ABSTRACT

ENHANCED COLD TOLERANCE OF -DESTINED VS. NON-DIAPAUSE- DESTINED LARVAL STAGES OF THE FLESH FLY, SARCOPHAGA CRASSIPALPIS (DIPTERA: SARCOPHAGIDAE)

by Kent J. Smith

The ontogenetic development of cold-hardening associated with diapause was examined in feeding and wandering larval stages of the flesh fly, Sarcophaga crassipalpis. Cold tolerance was greatest for diapause-destined wandering larvae, as 62% of cold-shocked (-9 ºC, 2 h) diapause-destined individuals progressed to adult development as compared to only 10% of wandering, non-diapause-destined larvae. The viability of Malpighian tubules and fat bodies of cold-shocked diapause-destined wandering larvae were significantly greater (91%, 90%) than in non-diapause-destined groups (64%, 75%). The diapause program influenced the composition of membrane fatty acids (FAs). The dominant unsaturated , oleic acid, accounted for only 18% of the total phospholipid FA proportion in diapause-destined groups, down from 36% in non-diapause-destined larvae. This shift in oleic acid, coupled with increases in the principal saturated FA species in diapause-destined larvae, resulted in an index of unsaturation significantly lower (0.57) than in non-diapause-destined larvae (1.08).

ENHANCED COLD TOLERANCE OF DIAPAUSE-DESTINED VS. NON-DIAPAUSE- DESTINED LARVAL STAGES OF THE FLESH FLY, SARCOPHAGA CRASSIPALPIS (DIPTERA: SARCOPHAGIDAE)

A Thesis

Submitted to the

Faculty of Miami University

in partial fulfillment of

the requirements for the degree of

Master of Science

Department of Zoology

by

Kent J. Smith

Miami University

Oxford, Ohio

2007

Advisor: ______Richard E. Lee, Jr.

Reader: ______Phyllis Callahan

Reader: ______Jon P. Costanzo

TABLE OF CONTENTS

Table of contents ii List of tables iii List of figures iii

Dedication iv

Introduction 1 Materials and Methods 3 Results 8 Discussion 11 Acknowledgements 16 Literature Cited 17 Tables 21 Figure Legends 23 Figures 24

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List of Tables

Table Page

1 Cell viability of tissues (in vivo) of non-diapause-destined and diapause- 21 destined feeding and wandering larvae.

2 Composition of phospholipid fatty acids (PLFAs) in third instar larvae 22 from non-diapause-destined and diapause-destined groups.

List of Figures

Figure Page

1 Effect of diapause induction on cold tolerance of feeding and wandering 24 larvae.

2 Effects of cold shock (-9 ºC, 2 h) on cell viability of isolated tissues 25 of non-diapause-destined vs. diapause-destined wandering third instar larvae.

3 Body water content, hemolymph osmolality, and concentration 26 of non-diapause-destined and diapause-destined larvae at feeding and wandering-stages.

4 Fatty acid changes due to diapause induction in third instar larvae. 27

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Dedication

This thesis is dedicated to my grandparents, James and Elizabeth Powell for their persistent encouragement and their unseen contributions to my personal development, and to my wife, my fire, Sarah.

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INTRODUCTION

Cold hardiness and diapause The onset of presents significant challenges for in temperate regions that are unable to avoid exposure to subzero temperatures and must cold-harden in order to survive. Cold-hardening is the facility of an to survive exposure to low temperature over prolonged periods by a variety of physiological processes which result in enhanced cold tolerance (Hanec & Beck 1960). A primary for overwintering survival in many insects is entry into an arrested developmental state known as diapause. Diapause occurs during different times, in different life stages, and is activated by various seasonal cues (e.g. day-length, temperature, food quality and availability) (Denlinger 1985). While it is not always the case, entry into diapause is often associated with enhanced cold tolerance (Denlinger, 1991). Diapausing pupae of the flesh fly, Sarcophaga crassipalpis, can survive long periods at temperatures a few degrees above its supercooling point (-23 ºC), whereas non-diapausing pupae die due to chilling following brief exposure to -10 ºC (Lee & Denlinger 1985). S. crassipalpis is a freeze- intolerant species that is a particularly useful model organism for experiments involving a comparison of non-diapausing vs. diapausing insects. Flesh fly pupae overwinter only a few centimeters underground where they are commonly exposed to sustained sub-zero temperatures (Denlinger 1981). While the overwintering, pupal stage exhibits the greatest increase in cold tolerance, there is evidence suggesting enhanced cold tolerance in larvae during diapause induction, prior to entering into diapause (Lee et al. 1987). The increase in cold tolerance of diapausing S. crassipalpis pupae is largely credited to increased concentrations of the glycerol. However, elevated glycerol levels do not always improve cold hardiness. Lee et al. (1987) demonstrated that non-diapause-destined larvae accumulate substantial amounts of glycerol, with or without low temperature exposure, but prove far less cold tolerant than diapause-destined larvae which do not accumulate significant levels of glycerol. While accumulation of glycerol appears to contribute to the cold-hardiness in diapausing pupae of S. crassipalpis, it is unknown precisely what other mechanisms are responsible for increased cold

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tolerance associated with diapause, and exactly when this capacity is acquired during larval development.

