiccation resistance led some authors to suggest that certain adaptations that increase cold-hardiness may also influence, or arose originally to promote, desiccation resistance (Ring and Danks, 1994; Block, 1996; Danks, 2000). Yet, links between cold- hardiness and desiccation resistance remain unclear.

Temperate insects experience a large variation in the severity of winter conditions based on the location, elevation, and latitude of their hibernaculum (Danks, 2004). Consequently, an insect's level of cold-hardiness varies depending on the severity of low temperatures it typically experiences. On a local scale, stem galling cynipid wasps that overwinter above the snow pack in southern Canada experience more severe cold and are more cold-hardy than congeners in the buffered and mild subnivean space (Williams et al., 2002). On a global scale, Addo-Bediako et al. (2000) found that cold tolerance generally increases with higher latitudes across insect taxa. However, few studies have examined intraspecific differences in desiccation resistance and cold tolerance of wide ranging insects that experience different overwintering temperatures.

The goldenrod gall fly, Eurosta solidaginis, ranges throughout much of North America, from southern Texas to southern Canada (Uhler, 1951). Gall flies overwinter as

59 freeze-tolerant, third instar larvae in dried stem galls on goldenrod plants (Solidago spp.). Larvae overwinter above the snow pack where they are subjected to winter extremes as dried gall tissue offers little protection against low temperature and desiccation stress (Uhler, 1951; Layne, 1993). Gall fly larvae likely experience much milder overwintering conditions in the southern portion of their range compared to the extremely low temperatures that occur at higher latitudes of the northern United States and Canada.

Throughout their range, E. solidaginis larvae seasonally increase their cold- tolerance by producing high levels (~1 molar) of the cryoprotectants glycerol and sorbitol (Lee, 1991). However, larvae collected from the southern portion of their range are less cold tolerant and produce considerably less glycerol under common garden conditions (Baust and Lee, 1982), suggesting that lower levels of cold tolerance in southern larvae may be the result of genetic differences between the populations. Latitudinal variation in desiccation resistance for E. solidaginis larvae is unknown. However, larvae from a mid-latitudinal collection site in Ohio seasonally lower rates of water loss by increasing epicuticular lipids (Nelson and Lee, 2004), by reducing respiratory transpiration as larvae enter diapause (see chapter 3), and possibly through the production of cryoprotectants (Williams et al., 2004).

A previous study focused on linking seasonal changes in levels of cold-tolerance, desiccation resistance and cryoprotectant production in single populations of E. solidaginis larvae (Williams et al., 2004). In contrast, the purpose of this study was to 1) determine if mid-winter collected larvae from northern latitudes were more cold-tolerant than larvae collected from a southern population and 2) to determine if cold-tolerance is positively linked to higher levels of resistance to desiccation. To investigate these questions we measured survival after freezing, hemolymph osmolality, cryoprotectant concentration, body water content, rate of total water loss, rate of cuticular water loss, metabolic rate, lipid content, and glycogen content of E. solidaginis larvae taken from three widely separated sites in Michigan, Ohio, and Alabama. Lower latitude larvae likely experience higher overwintering temperatures. Therefore, we measured several of the above parameters after acclimating larvae to a relatively high overwintering temperature.

60

Materials and Methods

Insect collection

All galls were collected from goldenrod (Solidago altissima) within two weeks of the plant senesce in the fall of 2004. Galls were collected from a relatively high-latitude, northern site, near Allendale, Michigan (42° 97’ 02” N, 85° 95’ 03” W), a mid-latitude site at the Ecology Research Center of Miami University, near Oxford, Ohio (39° 31’ 57” N, 84° 43’ 23” W), and a southern site near Auburn, Alabama (32° 61’ 59” N, 85° 26’ 01” W). The Michigan collection site was ~ 455 km north of the Ohio site and ~ 1182 km north of the Alabama collection site. After collection the larvae were removed from their galls and held in 24 well micro-titer plates at 5 °C over a saturated solution of sodium chloride to produce a relative humidity of 75%. Larvae were held at these conditions for two to eight weeks prior to analysis in mid-December. It is important to note that no morphometric or genetic analysis were performed to determine if larvae collected from the three sites were from the same species, however it is highly likely.

Measures of cold-tolerance

Larval cold-tolerance was assessed by measuring survival after exposure to a low subzero temperature. Fifty larvae per collection site were cooled at 1 °C•min-1 from 5 °C until reaching -40 °C. After a 96-h exposure to -40 °C, larvae were warmed to 5 °C at 1 °C•min-1 and then returned to 5 °C and 75% RH. Survival measurements were taken 24 h, 48 h, 2 weeks, 6 weeks and 16 weeks after the low temperature exposure by determining if larvae moved in response to being gently touched with a blunt probe. After the 16-week survival assessment, all larvae were placed at 23 °C, allowed to develop, and the number of larvae which pupated, eclosed, and became viable adults recorded. Adults were judged to be viable if they had fully formed appendages (i.e. wings) and were able to right themselves within 1 min of being turned on their dorsal side.

61

Hemolymph osmolality and cryoprotectant concentrations Hemolymph osmolality (n = 10 per collection site) was determined by drawing 7- 10 µl of hemolymph into a capillary tube through a small incision in the larval cuticle. The hemolymph was then analyzed in a Wescor Vapro 5520 Hemolymph Osmometer (Logan, Utah). To measure cryoprotectant concentrations, larvae (n = 10 per collection site) were frozen at -80°C until whole body measurements of glycerol were performed by enzymatic assay (Sigma Chemical Co., no. 337) as described by Hølmstrup et al. (1999). Sorbitol concentration was measured on the same individuals using the enzymatic assay described in Bergmeyer et al. (1974).

Measures of desiccation resistance To determine the overall rate of water loss, larvae (n = 10 per collection site) were weighed to ± 0.01 mg to obtain a fresh mass. Larvae were then re-weighed after being desiccated over Drierite (W.A. Hammond Drierite Co., Ohio, USA) at 4% RH and 5˚C until they lost ~5% of their fresh mass (120-240 h). Cuticular surface area was estimated from initial wet mass using an equation previously determined by Williams et al. (2004): y = 0.912x + 4.204, r2 = 0.804, where y = surface area in mm2 and x = mass in mg. Body water content was determined by placing the desiccated larvae in an oven at 65 ˚C until a constant dry mass was obtained. Hemolymph content was assessed by obtaining the fresh mass of the larvae prior to tearing open the cuticle with forceps and removing the hemolymph by gently blotting the larval body cavity with Kimwipes ® that were slightly wetted with a glycerol solution (~900 mOsm) that was iso-osmotic to larval hemolymph as described in Folk et al. (2001). The hemolymph-free carcass was then re-weighed before being placed in an oven at 65 ˚C until a constant dry mass was obtained. Hemolymph content was determined as the difference between initial wet mass and the hemolymph-free carcass mass.

62 Rate of cuticular water loss (µg•mm-2•h-1) was measured to determine the relative contributions of respiratory and cuticular components of overall organismal water loss. Rate of cuticular water loss was assessed by weighing larvae (n = 10) before and after exposure to 4% RH at 5 ˚C as described above, however, prior to testing, the spiracles of each larva were topically blocked with a small amount of Thomas Scientific Lubriseal stop cock grease (Swedesboro, New Jersey), to eliminate respiratory water loss.

Metabolic reserves

Since desiccation-selected Drosophila melanogaster preferentially store metabolic fuels in the form of glycogen, which when metabolized release higher levels of water (Folk et al., 2001), we measured glycogen content on 10 larvae per population using a calorimetric micromethod (Kemp and Kits Van Heijningen, 1954); glycogen concentration was expressed in glucosyl units. Lipid content was also determined on 10 larvae per population using the chloroform/methanol procedure described by Teitz (1970).

Measures of metabolic rate

To correlate rates of water loss and metabolism, we measured CO2 emission. Larvae (n = 8 per collection site) were weighed and individually placed into small glass respirometry chambers kept within a temperature controlled bath held at 5 °C. After the

larvae had equilibrated to the chamber for one hour, CO2 was measured using a flow through (50 ml•l-1) respirometer (TR-3 model, Sable Systems, Las Vegas, Nevada).

Metabolic rate data was converted into the units of microliters of CO2 emitted per milligram fresh mass per hour using DATACAN software (Sable Systems).

Effect of 20 °C acclimation on cold-hardiness and desiccation resistance

63 Low latitude larvae likely experience higher overwintering temperatures. Therefore, we measured several of the above parameters after acclimating larvae from all three collection sites to a relatively high overwintering temperature. We used the previously described techniques to measured hemolymph osmolality, cryoprotectant levels, overall rate of water loss, rate of cuticular water loss, hemolymph volume, body water content, and metabolic rate on larvae that were acclimated to 20 °C for one week. In contrast to previous methods, measurements of total rate of water loss, rate of cuticular water loss, and metabolic rate were assessed at 20 °C in these experiments.

Statistical analysis

To compare values within temperature treatments, larvae from the three collecting sites were analyzed using a one-way ANOVA followed by a Bonferroni multiple comparisons test. T-tests were used to determine differences between 5 °C and 20 °C temperature treatments from a single collection site. A one-way ANOVA followed by a Tukey’s multiple comparison exam, after the proportions were angularly transformed, was used to determine differences in survival between larvae at each assessment period. Significance for all analyses was set at α=0.05 and all data are reported as mean ± SEM.

