Goldenrod Gall Research Articles

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)

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall ( Solidago sp.). In late September, when the water content of the galls was high (~55%), more than half of the larvae froze within their galls when held at –2.5 °C for 24 h, and nearly all larvae froze at –4 or –6 °C. At this time, most larvae survived freezing at ≥ –4 °C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at –4 and –6 °C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures ≥ –6 °C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as –4 °C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization ( T c) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.

Cold winter microenvironments conserve energy and improve overwintering survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis

We studied the influence of two overwintering microenvironments on survival and potential fecundity of goldenrod gall flies, Eurosta solidaginis (Fitch) (Diptera, Tephritidae). These freeze‐tolerant larvae overwinter above the snow on standing goldenrod stems (elevated) or below the snow on broken stems (ground‐level). When covered by snow, the ground‐level larvae were well insulated and thus protected from the lowest temperatures of the winter, but, because they were warmer, they consumed more energy than their elevated counterparts. The ground‐level group also experienced greater warming from the soil during sunny spring days, and their galls were less prone to drying than their elevated counterparts. By winter's end the ground‐level larvae exhibited significantly lower rates of emergence (83.5% vs 93.0%) and reduced potential fecundity (274±11 eggs/female vs 336±17 eggs/female). Models of seasonal energy use indicate that these differences were due to higher metabolic rates in the ground‐level microenvironment due to insulation by snow and warming from the soil, which reduced the energy available for morphological development and egg production in the spring. We conclude that colder winter microenvironments can have a strong positive effect on overwintering ectotherms, particularly those that rely on energy stores accumulated during the autumn to produce eggs in spring. The enhanced reproductive output of insects overwintering in colder microenvironments may be a selective force promoting the evolution of increased cold‐hardiness.

Protein kinase A: purification and characterization of the enzyme from two cold-hardy goldenrod gall insects

The catalytic subunit of protein kinase A (PKAc) was purified to apparent homogeneity from two species of cold-hardy goldenrod gall insects, Epiblema scudderiana and Eurosta solidaginis. Final specific activity for both enzymes was ∼74.5 nmol of phosphate transferred per minute per milligram protein. Molecular weights were 41 and 40 kDa for E. scudderiana and E. solidaginis PKAc, respectively. K m values at 24°C for the artificial substrate, Kemptide, were 38.1±4.9 and 3.67±0.11 μM for E. scudderiana and E. solidaginis PKAc, respectively, whereas K m Mg-ATP values were 61.1±6.9 and 30.7±4.1 μM. Assay at 4°C lowered the K m for Kemptide of E. scudderiana PKAc by 55% and addition of 1 M glycerol further lowered the K m. Low assay temperature also enhanced holoenzyme dissociation in both species with the K a value for cyclic 3′5′-monophosphate at 4°C lowered to just 13–18% of the value at 24°C. Low temperature did not affect affinity for Mg-ATP or inhibition by PKA inhibitors (PKAi, H7, H89) but increased inhibition by some salts. PKAc from both species showed a break in the Arrhenius relationship at ∼10°C which suggests a conformational change at low temperature; activation energies ( E a) were 2.2–3 fold higher for the lower (<10°C) versus higher (>10°C) range. Addition of naturally occurring polyols, 1 M glycerol or 0.4 M sorbitol, affected E a in some cases. Temperature dependent regulation of holoenzyme dissociation and PKAc kinetic properties may have an role in regulating the enzymes involved in polyol synthesis in cold-hardy insects.

Energy and water conservation in frozen vs. supercooled larvae of the goldenrod gall fly, Eurosta solidaginis (fitch) (Diptera: Tephritidae)