Role of In addition to glycerol, compounds such as sorbitol, glucose, and trehalose function as colligative or noncolligative cryoprotectants, enhancing cold hardiness and, thus, winter survival (Lee 1991, Storey & Storey 1991). Traditional colligative-acting cryoprotectants enhance cold tolerance by depressing both the ’s hemolymph and supercooling points (Salt 1961, Zachariassen 1985), whereas noncolligative cryoprotectants act by stabilizing , phospholipid bilayers, and liposomes (Crowe et al. 1987, Crowe et al. 1983, Loomis et al. 1988). The synthesis of these cryoprotectants is initiated by external temperature cues, as with most freeze-tolerant insects, or by intrinsic insect development often associated with diapause induction (Morrissey & Baust 1976, Zachariassen 1977, Baust & Lee 1982, Nordin et al. 1984).

Cell membranes and cold As temperatures decrease, cell membranes become susceptible to significant chilling injury (Levitt 1980, Watson & Morris 1987, Drobnis et al. 1993). A significant cold shock may lead to an increase in rigidity, by which cellular dehydration associated with cold stress lowers the lamellar-to-hexagonal phase transition temperature, resulting in a deterioration of the membrane’s functional properties (Cossins 1983, Webb et al. 1994). Freeze-susceptible insects likely engage in membrane re- organization in order to prevent lethal, cold-induced phase transitions. Bennett et al. (1997) demonstrated that larvae of the goldenrod gall fly, Eurosta solidaginis, increased monoenic fatty acids and decreased saturated species, resulting in an overall desaturation of cell membranes during seasonal cold-hardening. Similarly, Ohtsu et al. (1993) found that five species of Drosophila showed increased proportions of unsaturated fatty acids in diapausing compared to non-diapausing flies; however, the effect this difference had on cold tolerance was not determined. Similar changes in membrane composition occur in the cold-hardy insect, Epiblema scudderiana, as it prepares to overwinter (Joanisse & Storey 1996). These reports suggest a link between desaturation

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of membrane and diapause associated with overwintering, however, no previous study has directly linked diapause with membrane restructuring in pre-diapausing larvae.

The purpose of this study was to investigate physiological differences in non- diapause-destined vs. diapause-destined larvae of S. crassipalpis. We examined cold tolerance at both organismal and tissue-levels, and measured several biological parameters often associated with diapause induction and increased cold tolerance. Taken together, this study has contributed further insight into the ontogenetic development of cold tolerance related to diapause.

MATERIALS AND METHODS

Insect rearing Diapausing and non-diapausing S. crassipalpis were reared according to the methods outlined by Lee & Denlinger (1985). Non-diapause-destined animals were maintained at 25 ºC under a long-day photoperiod (L/D 15:9 h), while diapaused-destined adults were maintained at 25 ºC under a short-day photoperiod (L/D 12:12 h). Table sugar and water were provided ad libitum. During the first 6 days post-emergence, adults were provided with a source (beef liver) to allow normal oogenesis and embryonic development. On day 11, post-emergence, a ~50 g package of liver was provided for larviposition. Following larviposition, larvae were transferred to fresh packets of liver and placed in plastic tubs lined with a 1-2 inch layer of sawdust, provided for pupariation. For diapause-destined colonies, newly larviposited larvae were transferred to a chamber maintained at 20 ºC under a short-day photoperiod (L/D 12:12 h) for the remainder of development. Third instar feeding larvae were collected on day 3, post-larviposition when they are approximately half the size of full grown, wandering third instar larvae and removed from the food source. Feeding stage larvae were allowed 24 h to evacuate their gut contents before experiments were run. Wandering stage larvae were collected after they left the food source, on day 5-6 post-larviposition.

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Temperature treatment Larvae were placed in 1.5 mL Eppendorf tubes and inserted in large glass tubes submerged in a cold bath. Bath temperature was cooled from 20 ºC to -9 ºC at a rate of 1 ºC/min and held at -9 ºC for 2 h. Following temperature treatment, larvae were removed and either analyzed immediately or stored frozen at -80 ºC.

Organismal cold tolerance The cold tolerance of larvae was assessed by three parameters following cold shock: motility, incidence of pupariation, and adult survival (n = 5 replicates of 10 larvae each). Motility was scored by lightly touching each larva with a blunt probe immediately following cold treatment to observe tactile responsiveness. Then larvae were returned to their respective incubators in order to continue development. Feeding stage larvae were returned to their food source. Incidence of pupariation was scored by observing whether the puparium was formed within 7 days, post-treatment for feeding groups and 5 days, post-treatment for wandering larvae. Adult survival for non-diapause-destined pupae was scored by observing the appearance or absence of adult flies by day 20, post-treatment. Because diapausing pupae may remain in the pupal stage for up to 180 days, 25 day-old diapausing pupae were submerged in hexane for 45 min (Denlinger et al. 1980) in order to terminate diapause. For those individuals that did not emerge, adult survival was scored 7 days later by removing the anterior portion of the puparium to screen for pharate adult development.