Results

Measures of cold-tolerance Northern larvae were more cold tolerant than ones from southern populations. Survival at the -40 °C exposure was significantly lower (p < 0.05) for larvae collected in Alabama compared to Ohio and Michigan larvae (Figs. 1A and 1B). Two weeks after the low temperature treatment, only 29 of 50 Alabama larvae responded to tactile stimulation, compared to 94% and 98%, respectively, of larvae in the Ohio and Michigan groups. The pattern of lower survival for Alabama larvae continued after being placed at room temperature as no larvae in the Alabama group eclosed (Fig. 1B). In contrast,

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eek s o ved Ohio larvae demonstrated moderate survival as 16% eclosed and 10% developed into viable adults. Survival was the greatest for Michigan-collected larvae (p < 0.05) which had the highest rates of eclosion (43%) and adult development (25%) (Fig. 1B). Interestingly, larvae collected from Michigan and Ohio responded to tactile stimulation much sooner than Alabama larvae. For example, 96% of the larvae from Michigan and Ohio that responded to tactile stimulation did so within 48 h of being removed from the low temperature exposure, while only 10% of larvae collected from Alabama responded at this time, even though 58% were responsive after two weeks (Fig. 1A). As with cold tolerance, levels of cryoprotectants varied among larval populations (Fig. 2). Michigan-collected larvae had significantly higher concentrations of glycerol (p < 0.05) after acclimation at 5 °C (500 ± 30 mmol) than Ohio and Alabama larvae (Fig. 2A). However, after acclimation to 20 °C larvae from the three collection sites had similar glycerol levels (~270 ± 24 mmol). Although 20 °C acclimation had no effect compared to the 5 °C treatment for Ohio and Alabama larvae, Michigan larvae at 5 °C had had higher glycerol levels than those at 20 °C. Unlike glycerol, sorbitol concentrations did not differ (p > 0.05) among the collection groups acclimated at 5 °C, averaging 328 ± 63 mmol (Fig. 2B), and between those acclimated at 20 °C, averaging 39 ± 17 mmol (Fig. 2B). However, sorbitol concentrations were significantly higher within all populations acclimated at 5 °C (p < 0.05) compared to those acclimated at 20 °C. Larvae collected in Michigan and Ohio averaged ~170 mOsm•kg-1 higher hemolymph osmolality than those from Alabama, which averaged 777 ± 16 mOsm•kg-1 when acclimated to 5 °C (Fig. 3). In contrast, hemolymph osmolalities did not differ among collection groups after acclimation to 20 °C, averaging 788 ± 19 mOms•kg-1.

Body water, glycogen, and lipid content

Body water contents did not differ among larval groups (p > 0.05) regardless of collection site or acclimation temperature, ranging between 1.6 and 1.8 mg water•mg dry mass-1 or 60.5 and 64.3 % body water (Fig. 4B). Similarly, hemolymph volume did not

66 Figure 2.(A)Meanglycerolco tem ANOVA f sharing thesam acclim and Alabamaacclimatedtoeither5°C (n =10perdatapoint),forlarvaeof 20 °Cdatafrom Mean Sorbitol Concentration Mean Glycerol Concentration p

erature treatm (mmol) (mmol) ated at5°Cnotsh 100 200 300 400 500 10 20 30 40 50 60 o 0 0 0 0 0 0 0 0 llowed withaBonf B A e larvaecollectedatthesam numberweresignificantly ent withanasteris a* a* aring th 5 ° a* erroni m b ncentration (n=10) C A e sam ccl

Eurosta solidaginis k indi e i u lette m a* b ltiple co at cate asignifican or20°C.Means(±SEM)oflarvae e 67 d sitewhenusingat-test. i r orm o i fferent whenan n T mparisons te eans oflarvaeacclim and(B)m e m p collectedfrom er 1 1 t d at u st. Valuesinthe5°C i alyzed withaone-way ean sorbitolconcentrations fference between5°Cand r 20 ° M Oh A e l i ab ch i 1 1 o am L i C g a an Michigan,O a L r ated at20°Cnot v L a a e a r r 1 v 1 v ae a e h io,

1200 Michigan Larvae Ohio Larvae Alabama Larvae 1000 a* a*

lality 1 o b 1 800 1 ) -1 h Osm kg p

m 600 m s ly O o

(m 400

Mean Hem 200

0 5 °C 20 °C

Acclimation Temperature

Figure 3. Mean hemolymph osmolality (n =10) for larvae of Eurosta solidaginis collected from Michigan, Ohio, and Alabama and acclimated to either 5 °C or 20 °C. Means (± SEM) of larvae acclimated at 5 °C not sharing the same letter or means of larvae acclimated at 20 °C not sharing the same number were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test. Values in the 5 °C temperature treatment with an asterisk indicate a significant difference between 5 °C and 20 °C data from larvae collected at the same site when using a t-test.

68 with aBonferronim num sharing thes acclim 10) forlarvaeof Figure 4.(A)Meanhemolym

Mean Body Water Content Mean Hemolymph Volume b

er weresignificantlydi -1 (% of total body water) ated toeither5°Cor20°C.Means (mg water mg dry mass ) 0. 0. 1. 1. 2. 10 20 30 40 0 5 0 5 0 0 a m B A e letteror Eurosta solidaginis u ltiple com a a m eans of fferent whenanalyzedwithaone-wayANOVA followe 5 ° ph volum a a p arisons test. C A larvae c collectedfro c l im e a (n a* acclim a (± S 69 =10), and(B)meanbodywatercontent(n= tio E n A Oh M M) oflarvaeacclim ated at20°Cnotsharingthesam T l i m a ch i b o Michigan, e am L i g m a an a L r p v L e a 1 11 a e r a r a r v v t a a u e Ohio,andAlabam e r 20 ° e 1 a C ted at5°Cnot 1 1 a and e

d

differ (p > 0.05) among collection groups acclimated at 5 °C, averaging 30.5 % of total water volume, or among those acclimated at 20 °C (30.7 %) (Fig. 4A). Alabama larvae acclimated at 20 °C did, however, have a higher hemolymph volume (p < 0.05) than larvae acclimated at 5 °C.

Larval glycogen contents were similar (p > 0.05) among the three collection sites with values ranging between 57 and 81 mg glycogen•g-1 dry mass, nor did concentrations of lipid differ among populations (Table 1). However these data suggest a trend with Alabama larvae having the lowest levels of glycogen and lipid compared to the more northern Ohio and Michigan larvae.

Measures of desiccation resistance

Overall rates of water loss were ~40% lower (p < 0.05) for larvae collected in Michigan (0.10 ± 0.01 µg•mm-2•h-1) compared to Ohio and Alabama collected larvae when acclimated to, and desiccated at, 5 °C (Fig. 5A). In contrast, rates of overall water loss did not differ among collection groups (p > 0.05) when acclimated and tested at 20 °C (Fig. 5A). Within each population, rates of water loss were significantly higher (p < 0.05) for larvae acclimated and tested at 20 °C compared to those at 5 °C (Fig. 5A).

Similar to rates of total water loss, Michigan larvae at 5 °C had a ~44% lower rate of cuticular water loss (0.037 ± 0.003 µg•mm-2•h-1) compared to the Ohio and Alabama larvae (Fig. 5B). In contrast, rates of cuticular water loss did not differ among populations at 20 °C, averaging 0.215 ± 0.019 µg•mm-2•h-1 (Fig. 5B). Unsurprisingly, rates of cuticular water loss were significantly increased, ~3.1 fold (p < 0.05), for all larval groups acclimated at 20 °C compared to those at 5 °C (Fig. 5B). Carbon dioxide production ranged from 0.017 to 0.028 µl•g-1•h-1 among larvae at 5

°C, with CO2 levels were significantly lower (p < 0.05) for larvae collected in Ohio compared to those from Michigan (Fig. 5C). Michigan and Ohio-collected larvae had significantly higher rates of CO2 production ( ~4.4 fold; p < 0.05) at 20 °C compared to those at 5 °C (Fig. 5C). Production of CO2 also increased significantly for

70

Table 1. Mean lipid content (n =10 per) and glycogen content (n = 10), for larvae of Eurosta solidaginis collected from Michigan, Ohio, and Alabama. Means (± SEM) within a column not sharing the same letter were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test.

Collection site Lipid Content Glycogen Content (mg lipid•g dry mass-1) (mg glycogen•g dry mass-1) Michigan 580 ± 45 a 72 ± 7a

Ohio 547 ± 55 a 81 ± 8 a

Alabama 470 ± 12 a 57 ± 7 a

71 1.2 1 1

s A 1.0 M ichigan Larvae ter Los Ohio Larvae ) Wa

-1 0.8

A labam a Larvae h

-2 te of 0.6 1 mm

ll Ra g a r µ

( 0.4 Ove

n b* b* a 0.2 e a* M 0.0 0.30 B 1

Loss 0.25 1

ter 1 a ) 0.20 -1 h

te of W -2 a 0.15 R r mm a

g b* cul

µ b*

i 0.10 (

0.05 a* Mean Cut 0.00 0.12 C 1 1 on i 0.08 )

-1 2 h

-1 Product 2 g

l µ CO ( n 0.04 a*

Mea ab* b*

0.00 5 °C 20 °C Acclim ation Tem perature

Figure 5. (A) Mean total rate of water loss (n =10), and (B) mean blocked spiracles rate of water loss (n = 10), and (C) mean CO2 production (n = 8) for larvae of Eurosta solidaginis collected from Michigan, Ohio, and Alabama and acclimated to either 5 °C or 20 °C. Means (± SEM) of larvae acclimated at 5 °C not sharing the same letter or means of larvae acclimated at 20 °C not sharing the same number were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test. Values in the 5 °C temperature treatment with an asterisk indicate a significant difference between 5 °C and 20 °C data from larvae collected at the same site when using a t-test.

72 Alabama-collected larvae at 20 °C compared to those at 5 °C (p < 0.05), however, CO2 levels only increased by 2.6-fold (Fig. 5C).