Insects that tolerate severe cold during winter may either supercool or tolerate ice forming within the tissues of the body. To compare the relative advantages of freezing and supercooling, we measured rates of CO(2) production and water loss in frozen and supercooled goldenrod gall fly larvae (Eurosta solidaginis). As an important first step, we measured the time required for ice content and metabolic rate to stabilize upon freezing. Ice content stabilized after only three hours of freezing at -5 degrees C, whereas CO(2) production required 12 hours to stabilize. Subsequent experiments found that freezing greatly reduced both water loss and metabolic rate. Comparisons of supercooled and frozen larvae at -5 degrees C indicated that CO(2) production fell 47% with freezing and water loss decreased 35%. As temperature decreased to -10 and -15 degrees C, CO(2) production fell exponentially and was no longer detectable at -20 degrees C with our measurement system. Our results demonstrate that freezing significantly reduces energy consumption during the winter and may therefore improve winter survival and spring fecundity. The advantages of freezing over supercooling would drive selection toward insect freeze tolerance and also toward higher supercooling points to increase the duration of freezing each winter.

Purification and characterization of protein phosphatase-1 from two cold-hardy goldenrod gall insects.

The catalytic subunit of protein phosphatase-1 (PP-1) was purified to homogeneity from final instar larvae (the overwintering stage) of freeze avoiding (Epiblema scudderiana) and freeze tolerant (Eurosta solidaginis) cold-hardy insects. Arrhenius plots showed that activity of PP-1 from both species was strongly suppressed at low temperature. Acidic shifts in pH optima and increased inhibition by okadaic acid were also observed when the enzymes were assayed at 4 degrees C compared with 24 degrees C. The data identify multiple ways by which PP-1 can be inhibited at low temperature and this inhibition appears to be key to sustaining high glycogen phosphorylase activity in support of polyol synthesis at low temperatures.

EsMlp, a Muscle-LIM Protein Gene, Is Up-regulated during Cold Exposure in the Freeze-Avoiding Larvae of Epiblema scudderiana

Screening of a cDNA library identified transcripts that were up-regulated by cold (4 or −20°C) exposure in larvae of the freeze-avoiding goldenrod gall moth, Epiblema scudderiana. One clone contained a full-length open reading frame encoding a protein of 94 amino acids. The gene product, with 79.1% of residues identical with the Drosophila LIM protein Mlp60A, was named EsMlp and contained a single LIM domain and consensus sequences characteristic of a LIM protein. Transcript levels rose approx twofold when larvae were shifted from 4 to −20°C and approx threefold over the midwinter months compared with larvae sampled in October or April. EsMlp expression was high in larval head (possibly due to expression in pharyngeal muscles) and body wall but was not detected in fat body. Immunoblotting revealed a three- to fourfold increase in EsMlp protein in midwinter larvae (January-February) compared with November-collected animals and a further rise to eightfold higher than November values in larvae collected in April. Cold up-regulation of EsMlp and the pattern of EsMlp levels in the larvae suggest possible roles for the protein, such as in muscle maintenance over the winter or as a preparative function that could facilitate the rapid resumption of development and metamorphosis when environmental temperatures rise in the spring.

Regulation of hexokinase in a freeze avoiding insect: role in the winter production of glycerol.

Hexokinase from larvae of the freeze-avoiding goldenrod gall moth, Epiblema scudderiana, was purified 20-fold using chromatography on DE52 Sephadex, phosphocellulose, and blue dextran. Final specific activity was 75.8 U/mg and SDS-PAGE gave a molecular weight of 94,000 for the monomer. Arrhenius plot showed a break at 16 degrees or 12 degrees C in the absence vs. presence of 10% v/v glycerol, indicating a conformational change in the enzyme at lower temperatures but suggesting a stabilizing effect of glycerol. Comparison of hexokinase kinetic properties at 22 degrees and 4 degrees C showed higher affinity for both glucose and ATP (Km values were 45-50% lower), as well as for the cofactor Mg(2+), at the lower temperature. Furthermore, product inhibition by glucose-6-phosphate and ADP was reduced at 4 degrees C. Glucose levels rise in E. scudderiana as an apparent by-product of high rates of glycogenolysis during glycerol synthesis. The temperature-dependent properties of hexokinase would facilitate the recycling of this glucose back into the pathway of glycerol synthesis and could help to achieve the near stoichiometric conversion of glycogen to glycerol that is seen during cold hardening. Arch.

Diapause development in frozen larvae of the goldenrod gall fly, Eurosta solidaginis fitch (diptera: tephritidae).