Tissue dissection and assessment of cell viability Immediately following each temperature treatment, midgut, fat bodies, and Malpighian tubules were dissected with the aid of a dissecting microscope and placed in 50 µL Coast’s solution (Coast 1988). Following dissection, cell viability of each tissue was assessed using the LIVE/DEAD sperm viability kit (Molecular Probes, Eugene, OR) as modified by Yi and Lee (2003). A recovery group was also included by administering the temperature treatment and returning the treated larvae to their appropriate incubator for 24 h before dissection. Live cells with intact cell membranes fluoresce green/yellow- green, while damaged/dead cells fluoresce red/red-orange. Cells were visualized using a

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fluorescent microscope at 100X total magnification. Each mean value is based on counts of 100 cells in triplicate for each tissue from four larvae. Means were compared using ANOVA Bonferroni-Dunn test (Statview from SAS Institute) with statistical significance set at P < 0.05.

Hemolymph osmolality and body water content Hemolymph osmolality (n = 12) was determined by drawing 8-10 µL of hemolymph into a capillary tube through a small incision in the larval cuticle. Hemolymph was analyzed in a Wescor Vapro 5520 Hemolymph Osmometer (Logan, Utah). Body water content and dry mass were measured by weighing (±0.1 mg) larvae (n = 10) before and after drying at 65 ºC until reaching a constant mass.

Cryoprotectant determinations Larvae were homogenized in perchloric acid and neutralized with an equal

volume of potassium bicarbonate (KHCO3) prior to determining glycerol and sorbitol content as described by Kelty & Lee (1999). Glycerol concentration was measured using the enzymatic assay (Sigma Chemical Co. #337-40A) described in Hølmstrup et al. (1999). For each standard or sample, 200 µL was added to 800 µL of GPO-Trinder Reagent A (Sigma) and allowed to incubate at room temperature for 15 min. Glycerol levels were determined spectrophotometrically by measuring sample absorbance (λ = 540 nm). Sorbitol content was determined using an enzymatic assay described in Bergmeyer et al. (1974). For each standard or sample, 480 µL was added to 950 µL sodium pyrophosphate buffer (0.1 M) and 50 µL of 30 mM nicotinamide-adenine dinucleotide (NAD) and the absorbance (λ = 340) was read. Then, 23 µL of sorbitol dehydrogenase (5 mg protein/mL) was added to standard and sample cuvettes and allowed to incubate at room temperature for 60 min. A second absorbance reading was taken following incubation and sorbitol levels were calculated by determining the difference in absorbance values (A2 – A1 = ΔA). Glycerol and sorbitol concentrations are expressed as µg / mg dry mass.

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Trehalose content was determined using an enzymatic assay as described in Chen et al. (2002). Larvae were homogenized in 0.25 mL of 0.25 M Na2CO3 and incubated in a water bath at 95 ºC for 2 h. Following incubation, 0.15 mL of 1 M acetic acid and 0.6 mL of 0.2 M sodium acetate was added to the homogenate and centrifuged at 16,000 X g for 10 min. The resulting supernatant was transferred to a clean centrifuge tube and re- centrifuged for 5 min. Ten µL of trehalase (0.10 IU/mL) was added to 200 µL of supernatant and incubated overnight at 37 ºC. Trehalose levels were determined spectrophotometrically by measuring sample absorbance (λ = 450 nm) and expressed as µg / mg dry mass.

Statistical analysis – osmolality & cryoprotectants A one-way ANOVA followed by a Bonferroni multiple comparisons test was used to determine differences between non-diapause-destined and diapause-destined larvae exposed to cold shock. Determination of significance for all analyses was set at P < 0.05 and all data are represented as mean ± SEM.

Extraction, isolation, and derivatization of fatty acid methyl esterases (FAMEs) Extraction, isolation, and derivatization of fatty acids were performed as described in Bligh and Dyer (1959). Larvae were homogenized in 1.9 mL of 1:2:0.8

chloroform:methanol:water mixture. Then 0.5 mL of dH2O and 1 mL of chloroform were added to the homogenate before it was filtered through a filter funnel with no. 2 Whatman filter paper under vacuum and captured in a glass test tube. Filtrates were allowed to settle for 15 min to extract the bottom, organic layer into a new test tube. Under a gentle nitrogen stream, the organic layer was blown down to dryness, (thin, slightly yellow residue ~ 4-8 mg) capped under nitrogen, and stored at -80 ºC. To isolate phospholipids, residues were reconstituted in 1 mL chloroform and poured into a chloroform-conditioned silicic acid powder separation column. Ten mL chloroform was poured through the column, followed by 15 mL of 1:9 methanol:acetone mixture. For elution of FAMEs, 10 mL of methanol was poured through the column and captured into a test tube. Under a gentle nitrogen stream, the samples were allowed to evaporate until dry, resulting in a thin, clear residue (< 1 mg). For fatty acid methyl esterase

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derivitization, residue samples were resuspended in 0.5 mL 1:1 methanol:toluene. Then, 0.5 mL of 0.2 N KOH in methanol was added and samples were incubated at 40 ºC for 30 min. Two mL of chloroform and 2 mL of dH2O were added and the mixture was allowed to settle for 15 min, separating into two phases. The bottom, organic layer was extracted and evaporated to dryness under a nitrogen stream resulting in a thin, clear layer (< 1 mg). The samples were then resuspended in 1 mL hexane and placed into gas chromatograph autosample vials under nitrogen and held at -20 ºC until FAME analysis.