Discussion

Levels of cold-hardiness

Larvae collected from Michigan were more cold-hardy than larvae from either Ohio or Alabama when using adult development and viability as the determinant (Figs. 1 A and 1B). There is no standard protocol for assessing insect cold tolerance and the ability to survive low temperatures can vary greatly depending on biotic factors such as age or acclimation status of the insect, as well as the methodology (c.f. cooling rate, exposure temperature, and duration) (Denlinger and Lee, 1998). However, from the parameters used in this study, it appears that adult development was the most ecologically relevant assessment of cold-hardiness. For example, Michigan and Ohio larvae had the same levels of cold-hardiness when using movement in response to tactile stimulation (Fig. 1A), even though Michigan larvae typically experience much lower overwintering temperatures (Figs. 6A and 6B). However, when using adult viability as the determinant, cold-hardiness of Michigan larvae was significantly greater than those from Ohio. In addition, the relatively low-latitude Alabama larvae, which rarely experience subzero temperatures in winter (Fig. 6A and 6B), had the same pupation rate as Ohio larvae (Fig. 1B). Yet, when using adult viability as the determinant, Alabama had the lowest level of cold-hardiness (Fig. 1B). The fact that response to external stimuli varied considerably compared to adult development in all species suggest this method of assessing cold-tolerance should not be used whenever possible. For example, two weeks after removal from the low temperature exposure, 58 % of Alabama larvae responded to external stimuli (Fig. 1A). If this were the only assessment of cold-tolerance for these larvae, then they would considered relatively tolerant to the low temperature exposure. However, the assessment

73

weather s Figure 6.(A At station. Allweatherstation datacan station, andAlabam Grand Rapidsairport,O sites. Mich from m i m ni Mean Daily Minimum Temperature (°C) Mean Weekly Temperature (°C) o m 1971to2000fromweatherstationslocate -1 -1 -1 spheric website: 10 15 20 25 -5 u -8 -6 -4 -2 0 0 5 2 0 0 2 4 6 m temperaturesf t D ations locatedwithin B A Oc e igan tem ) OctobertoMarchweeklym cem t b e r a temperaturedatawas p erature datawasrecord No http://www.ncdc.noaa.gov/oa/clim h v r io tem om Decembertom 15kmofthelarvalcollectio p De erature da c J beaccessedontheNatio a nua M r ean airtem y ont ta was Ja recorded attheAubur 74 ed from i n d- h February, thecoldestpartofwinter, d within15kmofthelarvalcollection recordedattheFairfield Mi Oh A l ab c i theweatherstationlocatedat o p h am

Fe erature from i g a b a n a te/clim n sites.(B)Averagedaily nal Oceanicand Fe Ma ateresources.htm n Agronom br 1971to2000from r u a r y weather y weather

l

of cold-hardiness is drastically different when using the more ecologically relevant determinant of cold-tolerance, adult development. No larvae collected from Alabama developed into an adult, suggesting the low temperature exposure was excessively severe for these larvae (Fig. 1B).

Mechanisms of cold-tolerance

Higher levels of cold-hardiness for Michigan larvae were likely due, at least in part, to higher concentrations of cryoprotectants which can mediate the effects of extracellular ice formation and cellular dehydration (Lee, 1991). As reported previously (Baust and Lee, 1982), the most northerly population of E. solidaginis, in this case the Michigan collected larvae, had higher concentrations of glycerol than the lower latitude collected larvae. In contrast to previous reports, Michigan larvae had lower concentrations of glycerol after acclimation to a higher temperature (20 °C) (Storey et al., 1981; Baust and Lee, 1982; Storey and Storey, 1986). However, few studies have examined the glycerol content of mid-winter E. solildaginis larvae acclimated at the relatively high temperature of 20 °C, and one report suggested that glycerol levels vary in midwinter for field collected larvae (Baust and Lee, 1981). Concentrations of the cryoprotectant sorbitol did not differ between larval groups acclimated at 5 °C and were similar to levels reported for Texas and Minnesota collected larvae (Baust and Lee, 1982). Sorbitol is catabolized into glycogen at temperatures above 10 °C (Storey and Storey, 1981) consistent with the observed lower sorbitol levels for all larvae acclimated to 20 °C.

Cryoprotectants function in a variety of ways to increase levels of insect freeze tolerance; however the exact mechanism(s) by which cryoprotectants influenced larval survival in this study is unknown. Elevated glycerol concentration could have reduced ice content to a greater extent in Michigan larvae by colligatively lowering the melting point of its hemolymph (Lee, 1991; Denlinger and Lee, 1998) or non-colligatively binding more water (Storey, 1983; Block, 1996) than in those from Ohio and Alabama. Higher concentrations of the membrane penetrating glycerol could have also have

75 reduced cellular dehydration to a greater extent in Michigan larvae during freezing (Zachariassen, 1991). Lastly, higher concentrations of cryoprotectants may have positively influenced cold-tolerance of Michigan larvae by stabilizing membranes and proteins during freezing and thawing (Capenter and Crowe, 1988; Crowe et al., 1990). Therefore, it is likely that higher concentrations of glycerol conferred increased cold- tolerance in the Michigan larvae. However, cryoprotectants are unable to explain all differences in cold tolerance between larval populations. For example, Ohio and Alabama larvae had similar concentrations of cryoprotectants (Figs. 3A and 3B), yet those from Ohio were more cold tolerant than those from Alabama (Fig. 1B).

Michigan and Ohio larvae were able to respond to tactile stimulation sooner than Alabama larvae following the freezing exposure (Fig. 1A), which was likely the result of their ability to restore the electrochemical gradients across their membranes more quickly. Ion electrochemical gradients across a cell membrane are needed for many cell and tissue functions, such as neural impulses and muscular contraction. However, transporters which maintain the electrochemical gradient can be perturbed during the low temperatures and high hemolymph solute concentrations associated with severe freezing, allowing ions to equilibrate across the cell membrane (Morris and Clarke, 1981). Upon thawing, pre-freeze ion gradients need to be established before insects can respond to external stimuli and contract muscle tissue. Re-establishment of the ion gradient can be a slow process and took over 24 h for the wood fly, Xylophagus cinctus, once it was removed from freezing temperatures (Kristiansen and Zachariasen, 2001). I suggest that Michigan and Ohio larvae responded sooner to tactile stimulation following the low temperature exposure because they may have had ion transporters that functioned more efficiently at the recovery temperature and established pre-freeze electrochemical gradients faster than those from Alabama. If ion transporters in the Michigan and Ohio larvae functioned more efficiently at low temperatures, then other membrane transporters that would directly effect cold-tolerance, such as sorbitol transporters, may also function more efficiently at low temperatures (Storey and Storey, 1996). However, more studies of transporter function at low temperature are needed to justify this claim.

76 Resistance of desiccation stress

To survive desiccation stress, insects can either tolerate water loss and low body water levels or limit the rate at which they lose water to the environment (Danks, 2000). Insects that tolerate water loss typically have higher levels of initial body water (Miller, 1968; Block et al., 1990), and/or greater hemolymph volume (Folk et al., 2001). Desiccation tolerant insects may also have higher concentrations of glycogen which would release more water when metabolized than lipid (Folk et al., 2001). Body water content (Fig. 4B), hemolymph volume (Fig. 4A), and glycogen levels (Table 1) were the same in all larvae, suggesting that differences in water balance between the larvae would primarily exist in the rate in which larvae lose water.

There were differences in desiccation resistance among collection groups as overall rates of water loss were ~40% lower for Michigan larvae than those from Ohio and Alabama at 5 °C (Fig. 5A). Overwintering and dormant insects have reduced avenues of water loss, and typically lose water only through cuticular and respiratory transpiration (Danks, 2000). Rates of cuticular water loss were ~44% lower in Michigan larvae acclimated at 5 °C compared to the other populations. Thus, it is likely that the lowered rates of cuticular water loss for the Michigan larvae were primarily responsible for their lowered overall rate of water loss at 5 °C (Figs. 5A and 5B).

Lower rates of cuticular water loss for Michigan larvae acclimated at 5 °C may be due to differences in epicuticular lipids. Epicuticular lipids are the primary barrier to cuticular water loss (Hadley, 1994; Gibbs, 1998). Insects can reduce cuticular water loss by increasing the overall amount of epicuticular lipids or by increasing the relative proportion of long chain, saturated, and non-methylbranched hydrocarbons comprising the lipids (see references in Gibbs, 1998). Compostion of epicuticular lipids can vary between insect populations of the same species (Howard, 1993) and many insects at risk of desiccation, such as dormant insects, increase their epicuticular lipids compared to non-dormant individuals (Hedekar, 1979; Coudron and Nelson, 1981; Yoder et al., 1992). In fact, E. solidaginis larvae from the Ohio collecting site increase their epicuticular lipids 40-fold from late summer to mid-winter (Nelson and Lee, 2004). However, it is

77 unclear if Michigan larvae had increased levels of epicuticular lipids or increased proportions of more water conserving hydrocarbons compared to Ohio and Alabama larvae. If lower cuticular water loss for Michigan larvae at 5 °C were due to differences in their epicuticular lipids, then they likely would have had lower rates of cuticular water loss at 20 °C as well (Fig. 5B). Michigan larvae acclimated at 20 °C may have reduced the amount of epicuticular lipids or composition of those lipids compared to those at 5 °C; however it seems unlikely they would reduce their amount of lipids or could substantially change their composition of lipids during only 7 days of acclimation to 20 °C (Gibbs, 1998).