Seasonal changes in metabolic rate and the potential for morphological development demonstrated that third-instar larvae of the goldenrod gall fly, Eurosta solidaginis Fitch, exhibit a distinct winter diapause. Metabolic rate (CO2 production) was significantly lower from 15 October to 9 February than in early autumn (9 September) and spring (1 March) samples. The induction of diapause coincided with the development of cold-hardening, maximum larval mass, and gall senescence, but our experiments did not identify specific cues triggering diapause induction. We examined the influence of exposure to 0 degrees C and -20 degrees C on diapause development. Diapause development in larvae stored at 0 degrees C occurred at approximately the same rate as in nature. Until 15 December the larvae were in the refractory phase of diapause (incapable of morphological development, even at permissive temperatures), but afterward moved to the activated phase within which diapause intensity decreased until termination in February. Diapause development occurred in larvae collected during the winter and stored at -20 degrees C for periods of 1 week to 3 months. Diapause intensity decreased in frozen larvae through the winter but at a slower rate than in larvae stored at 0 degrees C.

Mild winter temperatures reduce survival and potential fecundity of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae)

We tested the hypothesis that mild winter temperatures are detrimental to the survival and reproductive potential of insects. We measured survival, body size, and potential fecundity of a freeze tolerant insect, the goldenrod gall fly (Eurosta solidaginis), after overwintering in the laboratory for ~3 mo. frozen at −22°C, unfrozen at 0°C, or unfrozen at 12°C. Larvae held at 12°C suffered high mortality (70%) and relatively low potential fecundity as adults (mean±SEM=199±11 eggs/female), while those held at 0°C had both low mortality (11%) and high potential fecundity (256±15 eggs/female). Freezing (−22°C) increased mortality (30% overall) but did not significantly reduce fecundity (245±13 eggs/female). Egg length and width were constant regardless of treatment group or female body size. Analysis of covariance indicated that reduced fecundity in the 12°C group was related to reduced larval body weight following treatment. Patterns of larval weight loss in the experimental treatments were generally correlated with previous reports of latitudinal trends in weight loss through the winter. We conclude that mild winter temperatures may be detrimental to some overwintering insects, particularly species that do not feed following winter diapause. Low temperature and even freezing are beneficial, allowing conservation of energy reserves to maintain high survival and potential fecundity.

Extreme resistance to desiccation in overwintering larvae of the gall fly Eurosta solidaginis (Diptera, tephritidae).

During winter, larvae of the goldenrod gall fly Eurosta solidaginis are exposed for extended periods to severe low ambient temperatures and low humidities within plant galls. The resistance of these larvae to desiccation at various temperatures and humidities, the transition (critical) temperature, and the effects of treatment with organic solvents on the larval rates of water loss and on changes in osmolality during desiccation were examined. The water loss rates of the flesh fly Sarcophaga crassipalpis under desiccating conditions were also measured. The water permeability of the cuticle of E. solidaginis larvae was very low (0.038 microgram h(-1 )cm(-2 )Pa(-1) at 20 C and 4% relative humidity) compared with that of larvae of other species. The value for E. solidaginis is equivalent to that of the very drought-resistant larvae of the tenebrionid beetle Tenebrio molitor (0.038 microgram h(-1)cm(-2)Pa(-1) at 30 C). In contrast, the permeability of larvae of the flesh fly Sarcophaga crassipalpis at 20 C and 4 % relative humidity was 0.331 microgram h(-1) cm(-2)Pa(-1). The thermal dependence of the cuticular permeability increased with temperature by approximately 0.0010 +microgram h(-1) cm(-2)Pa(-1) C(-1) in the interval between 4 and 40 C. At the transition temperature of 40 C, the thermal dependence of the permeability increased abruptly to 0.0400 microgram h(-1)cm(-2)Pa(-1) C(-1). Larvae treated with hexane and acetone remained remarkably resistant to water loss. However, treatment with chloroform:methanol increased the water loss rate approximately 25-fold. During desiccation at 4 C and 4% relative humidity for 21 days, E. solidaginis larvae showed a mass loss of 18.5+/-4.4 % (mean +/- s.e.m., N=6). Animals dried under the same conditions over the same period showed a haemolymph osmolality of 851+/-75 mosmol kg(-1) (N=4). Larvae freshly removed from the galls showed a haemolymph osmolality of 918+/-67 mosmol kg(-1)(N=3). A higher osmolality in the dried compared with the fresh larvae would have been expected. The present observation suggests that important ions in the haemolymph may have been excreted or rendered osmotically inactive during desiccation.