FAME analysis: Gas chromatography – mass spectrometry Samples (n = 7) containing the FAMEs were injected into a Finnigan Trace GC- MS with a flame-ionization detector and subjected to chromatographic analysis by mass spectrometry. Samples were run through a Restek 30 m fused silica column (I.D. 25 mm, 95% dimethyl siloxane, 5% diphenyl) with helium gas as a carrier (50 ml/min). The identity of FAMEs in the samples was determined using retention times of standards.

Statistical analysis – lipids Identification of each fatty acid by gas chromatography-mass spectrometry was determined by comparing the spectrum of each peak with multiple chemical libraries (Xcalibur software, Thermo). Peaks with an SI value greater than 800 were identified with a reasonable degree of confidence. For each fatty acid detected, peak areas were computed by factoring in peak height and width with the equation A = 0.5*w*h. Areas were then converted to mean percentages of total content (mean relative proportions), arcsin transformed, and analyzed by a one-way ANOVA (P < 0.05). The index of unsaturation (UFA/SFA) was calculated for each sample and lipid class as the cumulative percentage of all unsaturated fatty acids (UFA) divided by cumulative percentage of all saturated fatty acids (SFA). Significance was determined by an unpaired t-test (P < 0.05).

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RESULTS

Greater cold tolerance of diapause-destined larvae Cold tolerance was assessed by three parameters following cold shock: motility, incidence of pupariation, and adult survival. Larvae reared in diapause-inducing conditions (20 ºC, L/D 12:12 h) were more cold tolerant (Fig. 1A, B) than larvae from the non-diapause-destined population (25 ºC, L/D 15:9 h). Feeding larvae of both non- diapause-destined and diapause-destined individuals were markedly less cold tolerant than wandering larvae for each parameter (Fig. 1A, B). Diapause-destined feeding larvae displayed greater motility and a higher rate of pupariation compared to non-diapause- destined individuals of a similar developmental stage (Fig. 1A). Only 12% of non- diapause-destined larvae were motile following cold shock, as compared to 28% of feeding diapause-destined larvae (ANOVA, unpaired t-tests, P < 0.05). After cold shock treatment and motility assessment, larvae were returned to the food source to permit continued development. Cold-shocked, non-diapause-destined feeding larvae were much less likely (8%) to continue to feed and proceed to pupariation than diapause-destined larvae (30%). Of those individuals that pupariated, none of the resulting non-diapause- destined, and only 13% of diapause-destined pupae emerged as adults. Diapause-destined, wandering larvae were more cold tolerant than non-diapause- destined wandering larvae for the motility and adult survival assessments (Fig. 1B). Immediately following cold shock, significantly more (82%) diapause-destined wandering larvae scored positive for motility, compared to only 28% of non-diapause- destined wandering larvae. Despite the difference in motility between non-diapause- destined and diapause-destined individuals, incidence of pupariation did not differ significantly between groups. Adult survival mirrored the larval motility rates, where 62% of diapause-destined larvae progressed to the pharate adult or adult stage as compared to only 10% of non-diapause-destined larvae. Thus, the lethal effects of cold shock in non-diapause-destined larvae were not expressed until two life stages following the stress.

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Greater cold tolerance of tissues from diapause-destined larvae The greater cold tolerance observed at the organismal level in diapause-destined larvae was confirmed at the tissue level. Tissues from diapause-destined larvae were more cold hardy than those from non-diapause-destined individuals. Malpighian tubules, fat bodies, and midgut were dissected from untreated control and cold shocked (-9 ºC, 2 h) larvae from both feeding and wandering stages of non-diapause-destined and diapause- destined groups before cell viability was assessed using fluorescent vital dyes (Table 1, Fig. 2). Between feeding populations there were no significant (P < 0.05) differences in any tissues between cold shocked non-diapause-destined and diapause-destined larvae (Table 1A). However, in wandering groups, specific tissues from diapause-destined larvae were significantly more cold tolerant than tissues from non-diapause-destined individuals (Table 1, Fig. 2). Figure 2 displays greater cell mortality in wandering non- diapause-destined larvae as a result of cold shock. Cold-shocked, non-diapause-destined larvae (D-F) show higher counts of dead (red) cells than do cold-shocked, diapause- destined larvae (J-L). The cell viability of Malpighian tubules and fat bodies of cold- shocked diapause-destined wandering larvae were significantly greater (91.3%, 89.9%) than in non-diapause-destined groups (63.5%, 75.3%), while no statistical difference was detected in the midgut. The cold-induced damage sustained by the Malpighian tubules and fat bodies in non-diapause-destined individuals suggest substantial damage to cell membranes, as nuclei are stained red, red/orange by propidium iodide. To determine whether cellular repair occurred following cold-induced injury, cold-treated larvae were returned to their respective rearing conditions immediately following cold shock before dissection and vital dye assessment, 24 h later. In all groups examined, increased cell mortality was observed, thus also confirming the initial viability assessment.