Cryoprotectants may have lowered rates of cuticular water loss in Michigan larvae acclimated at 5 °C compared to Ohio and Alabama collected larvae. After E. solidaginis larvae enter diapause, cryoprotectant production, as measured by hemolymph osmolality, is strongly correlated with reductions in rate of overall water loss (Williams et al., 2004). Williams et al. (2004) hypothesized that cryoprotectants may function in a non-colligative manner to reduce rates of water loss by binding water at the cuticular basement membrane which would increase the distance bulk water would have to travel to be lost to the environment. Michigan collected larvae acclimated at 5 °C had much higher concentrations of the cryoprotectant glycerol (Fig. 2A), and also significantly lower cuticular water loss compared to Ohio and Alabama larvae. In addition, after the seven day acclimation to 20 °C, Michigan larvae had similar concentrations of glycerol and also similar rates of cuticular water loss as larvae collected from Ohio and Alabama. Thus, these data suggest that cryoprotectants may function to reduce rates of cuticular and overall rates of water loss for Michigan collected larvae acclimated to 5 °C and may represent a link between cold-hardiness and desiccation resistance.

Since larvae from Alabama typically experience much warmer temperatures during winter than those from Michigan and Ohio (Figs. 6A and 6B), we measured parameters associated with desiccation resistance after larvae from each population were acclimated to 20 °C. It is likely that larvae from Michigan and Ohio never experience 20 °C during the winter, as average daily maximum temperatures in December and January taken at the weather stations in figure 6 ranged between 3.9 to -1.7 °C and 2.8 to 8.3 °C,

78 respectively (data not shown). Over the same time period, average daily maximum temperatures were much higher at the Alabama weather station, ranging between 12.2 and 16.1 °C (data not shown). Therefore, E. solidaginis larvae in Alabama likely experience 20 °C during the mid-winter, especially when considering the effect of solar radiation, which can warm the gall well above ambient air temperature (Layne, 1991).

After acclimation to 20 °C, larvae from Alabama had the same overall rate of water loss as those from Michigan (Fig. 5A). This is in contrast to the 5 °C acclimation, in which Alabama larvae had much higher overall rates of water loss than Michigan larvae. The relative lowering of overall rates of water loss for Alabama larvae acclimated at 20 °C appears to be the result of respiratory transpiration which increases relatively slowly with temperature. After acclimation to 5 °C, larvae from Michigan, Ohio, and Alabama lost 62%, 47%, and 52% of their rates of total water loss, respectively, through respiratory transpiration. I estimated respiratory transpiration by subtracting rates of cuticular water loss (Fig. 5B) from overall rates of water loss (Fig. 5A). After acclimation to 20 °C, percentages of total rates of water loss attributed to respiratory transpiration was considerably higher for Michigan and Ohio larvae, averaging ~75%. In contrast, the percentage of total rates of water loss attributed to respiratory transpiration for Alabama collected larvae acclimated to 20 °C remained relatively unchanged at 54%.

The lowered respiratory transpiration for Alabama larvae acclimated to 20 °C was likely the result of a reduced metabolic rate (Fig. 5C). Overall rate of water loss is positively linked to metabolic rate in insects performing high metabolic activities, such as flight (Nicolson and Louw, 1982) and in inactive insects that have such low rates of cuticular water loss that respiratory transpiration constitutes the majority of overall water loss (Zachariasen, 1996; Williams and Lee, unpublished data). Larvae of E. solidaginis from all three collection sites did have extremely low rates of cuticular water loss (see references in Hadley, 1994) which resulted in cuticular transpiration being the major avenue of overall water loss (see above paragraph). Thus, it is likely that the lowered metabolic rate of Alabama larvae at 20 °C would allow them to lower respiratory transpiration (Gibbs et al., 2003) compared to the other larvae.

79 If a lowered metabolic rate allowed Alabama larvae to reduce respiratory transpiration compared to other populations at 20 °C, then it may represent an adaptive link between water conservation and energy conservation in overwintering insects.

Levels of CO2 production at 5 °C indicate that larvae from all collection sites were in the state of diapause (Irwin et al., 2001). Diapause is defined as a genetically determine state of lowered metabolism and suppressed development induced by environmental factors which lasts longer than the adverse conditions (Tauber et al., 1986; Danks, 1987). The lowered metabolic rate of diapausing insects is typically associated with conserving metabolic reserves needed for post-diapause development and activities (Tauber et al., 1986; Danks, 1987). However, even in diapause, overwintering temperatures can influence metabolic reserves. Larvae of E. solidaginis and prepupae of cynipid wasps have lower potential fecundity when subjected to ecologically relevant, yet high overwintering temperatures (Irwin and Lee 2003, Williams et al., 2003). Alabama- collected larvae likely experience substantially higher overwintering temperatures compared the Michigan and Ohio populations (Fig. 6A and B) and undoubtedly, the lower metabolic rate at 20 °C would be necesary to conserve metabolic fuels during the winter. However, the lowered metabolic rate also would allow larvae to conserve body water by substantially lowering respiratory transpiration compared to the other larvae. In fact, if the respiratory transpiration of Alabama larvae increased at a similar rate as Michigan and Ohio larvae between 5 and 20 °C, then total rates of water loss for Alabama larvae could be as much as 42% higher than what was measured.

In summary, the higher latitude Michigan-collected larvae likely experience the lowest overwintering temperatures and were more cold tolerant than the more southerly Ohio and Alabama larvae. Cold-tolerance was partially linked to higher concentrations of the cryoprotectant glycerol and possibly membrane transporters which function better at lower temperatures. Cryoprotectants may also have functioned to reduce rates of cuticular and overall water loss for Michigan larvae at 5 °C. In contrast, low latitude Alabama larvae likely experience the highest overwintering temperatures and consequently were less affected by the 20 °C acclimation. They had the lowest metabolic rate at 20 °C, which likely resulted in a reduced respiratory transpiration compared to other larvae.

80

Acknowledgements

I thank Mike Elnitsky, Kent Smith, Joe Jacquot, William Rowden and Mickey Eubanks for help in gall collecting. I also thank Jim Oris, Alan Cady, and Jack Vaughn for help in the design of this project, and Robert Schaefer for aid with statistical analysis. Support for this project was provided by the National Science Foundation IOB# 0416720.

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81 Coudron, T. A. and Nelson, D. R. (1981). Characterization and distribution of the hydrocarbons found in diapausing pupae tissues of the tobacco hornworm, Manduca sexta (L). J. Lipid Res. 22, 103-112. Crowe, J. H., Carpenter, J. F., Crowe, L. M. and Anchordoguy, T. J. (1990). Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiol. 27, 219-231. Danks, H. V. (1987). Insect Dormancy: An Ecological Perspective. Ottawa: Biological Survey of Canada (Terrestrial Arthropods). Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46, 837-852. Danks, H. V. (2004). Seasonal adaptations in arctic insects. Integr. Comp. Biol. 44, 85- 94. Denlinger, D. L. and Lee, R. E. (1998). Physiology of cold sensitivity. In Temperature Sensitivity in Insects and Application in Integrated Pest Management, (eds. G. J. Hallman and D. L. Denlinger), pp. 55-95. Boulder: Westview Press. Folk, D. G., Han, C. and Bradley, T. J. (2001). Water acquisition and partitioning in Drosophila melanogaster: effects of selection for desiccation-resistance. J. Exp. Biol. 204, 3323-3331. Gibbs, A. G. (1998). The role of lipid physical properties in lipid barriers. Am. Zool. 38, 268-279. Gibbs, A. G., Fukuzato, F. and Matzkin, L. M. (2003). Evolution of water conservation mechanisms in Drosophila. J. Exp. Biol. 206, 1183-1192. Hadley, N. F. (1994). Water Relations of Terrestrial Arthropods. San Diego: Academic Press. Hegdekar, B. M. (1979). Epicuticular wax secretions in diapause and non-diapause pupae of the Bertha army worm Mamestra configurata. Annu. Entomol. Soc. Am. 72, 13-15. Holmstrup, M., Costanzo, J. P. and Lee, R. E. (1999). Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-tolerant and freeze-intolerant earthworms. J. Comp. Physiol. B 169, 207-214.

82 Howard, R. W. (1993). Cuticular hydrocarbons and chemical communication. In Insect Lipids: Chemistry, Biochemistry and Biology, (eds. D. W. Stanley-Samuelson and D. R. Nelson), pp. 179-226. Lincoln, Nebraska: University of Nebraska Press. Irwin, J. T., Bennett, V. A. and Lee, R. E. (2001). Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis fitch (Diptera: Tephritidae). J. Comp. Physiol. B 171, 181-188. Irwin, J. T. and Lee, R. E. (2003). Cold winter microenvironments conserve energy and improve overwintering survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis. Oikos 100, 71-78. Kemp, A. and Kits Van Heijningen, A. J. M. (1954). A colorimetric micro-method for the determination of glycogen in tissues. Biochem. J. 50, 646-648. Kristiansen, E. and Zachariassen, K. E. (2001). Effect of freezing on the transmembrane distribution of ions in freeze-tolerant larvae of the wood fly Xylophagus cinctus (Diptera, Xylophagidae). J. Insect Physiol. 47, 585-592. Layne, J. R. (1993). Winter microclimate of goldenrod spherical galls and its effects on the gall inhabitant Eurosta solidaginis (Diptera, Tephritidae). J. Therm. Biol. 18, 125- 130. Layne, J. R., Jr. (1991). Microclimate variability and the eurythermic nature of goldenrod gall fly (Eurosta solidaginis) larvae (Diptera: Tephritidae). Can. J. Zool. 69, 614-617. Lee, R. E., Jr. (1991). Principles of insect low temperature tolerance. In Insects at Low Temperature, (eds. R. E. Lee, Jr. and D. L. Denlinger), pp. 17-46. New York and London: Chapman and Hall. Mazur, P. (1984). Freezing of living cells. Am. J. Physiol. 16, C125-C142. Miller, P. L. (1968). On the occurrence and some characteristics of Cyrtopus fastuosus Bigot (Dipt. Stratiomyidae) and Polypedilum sp. (Dipt. Chironomidae) from temporary habitats in western Nigeria. Entomologists' Monthly Magazine, 233-238. Morris, G. J. and Clarke, A. (1981). Effects of Low Temperatures on Biological Membranes. Lodon: Academic Press.