Foraging Patterns of Eastern Gray Squirrels (Sciurus carolinensis) on Goldenrod Gall Insects, a Potentially Important Winter Food Resource

We document the use of goldenrod (Solidago spp.) gall insects by eastern gray squirrels (Sciurus carolinensis) as a winter food resource. We also examined the foraging patterns of gray squirrels on these gall insects by presenting goldenrod gall feeders at ten sites around Hamilton, New York, during the winters of 1996–1997 and 1997–1998. We predicted that squirrels would attack larger-than-average galls among those presented because larger galls are more likely to contain the larger fly larvae (Eurosta solidaginis), rather than the smaller parasitoid (Eurytoma gigantea) or nothing at all. Squirrels attacked galls at least once at 6 of the 10 sites where gall feeders were maintained. Galls at feeders were attacked from 11 February to 17 April, and the number of separate attacks on galls at a particular site ranged from 1 to 17. We detected no evidence that squirrels preferentially attacked galls of a particular size class (small, medium, large). There was no indication that squirrels attacked increasingly larger-than-average galls over the duration of the study, which would be expected if squirrels were learning by positive reinforcement. Our results suggest either (1) that squirrels are unable to discriminate among food rewards in galls of different sizes or (2) that all gall insects, regardless of size, provide an important dietary supplement for squirrels during the winter.

Oxidative stress and antioxidants in stress and recovery of cold-hardy insects

Antioxidants, pro-oxidants, and oxidative stress and damage were studied in larvae of cold-hardy goldenrod gall insects during freezing (−14°C, 24 h), anoxia (under N 2 gas, 24 h), and upon recovery from these stresses. Although oxidized glutathione levels increased during freezing, no oxidative damage was seen upon recovery from freezing and anoxia in freezing tolerant Eurosta solidaginis. Antioxidant enzyme activities were unchanged except for catalase activity which increased gradually during freezing to peak at 147% of control values after 30 min of recovery at 3.5°C. Xanthine oxidase activity was undetected in E. solidaginis. Anoxia and recovery in freeze avoiding Epiblema scudderiana also failed to result in oxidative damage, although a slight increase in the pro-oxidant xanthine oxidase (from 12.5 to 18% of total xanthine dehydrogenase + xanthine oxidase) was observed. No changes in antioxidant enzyme activities were observed in this species either during anoxia or upon aerobic recovery. The ability of these larvae to limit oxidative damage arising during oxygen reperfusion during freezing- or anoxia-exposures is suggested to play an important role in their capacity to survive these stresses.

Seasonal changes in fatty acid composition associated with cold-hardening in third instar larvae of Eurosta solidaginis

Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) survive extended periods in winter during which tissue water is frozen. Both low temperature and reduced water activity during freezing present challenges for the structural integrity of cellular lipids. Fatty acids of both phospholipids and triacylglycerols from fat body cells of E. solidaginis were analyzed throughout fall and early winter, a period that encompasses the acquisition of freeze-tolerance, to determine if adaptations to freezing include changes in fatty acid unsaturation. The five most abundant fatty acids from both fractions were (in decreasing order) oleic (40–65%), palmitoleic (18–20%), palmitic (12–17%), linoleic (5–10%), and stearic acids (4 –7%). This represents a typical complement of Dipteran fatty acids, although oleic acid levels were higher in E. solidaginis than those reported from other Dipterans (˜28%; Downer 1985). From September to November, monounsaturates increased from 59 to 70% in phospholipids at the expense of saturated fatty acids (25% –20%) suggesting activation of a Δ9-desaturase enzyme. These changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (U/S) from 3.0 to 4.2, although there was no change in the average number of double bonds per fatty acid (unsaturation index, UI ≈ 1.2 in phospholipids and 0.9 in triacylglycerols throughout the season). These changes were temporally correlated to decreasing ambient temperatures and increasing larval and fat body cell freeze-tolerance.