Body water content, hemolymph osmolality, and cryoprotectants Body water content did not differ between non-diapause-destined and diapause- destined groups for either feeding or wandering stages (Fig. 3A). In non-diapause- destined larvae, hemolymph osmolality decreased significantly (P < 0.05) from 565 ± 15

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mOsm·kg-1 in feeding groups, to 407 ± 14 mOsm·kg-1 in wandering individuals. Similarly, for diapause-destined larvae hemolymph osmolalities decreased significantly (P < 0.05) from 641 ± 23 mOsm·kg-1 (feeding) to 512 ± 11 mOsm·kg-1 (wandering). In both larval stages, hemolymph osmolality was significantly (P < 0.05) higher in diapause-destined groups compared to non-diapause-destined larvae (Fig. 3B). Accumulation of low molecular mass cryoprotectants is commonly employed by insects to improve their cold tolerance. Whole body glycerol levels decreased significantly (P < 0.05) from feeding to wandering stages for both non-diapause-destined and diapause-destined larvae (Fig. 3C). The mean glycerol concentration for feeding non-diapause-destined larvae was 3.64 µg·mg dry mass-1 as compared to only 1.35 µg·mg dry mass-1 in wandering individuals. Similarly, glycerol concentrations in diapause- destined groups declined from 5.48 (feeding) to 1.26 (wandering) µg·mg dry mass-1. In addition to glycerol, we tested for the presence of two other possible cryoprotectants. Sorbitol and trehalose were measured but were found only in trace amounts (< 0.7 µg/mg) and did not differ significantly (P > 0.05) for any of the experimental groups. These data suggest that cryoprotectant accumulation is not responsible for the enhanced cold tolerance of diapause-destined larvae.

Cell membrane phospholipid composition and membrane saturation Ten fatty acids comprised the majority of the membrane phospholipids found in whole-body FAME isolations of wandering larvae from non-diapause-destined and diapause-destined populations. Other fatty acids found in trace amounts (< 0.5%) were excluded from compositional analysis. The predominate saturated fatty acids, stearic acid (18:0), margaric acid (17:0), and palmitic acid (16:0) comprised 58.1% of the total fatty acid composition in diapause-destined larvae as compared to only 46.1% of the non- diapause-destined larvae (Table 2, Fig. 4). Diapause-destined larvae displayed significantly higher concentrations of stearic acid (18:0) and margaric acid (17:0). However, diapause-destined larvae showed decreased levels (16.9%) of palmitic acid (16:0) as compared to non-diapause-destined individuals (23.8%). The dominant unsaturated fatty acid, oleic acid [18:1(n-9)] accounted for 36.1% of the total phospholipid fatty acid proportion in non-diapause-destined groups as

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compared to only 18.4% in diapause-destined individuals. Other unsaturated fatty acids, 18:1(m), 16:1(n-9), 18:2(n-7), and 20:3(n-7) did not differ significantly between non- diapause-destined and diapause-destined groups. These results were also analyzed by considering the structural class of the specific fatty acids. Saturates composed 48.1% of non-diapause-destined larvae compared to 64.0% of diapause-destined larvae for total phospholipids; monoenes, 42.2% to 23.8%; and polyunsaturates, 9.7% to 12.2%. These data indicate a significant (P < 0.05) increase of saturates and decrease of monoenes in diapause-destined larvae. An index of unsaturation was calculated to summarize the overall difference in saturation among non-diapause-destined and diapause-destined larvae. The UFA/SFA ratio, which was determined by calculating the cumulative percent of all unsaturated fatty acids including both monoenes and polyunsaturates, divided by the cumulative percent of all saturated fatty acid species, was significantly (P < 0.05) lower (0.566) in the diapause-destined larvae as compared to non-diapause-destined groups (1.084) (Table 2). This difference in UFA/SFA reflects the increase in saturated fatty acid species and relative decrease in monoenes found in diapause-destined individuals.