83 Nelson, D. R. and Lee, R. E. (2004). Cuticular lipids and desiccation resistance in overwintering larvae of the goldenrod gall fly, Eurosta solidaginis (Diptera : Tephritidae). Comp Biochem. Physiol. B 138, 313-320. Nicolson, S. W. and Louw, G. N. (1982). Simultaneous measurement of evaporative water-Loss, oxygen-consumption, and thoracic temperature during flight in a carpenter bee. J. Exp. Zool. 222, 287-296. Ring, R. A. and Danks, H. V. (1994). Desiccation and cryoprotection: overlapping adaptations. Cryo-Lett. 15, 181-190. Sjursen, H., Bayley, M. and Holmstrup, M. (2001). Enhanced drought tolerance of a soil-dwelling springtail by pre-acclimation to a mild drought stress. J. Insect Physiol. 47, 1201-1207. Storey, K. B. (1983). Metabolism and bound water in overwintering insects. Cryobiol. 20, 365-379. Storey, K. B., Baust, J. G. and Storey, J. M. (1981). Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Eurosta solidaginis. J. Comp. Physiol. 144, 183-190. Storey, K. B., McDonald, D. G. and Booth, C. E. (1986). Effect of temperature acclimation on haemolymph composition in the freeze-tolerant larvae of Eurosta solidaginis. J. Insect Physiol. 10, 897-902. Storey, K. B. and Storey, J. M. (1992). Biochemical adaptations for winter survival in insects. In Advances in Low-Temperature Biology, vol. 1, (ed. P. L. Steponkus), pp. 101-140. London: Jai Press Limited. Storey, K. B. and Storey, J. M. (1996). Natural freezing survival in animals. Annu. Rev. Ecol. Syst. 27, 365-386. Tauber, M. J., Tauber, C. A. and Masaki, S. (1986). Seasonal Adaptations of Insects. New York: Oxford University Press. Teitz, N. W. (1970). Fundamentals of Clinical Chemistry. Philadelphia: W.B. Saunders. Uhler, L. D. (1951). Biology and Ecology of the Goldenrod Gall Fly, Eurosta solidaginis (Fitch). In Cornell Experiment Station Memoir 300, pp. 1-51. New York: New York State College of Agriculture.

84 Williams, J. B., Ruehl, N. C. and Lee, R. E. (2004). Partial link between the seasonal acquisition of cold-tolerance and desiccation resistance in the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae). J. Exp. Biol. 207, 4407-4414. Williams, J. B., Shorthhouse, J. D. and Lee, R. E. (2002). Extreme resistance to desiccation and microclimate-related differences in cold-hardiness of gall wasps (Hymenoptera: Cynipidae) overwintering on roses in southern Canada. J. Exp. Biol. 205, 2115-2124. Williams, J. B., Shorthouse, J. D. and Lee, R. E. (2003). Deleterious effects of mild simulated overwintering temperatures on survival and potential fecundity of rose- galling Diplolepis wasps (Hymenoptera : Cynipidae). J. Exp. Zool. 298A, 23-31. Yoder, J. A., Denlinger, D. L., Dennis, M. W. and Kolattukudy, P. E. (1992). Enhancement of diapausing flesh fly puparia with additional hydrocarbons and evidence for alkane biosynthesis by a decarbonylation mechanism. Insect Biochem. Mol. Biol. 22, 237-243. Zachariassen, K. E. (1991). The water relations of overwintering insects. In Insects at Low Temperature, (eds. R. E. Lee, Jr. and D. L. Denlinger), pp. 47-63. New York and London: Chapman and Hall. Zachariassen, K. E. (1996). The water conserving physiological compromise of desert insects. Eur. J. Entomol. 93, 359-367.

85 Chapter 5

Effect of freezing and dehydration on hemolymph volume and the distribution of ions and cryoprotectants in the goldenrod gall fly, Eurosta solidaginis

86 Introduction

Insects are intrinsically at risk of dehydration because of their small size and high surface area to volume ratio. As insects dehydrate, they primarily lose water from the hemolymph, which concentrates solutes in the remaining extracellular fluids (Hadley, 1994; Danks, 2000). If insects are unable to replace the lost body water or continue to dehydrate, hemolymph solute concentrations increase and water is osmotically removed from their cells. Osmotic dehydration can be severely damaging if cell water content or cell volume fall below critical levels (Mazur, 1984). To maintain a critical cell volume during dehydration, insects osmoregulate by removing excess solutes from their extracellular fluids by polymerization of amino acids into peptides (Coutchie and Crowe, 1979), chelating ions (Treherne et al., 1975), transferring extracellular solutes into the intracellular compartment (Tucker, 1977a, b; Pederson and Zachariassen, 2002), or by excreting the excess solutes (Zachariassen and Einarson, 1993). The osmoregulatory response may differ depending on the type of solute or the life history of the insect. For example, the cockroach Periplaneta americana retains Na+ in the fat body during dehydration while excreting excess K+ (Tucker, 1977a, b). In addition, sodium is abundant in the diet of the carnivorous beetle Phrynocolus petrosus and is excreted during bouts of dehydration. In contrast, sodium is limited in the diet of a herbivorous carabid beetle in the genus Cypholoba and is removed to the intracellular space during dehydration (Pederson and Zachariassen, 2002).

Dehydration and freezing subject insects to similar stresses. During extracellular ice formation hemolymph solutes are concentrated as only water joins the growing ice lattice. Thus, to survive the osmotic gradient produced by solute concentration, freeze- tolerant insects must limit cellular water loss to avoid shrinking beyond a critical minimum cell volume (Lee, 1991; Denlinger and Lee, 1998). To reduce the risk of excessive cellular dehydration, freeze-tolerant insects produce low-molecular-mass polyols and sugars, termed cryoprotectants. During freezing, cryoprotectants reduce the osmotic gradient between the intracellular and extracellular fluids by reducing the amount of hemolymph that freezes at a given temperature or by increasing intracellular osmolality (Lee, 1991; Storey and Storey, 1991). However, it is unknown if solutes are

87 actively moved between extracellular and intracellular fluids to reduce potential damage caused by dehydration or extracellular freezing.

Larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera: Tephritidae), are a common model for studying insect freeze tolerance. The gall fly ranges from Texas to southern Canada and overwinters as third instar larvae in stem galls on goldenrod (Solidago spp.) (Uhler, 1951). Stem galls typically extend above the insulating snowpack where they are exposed to extreme cold and desiccation during winter (Uhler, 1951; Layne, 1993). In addition, larvae experience large fluctuations in daily temperature and must endure multiple freeze/thaw cycles throughout the winter (Layne, 1991).

Larvae of E. solidaginis are extremely freeze tolerant and can survive at -40 °C for 96 h (Williams and Lee, unpublished data). Larvae also have measurable respiration rates and demonstrate diapause development while frozen (Irwin et al., 2001; Irwin and Lee, 2003), suggesting that cellular function and osmo-regulatory mechanisms continue to function in frozen larvae. To increase cold tolerance, larvae seasonally produce high concentrations of the cryoprotectants glycerol and sorbitol (~1 M) (Lee, 1991). Glycerol freely penetrates across membranes and likely moves into the cell during freeze concentration, reducing cellular water loss by lowering the osmotic gradient created by extracellular ice formation (Storey and Storey, 1991; Storey and Storey, 1996). Less is known about how the larger non-membrane-penetrating sorbitol molecule functions in increasing freeze tolerance, however it is a major cryoprotectant in many freeze-tolerant insects (Storey and Storey, 1991).

The purpose of this study was to determine (1) if larvae of E. solidaginis reduce hemolymph solutes in response to both freezing and dehydration and (2) which solutes are being reduced during these stresses. To answer these questions, we measured body water content, hemolymph volume, whole body and extracellular concentrations of cryoprotectants (glycerol and sorbitol), and ions (Na+, K+, Mg++) in larvae after being dehydrated, held unfrozen at -5 °C, frozen at -5 °C, or frozen at -20 °C. To determine whether ions are removed from the hemolymph and sequestered in fat body tissue, as in the cockroach P. americana (Tucker, 1977b), we measured Na+, K+, and Mg++ ion

88 concentrations in fat body tissue and remaining carcass tissue after larvae were subjected to the above treatments.

Materials and Methods

Insect collection

Galls containing cold-hardened larvae were collected from the Miami University Ecology Research Center near Oxford, Ohio in early December, 2003. Larvae were immediately removed from the gall after collection and held at 5 °C and 76% RH (over a saturated solution of sodium chloride) for at least one week prior to being subjected to the experimental conditions.

Experimental treatments

After the holding period randomly selected larvae were either analyzed immediately, as a control group, or exposed to one of four experimental treatments prior to analysis. Larvae in two of the four treatments were placed within 1.6 ml microcentrifuge tubes and cooled in an alcohol bath from 5 °C at 0.25 °C•min-1 to -5 °C. Larvae were held either unfrozen (supercooled) on dry filter paper or inoculatively frozen on filter paper saturated with distilled water. To ensure freezing of the saturated filter paper, a corner of the filter paper was inoculated by spraying it with Super Friendly Freeze It coolant (Fisher Scientific; Pennsylvania, USA) when the bath temperature was approximately -2 °C. To examine the effect of higher extracellular ice contents, larvae in the third experimental treatment were cooled from 5 °C at 0.25 °C•min-1 to -20 °C and allowed to freeze at their supercooling point, ~ -9 °C (Lee, 1991). Larvae were held at their respective subzero temperature for 96 h before being removed, checked to see if they were frozen or supercooled, and placed at 5 °C for 15 min prior to analysis. In the fourth experimental treatment, larvae were desiccated at 5 °C over Drierite (W.A.

