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