DISCUSSION

Greater cold tolerance of diapause-destined larvae Entry into pupal diapause greatly enhances cold tolerance of the freeze-intolerant flesh fly, Sarcophaga crassipalpis (Lee & Denlinger 1985, Lee et al. 1987, Kelty & Lee 2000). While studies have focused on the cold tolerance of overwintering pupae, little is known about the pre-pupal ontogenetic acquisition of cold hardiness. Adedokun and Denlinger (1984) provided the first evidence of greater cold tolerance in larvae, observing that more wandering diapause-destined larvae pupariated after cold shock (-10 ºC) than non-diapause-destined larvae. Our study examines the mechanisms responsible for increased cold hardiness in diapause-destined larvae prior to entering into pupal diapause. Feeding and wandering stages of non-diapause-destined and diapause-destined larvae were examined to determine the ontogeny of cold tolerance associated with diapause induction. Interestingly, feeding diapause-destined larvae showed enhanced

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cold tolerance in both motility and rate of pupariation measures (Fig. 1A), suggesting the diapause program (12:12 L/D, 20 ºC) initiates physiological adjustments to increase cold tolerance as early as day 3 post-larviposition. Although significant differences were detected, cold tolerance in feeding larvae of both non-diapause-destined and diapause- destined groups was dramatically lower than in wandering larvae for each survival parameter (Fig. 1). Diapause-destined wandering larvae, as in the feeding stage, showed greater motility immediately following cold shock than non-diapause-destined larvae. However, the rate of pupariation did not differ significantly between wandering non-diapause- destined and diapause-destined groups. The immediate cold-induced injury, as measured by the motility assessment, was expressed later in development as evidenced by the significantly greater rate of adult survival in diapause-destined groups (Fig. 1B). These results are similar to those reported by Turnock et al. (1983 & 1985), where early stage diapausing individuals of Mamestra configurata and Delia radicum were exposed to sub zero temperatures. The damage induced by cold shock in M. configurata and to a lesser degree in D. radicum, was revealed only following adult emergence, as the proportion of malformed adults was significantly greater than of non-stressed controls.

Greater cold tolerance of tissues from diapause-destined larvae The greater cold tolerance of diapause-destined larvae was mirrored at the tissue level. Of the three tissues examined, Malpighian tubules were the most susceptible to cold shock injury in all groups (Table 1, Fig. 2). Feeding larvae of both non-diapause- destined and diapause-destined groups exhibited high cell mortality, matching the relatively poor rates of organismal survival (Fig. 1A). However, in cold-shocked wandering groups, larvae programmed for diapause maintained cell viability near control values (> 90%) for each tissue. For example, Malpighian tubules of cold-shocked, non- diapause-destined wandering larvae exhibited 36.5% cell mortality, as opposed to only 8.7% in diapause-destined individuals. Viability of fat body was also greater in diapause- destined larvae (10.1% mortality) in comparison to non-diapause-destined (24.7%) groups. In contrast, gut tissues from all groups displayed exceptional cold tolerance (> 89% survival), as previously reported in adult S. crassipalpis (Yi and Lee, 2004).

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While Malpighian tubules were the least cold tolerant in this study, it is likely that nervous tissue is the most cold-shock susceptible and the most likely to lead to organismal death. The poor performance of cold-shocked, non-diapause-destined larvae on the motility test (Fig. 1) may suggest significant impairment of neuromuscular function. Of the tissues examined in the freeze-tolerant gall fly, Eurosta solidaginis, the brain was the most susceptible tissue to cold-induced injury (Yi and Lee, 2003). Moreover, in S. crassipalpis adults, Kelty et al. (1996) demonstrated that cold shock decreases the conduction velocity of motor neurons and neuromuscular transmission. Whether diapause induction has the capacity to enhance the cold tolerance of nerve tissue is unknown.

Role of cryoprotectants A common strategy insects employ for winter survival is the accumulation of small molecular weight cryoprotectants. E. solidaginis larvae increase the synthesis of glycerol, sorbitol, and to a lesser extent, trehalose as a reaction to decreasing temperature in the autumn, allowing it to survive temperatures as low at -40 ºC (Baust & Lee 1982). The apple leaf miner, Phyllonorcter ringoniella, accumulates trehalose at the onset of diapause regardless of cold (Li et al. 2002). Likewise, Izumi et al (2005) demonstrated that diapausing pupae of the cotton boll worm, Helicoverpa armigera, contain higher concentrations of trehalose and glucose than non-diapausing pupae. As a measure of total solute concentration, hemolymph osmolalities were examined between non-diapause-destined and diapause-destined groups. In both non- diapause-destined and diapause-destined larvae there is a significant reduction in hemolymph osmolality from feeding to wandering stages, likely reflecting the constant intake of high dietary solutes. Interestingly, hemolymph osmolalities of diapause- destined larvae of both feeding and wandering stages were significantly higher than non- diapause-destined groups (Fig. 3B), suggesting the accumulation of possible cryoprotectants in the more cold tolerant, diapause-destined larvae. The synthesis and accumulation of glycerol is of particular importance to overwintering insects (Zachariassen 1985). Several studies (Lee et al. 1987, Izumi et al. 2005) have demonstrated a solid link between the accumulation of glycerol during

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overwintering and increased cold tolerance. However, Lee et al. (1987) reported that in S. crassipalpis, the less cold tolerant, non-diapause-destined larvae had substantially higher glycerol levels than diapause-destined larvae, and not until post-pupariation did the diapause-destined groups exhibit an extreme increase in glycerol. Glycerol was the only low molecular weight cryoprotectant detected in relatively high concentrations (Fig. 3C). Whole-body glycerol levels reflected osmolality values as concentrations from feeding groups were much higher than in wandering larvae for both non-diapause-destined and diapause-destined larvae. However, there were no significant differences between glycerol concentrations of non-diapause-destined and diapause- destined groups as suggested by the hemolymph osmolality values. Thus, the greater cold tolerance exhibited by diapause-destined larvae cannot be attributed to glycerol accumulation.