89 Hammond Drierite Co., Ohio, USA) producing a relative humidity of ~4% (Ramløv and Lee, 2000) until they lost between 9-11% of their initial wet mass prior to analysis.

Whole body ion analysis

After being subjected to the above treatment conditions, body water content and dry mass were determined by weighing (±0.1 mg) larvae (n = 15) before and after being dried at 65 °C until reaching a constant mass. The dried larvae were then completely

digested in 0.5 ml of 65% HNO3 before evaporating the acid at 90 °C for 24 h. The remaining solids were re-dissolved and diluted in a known volume of 0.1 M HNO3 containing 0.1 % cesium chloride as an ionization suppressant, and analyzed for Na+, K+, and Mg++ contents using a Varian Spectra AA 220FS atomic absorption flame spectrophotometer (Mulgrave, Australia). Whole body ion concentrations were expressed in: µg of ion•mg whole body dry mass-1.

Hemolymph, fat body, and remaining carcass ion concentrations

To determine hemolymph ion concentrations, larvae (n = 15) were weighed, lanced open, and 4 µl of hemolymph was drawn into a microcapillary tube for storage at -80 °C until analysis. Hemolymph volume was then determined by re-weighing the above larvae after the remaining hemolymph was removed by gently swabbing the viscera with a rolled kimwipe wetted with a 865 mOsm•kg-1 glycerol solution that was iso-osmotic to the hemolymph of control larvae (Folk et al., 2001). Next, each larva was quickly submerged in the iso-osmotic glycerol solution to aide in separating and removing the fat body tissue from the remaining carcass. Both the fat body tissue and carcass tissue were dried at 65 °C until reaching a steady mass prior to acid digestion and analysis for Na+, K+, and Mg++ ions as described above for whole body larvae. Extracellular ion concentrations were determined after diluting the 4 µl of hemolymph in a known volume

of 0.1 M HNO3 containing 0.1 % cesium chloride as described above.

90

Cryoprotectant concentrations and hemolymph osmolality

After exposure to the various treatment conditions, larvae (n = 10) were weighed and immediately frozen at -80 °C until whole body concentrations of cryoprotectants were determined. Upon removal from storage, larvae were homogenized in perchloric acid and neutralized with equal volumes of potassium hydrogen carbonate prior to determining glycerol content using the enzymatic assay (Sigma Chemical Co., no. 337) described by Hølmstrup et al. (1999). Sorbitol concentrations were measured on the same individuals using the enzymatic assay described in Bergmeyer et al. (1974). Hemolymph cryoprotectant concentrations were determined by weighing larvae (n = 10) prior to removing and analyzing 10 µl of hemolymph for glycerol and sorbitol content using the same enzymatic assays described above. After removal of the 10 µl, the remaining extracellular fluid was removed to determine hemolymph volume as described above. Hemolymph osmolality (n = 10) was determined by drawing 7-10 µl of hemolymph into a capillary tube through a small incision in the larval cuticle. The hemolymph was then analyzed in a Wescor Vapro 5520 Hemolymph Osmometer (Logan, Utah).

Statistical analysis

A one-way ANOVA followed by a Bonferroni multiple comparisons test was used to determine differences between larvae exposed to the five treatment conditions. Determination of significance for all analyses was α=0.05 and all data are represent as mean ± SEM.

91 Results

Body water content and hemolymph osmolality

Body water content was significantly lower (p < 0.05) for larvae in the desiccated treatment (1.24 ± 0.07 mg water • mg dry mass-1or 54% body water) compared to larvae in the remaining treatments, which averaged 1.56 ± 0.04 mg water•mg dry mass-1 or 61% body water (Table 1). Larval dry mass was similar in all treatments (p > 0.05), averaging 20.3 ± 0.4 mg (Table 1), indicating that the reduced body mass of the desiccated larvae was due completely to loss of body water. Interestingly, larvae in both the desiccated and low temperature treatment groups reduced the proportion of total body water in their hemolymph by 3.4 to 4.5 % (1.5 to 4.0 ml), compared to control larvae which had on average 30.5 % (16.4 µl) of total body water in the extracellular compartment (Table 1).

Even though hemolymph volume was markedly reduced in the desiccated and low temperature treatment groups, hemolymph osmolality was similar (p > 0.05) for larvae in the control, -5 °C frozen, and -20 °C frozen treatments averaging 908 ± 20 mOsm•kg-1 (Table 1). Larvae in the desiccated and -5 °C unfrozen treatments had hemolymph osmolalities that were similar to the two frozen treatment groups (p > 0.05); however, they were higher (p < 0.05), averaging ~100 mOsm•kg-1 more than control larvae (Table 1).

Whole body and hemolymph cryoprotectant concentrations Whole body glycerol concentrations were highest (p < 0.05) in desiccated larvae (554 ± 39 mmol) compared to the remaining groups, which averaged 402 ± 22 mmol (Fig. 1A). However, the increase in osmolarity in desiccated larvae was not due to a production of the polyol itself, but rather a reduction in body water, as larvae in all treatments had similar levels of glycerol (p > 0.05) on a per gram dry mass basis (ranging between 50.3 ± 3.1 and 62.3 ± 2.4 µg glycerol•mg dry mass-1;data not shown). Interestingly, hemolymph glycerol concentrations were 56 mmol lower in the -20 °C frozen larvae compared to controls (p > 0.05) (Fig. 1A) even though the -20 °C frozen larvae had reduced hemolymph volumes

92

Table 1. Mean body water content (n = 15), mean dry mass (n = 15), hemolymph content (n = 25) and mean hemolymph osmolality (n = 10), for mid-winter-collected larvae of Eurosta solidaginis subjected to five different treatment conditions. Means (± SEM) within a column not sharing the same letter were significantly different when analyzed with a one-way ANOVA followed with a Bonferroni multiple comparisons test.

Treatment Body Water Content Dry Mass Hemolymph Volume Hemolymph Osmolality (mg water • mg dry mass-1) (mg) (% total water content) (mOsm•kg1) Control 1.55 ± 0.04 a 20.6 ± 0.5a 30.5 ± 0.7 a 869 ± 18 a

Desiccated 1.24 ± 0.07 b 19.9 ± 0.3 a 26.0 ± 0.8 b 986 ± 11 b

- 5 °C 1.60 ± 0.04 a 20.6 ± 0.5 a 27.4 ± 0.4 b 959 ± 21 b Unfrozen

-5 °C 1.57 ± 0.02 a 19.8 ± 0.3 a 27.1 ± 0.5b 945 ± 27 ab Frozen

-20 °C 1.58 ± 0.03 a 20.4 ± 0.5 a 26.6 ± 0.7 b 913 ± 22 ab Frozen

93 B ofthisfigureanddeviding theextr cryoprotectants inthehemolym exposed tov f not sharinganum cocentrations. W located inth (B) m Figure 1.Wholebodyandextracellularconcentr

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llowed withaBonf Cryoprotectants in Hemolymph Sorbitol Concentration Glycerol Concentration (% of total) (mmol) (mmol) ean sorbitol(n=10),and(C)relative 100 100 200 300 400 500 600 700 200 400 600 800 10 20 30 40 50 60 70 0 0 0 0 e hem a C A rious treatm B a C a ont hole bodym ber aresignificantlydiffe o 1 r lym o 1 l erroni m p h f ent conditions. D a o e b s r m i cc u eans (±SEM)not 1, ltiple c at 2 T ph bycom i 1 d-winter-collected larvaeof e r d ea t acellular componenet m o U a m en a n -5 E W Iestimatedthep p fro t ° b x arisons test. G hol 2 rent whenanalyzedusing aone-wayANOVA t 1, C ining z 94 r of proportiontotalglycerolandsorbitol e a 2 r n c e o e B l u sharingaletterandextracellular m l o u p ations of(A)m sorbitol andglyceroldatainpartsA d l a y r a C a Fr -5 o o m ° z 2 1, C e pone n 2 r from ecentage oftotal nt Eurosta solidaginis a thewholebody a F -2 ean glycerol(n=10), r o 0 zen ° 1 2 C

larv e ans ae (Table 1).

Whole body sorbitol concentrations were highly variable and did not differ significantly (p > 0.05) between larvae in the control and experimental treatments (Fig. 1B). However, larvae from the desiccated and low temperature treatments tended to be higher than controls, averaging 604 ± 87 mmol compared to controls (329 ± 36 mmol). Extracellular sorbitol concentrations ranged between 168 ± 13 mmol and 330 ± 22 mmol (Fig. 1B) and were higher (p > 0.05) in the - 5 °C treatment groups compared to controls. Interestingly, 54% of total glycerol and sorbitol concentrations were extracellular in the control larvae (Fig. 1C). However, larvae in the desiccated and -20 °C frozen treatments were at the highest risk of cellular dehydration, and only had 37% and 32%, respectively, of total cryoprotectants in their extracellular fluids (Fig. 1C).

Whole body, hemolymph, carcass, and fat body ion concentrations

Whole body concentrations of Na+, K+, and Mg++ did not differ (p > 0.05) among treatment groups (Figs. 2A, 2B, and 2C), suggesting that E. solidaginis larvae retain these ions in response to desiccation, low temperature, and freezing. Extracellular, or hemolymph concentrations of Na+ and K+ in the low temperature treatments did not significantly differ from control values (p > 0.05), averaging 0.24 ± 0.01 µg Na+•mg dry mass-1 and 1.20 ± 0.04 µg K+•mg dry mass-1, respectively (Figs. 3A and 3B). However, extracellular concentrations of Mg++ were slightly reduced (p > 0.05) in the two treatments subjected to freezing, -5 °C frozen (0.24 ± 0.01 µg Mg++•mg dry mass-1) and - 20 °C frozen (0.18 ± 0.01 µg Mg++•mg dry mass-1) compared to larvae in other treatments which averaged 0.29 ± 0.01 µg Mg++•mg dry mass-1 (Fig. 2C). Larval carcass concentrations of sodium and magnesium did not differ significantly between treatment groups and controls (p > 0.05) and all groups averaged 0.11 ± .01 µg Na+•mg dry mass-1 and 2.04 ± 0.39 µg Mg++•mg dry mass-1, respectively (Figs. 3A and 3C). Carcass concentrations of potassium were significantly lower (p < 0.05) in the three low temperature treatments compared to the desiccated and control

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Figure 2. Mean whole body and extracellular ion concentrations (n = 15) of (A) sodium, (B) potassium, and (C) magnesium for mid-winter collected larvae of Eurosta solidaginis larvae exposed to various treatment conditions. Whole body means (± SEM) not sharing a letter and extracellular means not sharing a number are significantly different when analyzed using a one-way ANOVA followed with a Bonferroni multiple comparisons test.

96 Bonf letter aresignificantlydifferent whenan various treatm (C) m Figure 3.Meancarcassionconcentrations(n

Mean Magnesium Concentration Mean Sodium Concentration Mean Potassium Concentration -1

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gnesium (µg mg dry mass ) 0. 0. 0. 0. 0. 0. 0. 1. 1. 2. 2. 3. 3. 0 0 0 1 1 10 12 14 0 5 0 5 0 5 0 5 0 4 8 2 6 0 2 4 6 8 ent conditions.W C A B form C ont ab a a r i o d- mparisons te l winter co D e s i ab a cc hole bodyand a llected larvaeo at T ed st. r e a t alyzed usingaone-wayANOVA followedwitha m U e n -5 nt 97 fro a a ° b =15)of(A)sodium G C z e extracellular m n r f oup

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, and larval groups, with the -5 °C frozen and -20 °C frozen larvae having the lowest values, averaging 8.66 ± 0.21 and 7.95 ± 0.20 µg K+•mg dry mass-1, respectively (Fig. 3B).

Mean values of ion concentrations in fat body were wide ranging for Na+ (0.01 ± 0.01 to 0.04 ± 0.02 µg•mg dry mass-1), K+ (2.70 ± 0.23 to 3.38 ± 0.16 µg•mg dry mass-1), and Mg++ (0.62 ± 0.14 to 1.12 ± 0.28 µg•mg dry mass-1) (Figs. 4A, 4B, and 4C). However, perhaps because of high variability, no values were significantly different (p > 0.05) between treatments for any of the measured ions in fat body.

Discussion

In the absence of osmo-regulation, hemolymph solute concentrations will increase with organismal dehydration or extracellular ice formation. Our data indicate however, that E. solidaginis larvae were able to osmoregulate and reduce hemolymph solutes in response to both dehydration and freezing. Larvae in the desiccation and low temperature treatments had reduced hemolymph volumes, ~12 to 25% lower on a mg basis, compared to controls (Table 1). If the hemolymph of the control larvae behaved as an ideal solution, and was reduced to the similar volumes that were measured in larvae from the experimental treatments, then hemolymph osmolality of those control larvae would increase by an average of 136 to 1022 mOsm•kg-1. The 1022 mOsm•kg-1 predicted value is greater than any measured in the experimental groups, suggesting that E. solidaginis larvae remove hemolymph solutes when dehydrated or subjected to low temperature. Reduced hemolymph volumes for larvae subjected to desiccation and low temperatures suggest intracellular osmolality was higher than hemolymph osmolality after the stress (Table 1). During freezing at -4 °C, Malpighian tubule cells of the freeze-tolerant orthopteran, Hemideina maori, are reduced in volume by ~35% (Neufeld and Leader, 1998). Upon thawing, however, cell volume swelled to approximately 10%

98 0.06 A a ation ) a -1 0.04 a mass y r a mg d

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Figure 4. Mean fat body ion concentrations (n = 15) of (A) sodium, (B) potassium, and (C) magnesium for mid-winter collected larvae of Eurosta solidaginis larvae exposed to various treatment conditions. Whole body and extracellular means (± SEM) not sharing a letter are significantly different when analyzed using a one-way ANOVA followed with a Bonferroni multiple comparisons test.

99

above controls, suggesting solutes were moved from the bathing solution into the cells during freezing (Neufeld and Leader, 1998). Solute loading during freezing may occur in many different tissues and even in cells that normally do not experience freezing such as mammalian oocytes (Griffiths et al., 1974). Therefore, it is likely that solutes were moved from the hemolymph to the intracellular fluids of E. solidaginis larvae during freezing. Upon thawing, osmolality of the hemolymph would be much lower than in the intracellular fluids, causing water to move osmotically into the cell, consequently reducing hemolymph volume. Interestingly, reduction of hemolymph volume by moving solutes into the intracellular fluids may also occur in preparation for freezing, as larvae held unfrozen at -5 °C had relatively lower hemolymph volumes compared to controls (Table 1). Reduction of hemolymph volume also occurred in larvae subjected to dehydration, suggesting that these larvae also increased intracellular osmolality relative to hemolymph osmolality during the stress.

It is unlikely that E. solidaginis larvae exposed to low temperature and dehydration reduced hemolymph volume compared to controls by lowering extracellular ion concentrations. Insects that regulate ion concentrations during dehydration, such as the cockroach P. americana and the tenebrionid beetle P. petrosus, do so because those ions constitute a large portion of the osmotically active solutes in the hemolymph (Tucker, 1977a, b; Pederson and Zachariassen, 2002). Hemolymph concentrations of Na+, K+, and Mg++ measured for E. solidaginis larvae were comparable to other reports for phytophagus insects (Nation, 2002). However, hemolymph osmolality for E. solidaginis larvae (886-989 mOsm•kg-1) was considerably higher, due to high concentrations of cryoprotectants, than the osmotic pressure for the typical insect, ~300 mOsm•kg-1 (Edney, 1977). Therefore, the relative contribution of Na+, K+, and Mg++ to the overall hemolymph osmolality of E. solidaginis is relatively low (1 to 2 mmol for Na+, 39 to 52 mmol for K+, and 13 to 16 mmol for Mg++) and reductions in extracellular concentrations of these ions would have a minimal effect on regulating osmolality and water movement between the intracellular and extracellular compartments. Thus, it is not

100 surprising that extracellular ion concentrations were unchanged or only slightly reduced in larvae subjected to dehydration, low temperature, and freezing (Figs. 2A, B, C).

Even though Na+, K+, and Mg++ ions were apparently unimportant for regulating hemolymph osmolality, they were maintained at homeostatic levels even during severe freezing. When the freeze-tolerant wood fly, Xylophagus cinctus, is exposed to a survivable temperature of -10 °C for 24 h, ion transporters are impaired and Na+, K+, and Mg++ ions redistribute and nearly reach equilibrium across the cell membrane (Kristiansen and Zachariassen, 2002). Hemolymph concentrations of K+ also increase dramatically in E. solidaginis larvae subjected to lethal freezing at -80 °C, suggesting that severe membrane damage occurrs at this temperature (Michael Elnitsky and Jack R. Layne, personal communication). In contrast, Na+, K+, and Mg++ intracellular and extracellular gradients were maintained in E. solidaginis subjected to -20 °C for 96 h (Fig. 3), indicating that cell membranes were undamaged and ion transporters were able to function during that exposure.

Larvae of E. solidaginis exposed to low temperature and dehydration likely reduce hemolymph volume compared to controls by removing extracellular cryoprotectants. In control larvae, ~ 42 % of the hemolymph solutes are either glycerol or sorbitol (Figs. 1 and 2). Thus, movement or transport of cryoprotectants into the intracellular fluid could have a profound impact on maintaining cell water volume during dehydration or freezing in E. solidaginis larvae (Zachariassen, 1991) and might even increase the relative proportion of intracellular water at the expense of hemolymph volume (Table 1). When examining the intracellular and extracellular distribution of cryoprotectants in control larvae, ~ 54% of sorbitol and glycerol were located in the hemolymph (Fig. 1C). However, all experimental larvae had less than half of the relative proportion of total cryoprotectants in their hemolymph. In addition, larvae in the desiccation and -20 °C frozen treatments, which were likely subjected to the highest osmotic stress, had on average only 33% of their cryoprotectants located in the hemolymph. These results suggest cryoprotectants were transported into the intracellular fluids during these stresses (Fig. 1C). Thus, there is evidence that the lower hemolymph volumes of the experimental groups were due to movement of cryoprotectants into the

101 intracellular compartment. However, the results shown in Figure 1C should be viewed with caution, because they were derived onlt from sorbitol and glycerol data (Figs. 1A and 1B), in which very few values differed from controls.

Cryoprotectants could be accumulated intracellularly through the production of or transport of sorbitol from the hemolymph into the intracellular fluids. Although little is known about the movement of non-membrane penetrating cryoprotectants due to dehydration or freezing in insects, the wood frog Rana sylvatica utilizes glucose as a cryoprotectant and readily transports this sugar between compartments during freezing (King et al., 1993). In addition, mild freezing at -4 °C initiates wood frog hepatocytes to accumulate glucose, which in turn reduces shrinkage when the cells are subsequently subjected to -20 °C (Storey et al., 1992). Therefore, it is possible that E. solidaginis larvae exposed to dehydrating conditions or low temperatures accumulated intracellular sorbitol by transporting the cryoprotectant into the cell, or through the production of intracellular sorbitol, which readily occurs at low temperature and in frozen larvae (Storey and Storey, 1981). Once intracellular sorbitol concentrations are increased, regardless of the mechanism, hemolymph volume would likely be reduced as water moves into the cells. A reduced hemolymph volume would concetrate extracellular glycerol and would cause this polyol to enter cells by moving down its concetration gradient and would increase intracellular cryoprotectant concentrations further (Storey and Storey, 1991).