Phospholipid fatty acid composition of cell membranes and membrane fluidity Ten fatty acids comprised the majority of the phospholipids found in S. crassipalpis wandering larvae. In diapause-destined groups, the proportion of the major saturated fatty acids (stearic, margaric, and palmitic acids) were greater than in non- diapause-destined groups, and the dominant unsaturated fatty acid (oleic acid) was nearly doubled in non-diapause-destined individuals (Table 2, Fig. 4). Analysis of species composition showed the unsaturation index (UFA/SFA) of non-diapause-destined wandering larvae was greater than in the more cold tolerant diapause-destined group, rebutting our hypothesis that the increase in cold tolerance of diapause-destined larvae was due to increased membrane fluidity by the insects’ desaturating of cell membrane components in response to the diapause program. One prevailing hypothesis why membrane fatty acid restructuring is not solidly linked to improved cold tolerance is that it may be possible that membranes are sufficiently fluid, so that a decrease in overall saturatedness is simply not required to prevent harmful gel phase transitions that result from exposure to significant cold shock. As in our study, Ohtsu et al. (1998, 1999) found in seven Drosophila species a correlation between increased cold hardiness and decreased unsaturation index. Similarly, the diapausing, adult fire bug, Pyrrhocoris apterus, has a greater proportion of saturated

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fatty acid species and yet is more cold tolerant than non-diapausing groups with a higher unsaturation index (Hodková & Hodek, 1997). While there are many examples linking cold hardening and diapause-induction to the seemingly protective response of de-saturating cell membranes, it appears that fatty acid re-organization is not the only mode of maintaining adequate membrane fluidity. One area deserving attention is the restructuring of membrane phospholipid headgroups to favor phosphatidylethanolamines (PE) over phosphatidylcholines (PC) (Hazel 1995, McCoy 2003). A membrane rich in PE headgroups is more fluid as they carry less molecular bulk and have a lower capacity for hydrogen bonding to themselves than PC headgroups (Silvius et al. 1986). Conversely, cell membranes containing a greater proportion of bulkier PC headgroups are less fluid, as the gel transition temperatures for PCs are generally 20ºC higher than those of PE headgroups (Hazel 1995). Also, the proportion of membrane cholesterol influences membrane fluidity. Membranes rich in cholesterol are more stable and less likely to undergo damaging lipid phase transitions in response to lowering temperatures (Drobnis et al. 1993). This effectively means the PE/PC ratio and cholesterol content may influence a membrane’s overall fluidity as much or more than acyl chain structure.

In this study we provide further insight into the ontogeny of cold tolerance in pre- diapausing larvae of S. crassipalpis. We also reinforce the fact that the acquisition of cold tolerance associated with diapause is not the result of a singular physiological change, but likely involves a combination of cellular and molecular adjustments, of which many remain unknown.

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ACKNOWLEDGEMENTS

I wish to thank my committee, Richard E. Lee Jr., Jon P. Costanzo, and Phyllis Callahan for both their guidance and patience throughout my graduate career. Special thanks to collaborators David L. Denlinger and M. Robert Michaud of Ohio State University for their assistance in processing lipid samples by GC-MS. I would also like to thank all my friends and family for their constant support. Support for this project was provided by the National Science Foundation # IOB-0416720.

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Table 1. Cell viability of tissues (in vivo) of non-diapause-destined and diapause- destined (A) feeding and (B) wandering larvae. Untreated groups were maintained at their rearing conditions (25 ºC for non-diapause-destined, 20 ºC for diapause-destined). Cold shock groups were cooled 1 ºC/min and held at -9 ºC for 2 h. Each value is a mean based on four counts of 100 cells done in triplicate. Within a tissue, means marked with different letters represent statistical significance (P < 0.05, ANOVA, Bonferroni/Dunn).

A – Feeding larvae Cell viability (% mean ± SEM)

Tissue type Untreated Cold shock Non-diapause Diapause-destined Non-diapause Diapause-destined

Malpighian tubule 88.8 ± 2.7a 84.6 ± 2.8a 53.9 ± 9.2b 67.3 ± 4.5b Fat body 85.6 ± 3.1a 86.9 ± 4.0a 69.0 ± 8.4b 71.9 ± 9.4b Midgut 97.5 ± 4.6a 97.5 ± 4.1a 89.0 ± 8.1a 95.9 ± 5.0a

B – Wandering larvae

Tissue type Untreated Cold shock Non-diapause Diapause-destined Non-diapause Diapause-destined

Malpighian tubule 93.7 ± 2.4a 93.0 ± 2.3a 63.5 ± 5.7b 91.3 ± 2.5a Fat body 93.6 ± 0.8a 94.3 ± 0.9a 75.3 ± 4.5b 89.9 ± 2.1a Midgut 99.1 ± 1.5a 99.0 ± 1.5a 95.8 ± 3.2a 95.8 ± 6.2a

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Table 2. Composition of phospholipid fatty acids (PLFAs) in third instar larvae from non-diapause and diapause-destined groups (n = 7).

Non-diapause-destined Diapause-destined Lipid species 16:0 23.76 ± 2.5 16.91 ± 2.5 17:0 11.24 ± 2.9 16.73 ± 2.8*

18:0a 1.26 ± 1.5 4.21 ± 2.6*

18:0b 0.73 ± 0.9 1.70 ± 1.2

18:0c 11.09 ± 2.5 24.46 ± 2.2* 16:1(n-9) 4.25 ± 1.8 2.81 ± 0.9 18:1(n-9) 36.12 ± 2.3 18.44 ± 1.0* 18:1(m) 1.82 ± 1.5 2.59 ± 1.3 18:2(n-7) 5.32 ± 1.3 6.42 ± 0.9 20:3(n-7) 4.41 ± 2.2 5.75 ± 1.0

Class of fatty acid Saturates 48.48 ± 2.0 64.00 ± 1.2* Monoenes 41.64 ± 2.3 23.84 ± 1.1* Polyunsaturates 9.88 ± 1.5 12.16 ± 0.9

Index of unsaturation UFA/SFA 1.08 ± 0.09 0.57 ± 0.03*

PFLAs are presented as percentages of total fatty acid composition. All values are mean ± SEM. * denotes significant (data arcsin transformed, ANOVA, P < 0.05) differences between non-diapause- destined and diapause-destined groups.

UFA/SFA is the sum of the percentage of unsaturated fatty acids/sum percentage of saturated fatty acids.

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FIGURE LEGENDS

Figure 1. Effect of diapause induction on cold tolerance of (A) feeding and (B) wandering larvae. Motility was scored immediately following cold shock (-9 ºC, 2 h), incidence of pupariation was scored by day 5 (wandering) or day 7 (feeding), and adult survival was scored by day 20 (non-diapause) or day 25 (diapause-destined), post- treatment. Values are mean ± SEM based on 5 replicates of 10 individuals. * denotes a significant (P < 0.05) difference between non-diapause-destined and diapause-destined groups.

Figure 2. Effects of cold shock (-9 ºC, 2 h) on cell viability of isolated tissues of non- diapause-destined (A-F) vs. diapause-destined (G-L) wandering third instar larvae. Green-dyed nuclei represent live cells; red/red-orange-dyed nuclei represent dead cells.

Figure 3. (A) Body water content, (B) hemolymph osmolality, and (C) glycerol concentration of non-diapause-destined and diapause-destined larvae at feeding and wandering-stages. Data represent mean ± SEM (n = 12). * denotes a significant (ANOVA, P < 0.05) difference between non-diapause-destined and diapause-destined groups.

Figure 4. Fatty acid changes due to diapause induction in third instar larvae. Phospholipid fatty acids were isolated from whole-body larvae of non-diapause-destined and diapause-destined groups and analyzed by gas chromatography-mass spectrometry. Data represent means ± SEM. (n = 7) of the proportion of a given fatty acid species. Fatty acid species are designated by the form X:Y(n-Z), where X represents the fatty acyl chain length; Y represents the number of double bonds; and n-Z represents the location of the first double bond from the methyl end of the acyl chain or the presence of a methyl group (m). * denotes significant (ANOVA, P < 0.05) differences between non-diapause- destined and diapause-destined groups for a given fatty acid species.

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Fig. 1

100 A FeedingFeeding larvaelarvae

80 non-diapause-destinednon-diapause-destined diapause-destineddiapause-destined

60

40 * *

20

(0%)(0%) 0

Wandering larvae Wandering larvae Response (%) 100 * B

80 *

Incidence of

60

% Positive

40

20

0 motilitymotility pupariation pupariation adult emergence survival

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Fig. 2

Malpighian tubules Fat bodies Midgut

Non-diapause-destined Untreated

Non-diapause-destined Cold shock

Diapause-destined Untreated

Diapause-destined Cold shock

50µm

25

Fig. 3

3.0 A non-diapausenon-diapause-destined diapause-destineddiapause-destined ) -1 2.5

2.0

O · mg dry · mass O mg 2 1.5 Mean Body Water Content Body Mean H (mg

1.0

700 B *

600

) -1 *

500

· kg (mOsm 400

Osmolality Hemolymph Mean

300 6 C

5

) -1 4

3

2

(µg dry · mass mg 1

Mean GlycerolMean Concentration

0 feeding feeding wanderingwandering

26

Fig. 4

50 non-diapausenon-diapause-destined diapause-destineddiapause-destined 40

30 *

* 20 * * * % Fatty Acid Signal 10

0 ) 0a 0b 0c 0 0 m 9) 9) 7) 7) 17: 16: 1( n- n- n- n- 18: 18: 18: :1( :1( :2( :3( 18: 18 16 18 20

FattyFatty Acid Acid

27