The similar stresses imposed by dehydration and extracellular ice formation has led several authors to speculate that traits associated with dehydration tolerance may represent pre-adaptations which facilitated the evolution of animal freeze tolerance (Costanzo et al., 1993; Storey, 1997). Larvae of E. solidaginis exposed to dehydration as well as freezing had reduced hemolymph volumes and likely had increased intracellular cryoprotectants, suggesting that larvae may use a similar mechanisms to survive both stresses. Several tropical insects, which never experience sub-zero temperatures, produce cryoprotectants during periods of dehydration risk (see references in Ring and Danks, 1994). The presence of the biochemical pathways for producing cryoprotectants and possible use of these solutes to survive dehydration in tropical insects could have been

102 utilized as insects radiated northward and experienced seasonal desiccation stress in winter. Consequently, high concentrations of these solutes along with other physiological and morphological features, such as endogenous ice nucleators (Lee, 1991), may have facilitated the evolution of freeze tolerance in insects.

Acknowledgements

I thank Steve Dinkelacker, Mike Polin, Shuxia Yi, and Patrick Baker for helping to collect galls. Jon Costanzo assisted with the glycogen protocol. Aaron Roberts, Jim Oris and Paul Drevnick trained me in the use of the flame spectrophotometer. I also thank Jim Oris, Alan Cady, and Jack Vaughn for help in the design of this project, and Robert Schaefer for aid with statistical analysis. Support for this project was provided by the National Science Foundation IOB# 0416720 to REL.

Literature Cited

Bergmeyer, H. U., Gruber, W. and Gutmann, I. (1974). D-Sorbitol. In Methods of Enzymatic Analysis, vol. 3 (ed. H. U. Bergmeyer), pp. 1323-1326. New York: Academic Press. Costanzo, J. P., Lee, R. E. and Lortz, P. H. (1993). Physiological responses of freeze- tolerant and intolerant frogs: clues to evolution of anuran freeze tolerance. Am. Physiol. Soc. 265, R721-R725. Coutchie, P. A. and Crowe, J. H. (1979). Transport of water vapor by tenebrionid beetles. II. Regulation of the osmolality and composition of the hemolymph. Physiol. Zool. 52, 88-100. Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46, 837-852.

103 Denlinger, D. L. and Lee, R. E. (1998). Physiology of cold sensitivity. In Temperature Sensitivity in Insects and Application in Integrated Pest Management, (eds. G. J. Hallman and D. L. Denlinger), pp. 55-95. Boulder: Westview Press. Edney, E. B. (1977). Water Balance in Land Arthropods. New York: Springer-Verlag. Folk, D. G., Han, C. and Bradley, T. J. (2001). Water acquisition and partitioning in Drosophila melanogaster: effects of selection for desiccation-resistance. J. Exp. Biol. 204, 3323-3331. Griffiths, J. B., Cox, C. S., Beadle, D. J., Hunt, C. J. and Reid, D. S. (1979). Changes in cell size during the cooling, warming and post-thawing periods of the freeze-thaw cycle. Cryobiol. 16, 141-151. Hadley, N. F. (1994). Water Relations of Terrestrial Arthropods. San Diego: Academic Press. Holmstrup, M., Costanzo, J. P. and Lee, R. E. (1999). Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-tolerant and freeze-intolerant earthworms. J. Comp. Physiol. B 169, 207-214. Irwin, J. T., Bennett, V. A. and Lee, R. E. (2001). Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis fitch (Diptera: Tephritidae). J. Comp. Physiol. B 171, 181-188. Irwin, J. T. and Lee, R. E. (2003). Cold winter microenvironments conserve energy and improve overwintering survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis. Oikos 100, 71-78. King, P. A., Roshot, M. N. and Storey, K. B. (1993). Adaptations of plasma membrane glucose transport facilitate cryoprotectant distribution in freeze-tolerant frogs. Am. J. Physiol. 265, R1036-R1042. Kristiansen, E. and Zachariassen, K. E. (2001). Effect of freezing on the transmembrane distribution of ions in freeze-tolerant larvae of the wood fly Xylophagus cinctus (Diptera, Xylophagidae). J. Insect Physiol. 47, 585-592. Layne, J. R. (1993). Winter microclimate of goldenrod spherical galls and its effects on the gall inhabitant Eurosta solidaginis (Diptera, Tephritidae). J. Therm. Biol. 18, 125- 130.

104 Layne, J. R. (1991). Microclimate variability and the eurythermic nature of goldenrod gall fly (Eurosta solidaginis) larvae (Diptera: Tephritidae). Can. J. Zool. 69, 614-617. Mazur, P. (1984). Freezing of living cells. Am. J. Physiol. 16, C125-C142. Nation, J. L. (2002). Insect Physiology and Biochemistry. New York: CRC Press. Neufeld, D. S. and Leader, J. P. (1998). Cold inhibition of cell volume regulation during the freezing of insect malpighian tubules. J. Exp. Biol. 201, 2195-2204. Pedersen, S. A. and Zachariassen, K. E. (2002). Sodium regulation during dehydration of a herbivorous and a carnivorous beetle from African dry savannah. J. Insect Physiol. 48, 925-932. Ramløv, H. and Lee, R. E. (2000). Extreme resistance to desiccation in overwintering larvae of the gall fly Eurosta solidaginis (Diptera: Tephritidae). J. Exp. Biol. 203, 983-789. Ring, R. A. and Danks, H. V. (1994). Desiccation and cryoprotection: overlapping adaptations. Cryo-Lett. 15, 181-190. Storey, K. B. (1997). Organic solutes in freezing tolerance. Comp. Biochem. Physiol. 117, 319-326. Storey, K. B., Baust, J. G. and Storey, J. M. (1981). Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Eurosta solidaginis. J. Comp. Physiol. 144, 183-190. Storey, K. B., Bischof, J. and Rubinsky, B. (1992). Cryomicroscopic analysis of freezing in the liver of the freeze-tolerant wood frog. Am. J. Physiol. 263, R185- R194. Storey, K. B. and Storey, J. M. (1991). Biochemistry of cryoprotectants. In Insects at Low Temperature, (eds. R. E. Lee, Jr. and D. L. Denlinger), pp. 64-93. New York and London: Chapman and Hall. Storey, K. B. and Storey, J. M. (1996). Natural freezing survival in animals. Annu. Rev. Ecol. Syst. 27, 365-386. Treherne, J. E., Buchan, P. B. and Bennet, R. R. (1975). Sodium activity of insect blood: Physiological significance and relevance to the design of physiological saline. J. Exp. Biol. 62, 721-732.

105 Tucker, L. E. (1977a). Effect of dehydration and rehydration on the water content and Na+ and K+ balance in adult male Periplaneta americana. J. Exp. Biol. 71, 49-66. Tucker, L. E. (1977b). The influence of diet, age and state of hydration on Na+, K+ and urate balance in the fat body of the cockroach Periplaneta americana. J. Exp. Biol. 71, 67-79. Uhler, L. D. (1951). Biology and Ecology of the Goldenrod Gall Fly, Eurosta solidaginis (Fitch). In Cornell Experiment Station Memoir 300, pp. 1-51. New York: New York State College of Agriculture. Zachariassen, K. E. (1991). The water relations of overwintering insects. In Insects at Low Temperature, (eds. R. E. Lee, Jr. and D. L. Denlinger), pp. 47-63. New York and London: Chapman and Hall.

Zachariassen, K. E., Einarson, S. (1993). Regulation of body fluid compartments during dehydration of the tenebrionid beetle Rhytinota praelonga. J. of Exp. Biol. 182, 283-289.

106 Chapter 6

Concluding Remarks

The underlying hypothesis of this dissertations stated that cryoprotectants increased cold tolerance as well as water retention in overwintering insects.

The fact that seasonal increases in cryoprotectants for E. solidaginis larvae correlated well with both increased cold-tolerance and desiccation resistance support the hypothesis (Chapter two). In addition, E. solidaginis larvae collected from Michigan were more cold tolerant, had higher resistance to desiccation, and had higher concentrations of the cryoprotectant glycerol compared to Ohio and Alabama-collected larvae also support the underlying hypothesis (Chapter four).

Taken together, the results of the above studies suggest that cryoprotectants influenced water balance in E. solidaginis larvae and represent a link between desiccation resistance and cold tolerance. Such a link lends credence to the idea that production of cryoprotectants had originally evolved to limit water loss. Consequently, tropical insects could have exploited the biochemical pathways of cryoprotectant production to increase cold hardiness, allowing insects to radiate northward.

Data from chapter three illustrates that rates of water loss were substantially reduced as E. solidaginis larvae entered diapause. Thus, it appears that diapause is essential for maintaining sufficient metabolic reserves as well as water balance of overwintering insects. The phenomenon of diapause also likely evolved in tropical insects in response to dehydration stress (See in previous chapters Danks, 1987).

107 Therefore, diapause may represent another adaptation, such as cryoprotectant production,

which was originally used to increase desiccation resistance in tropical insects but was

exploited to survive winter conditions as insects radiated northward.

Finally, cryoprotectants were likely moved from the hemolymph to the

intracellular fluids to maintain cell volume during both dehydration and freezing in E.

solidaginis larvae (Chapter five). Movements of these solutes in response to both stresses

suggest that these larvae use a similar mechanism to survive both dehydration and

freezing. This is a significant finding because it illustrates that cryoprotectants are used

to not only increase desiccation resistance in this species, as noted above, but also reduce

the effects of dehydration as well. Also, insects may have again exploited cryoprotectant production to increase cold tolerance and possible even allow insects to evolve the ability

to survive extracellular freezing.

108