Oxidative stress and antioxidants in overwintering larvae of cold-hardy goldenrod gall insects

Antioxidant and pro-oxidant systems were studied in overwintering larvae of two cold-hardy gall insect species, the freeze-tolerant fly Eurosta solidaginis and the freeze-avoiding moth Epiblema scudderiana. An increase in the levels of the oxidized form of glutathione suggested slight oxidative stress in both species during the winter. Freeze-tolerant Eurosta solidaginis larvae generally had decreased activities of antioxidant enzymes in the winter, indicating that these larvae do not face increased challenge from oxidative stress during the numerous freeze-thaw events they experience. Instead, existing defences must be sufficient to prevent any damage. By contrast, increased winter activities of antioxidant enzymes in freeze-avoiding Epiblema scudderiana suggest that these larvae must defend against the formation of reactive oxygen species. This may result from the oxidative nature of winter metabolism in these larvae, as well as a dependence on lipid oxidation as their fuel over this season. Xanthine dehydrogenase activity decreased dramatically in both species during the autumn, reducing the potential for the formation of the pro-oxidant xanthine oxidase. Indeed, xanthine oxidase activity fell to undetectable levels by winter in Epiblema scudderiana and was not detectable at any time in Eurosta solidaginis.

The effects of temperature and season on the O2consumption of third-instar larvae of the goldenrod gall fly (Eurosta solidaginis)

. Third-instar larvae of the goldenrod gall fly (Eurosta solidaginis Fitch) live inside ball galls on goldenrod plants from summer to the following spring.Because galls are highly exposed to the weather, larvae experience substantial variations in body temperature.This study documents the oxygen consumption of gall fly larvae with regard to the effects of ambient temperature, seasonal conditioning, and prior exposure to subzero temperature.The body mass of larvae doubles between the late summer and the autumn; it subsequently undergoes a modest decline by early winter.The O2, consumption of field-acclimatized larvae increases with ambient temperature, especially between 0 and 10°C (Q10= 2.6-3.4).The thermal sensitivity of metabolism declines at higher ambient temperatures, most notably during the autumn/early winter.After exposure to 15°C for 1 week, autumn and early winter larvae maintain much lower rates of O2 consumption than do late summer specimens.Prior exposure to -5°C for 24 h did not influence the O2 consumption of larvae.Low thermal sensitivity of O2 consumption, especially at higher ambient temperatures, is an energy-sparing mechanism during seasonal inactivity.Indeed, the persistence of this metabolic pattern in larvae exposed to 15°C suggests that they have entered a state of diapause.

Cryobiology of the freeze-tolerant gall fly Eurosta solidaginis: overwintering energetics and heat shock proteins

CR Climate Research Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials CR 05:61-67 (1995) - DOI: https://doi.org/10.3354/cr005061 Cryobiology of the freeze-tolerant gall fly Eurosta solidaginis: overwintering energetics and heat shock proteins Lee RE Jr, Dommel RA, Joplin KH, Denlinger DL ABSTRACT: The goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) ranges from the southern U.S. northward into Canada. The larva overwinters within a ball gall on the stem of goldenrod Solidago spp. The galls often extend above the snowpack, exposing the larva to a wide range of environmental extremes in winter. This species has received extensive attention as a freeze-tolerant insect model. A seasonal study of the overwintering bioenergetics of an Ohio, USA, population revealed marked decreases in body weight, lipid and total caloric content in October and November when environmental temperatures were the highest. Overwintering larvae produce heat shock proteins in response to high-temperature exposure. However, unlike other insects E. solidaginis does not appear to synthesize heat shock proteins in response to low-temperature exposure. KEY WORDS: Cold-hardiness · Insect · Low temperature adaptation . Respiration · Stress proteins Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in CR Vol. 05, No. 1. Publication date: February 23, 1995 Print ISSN: 0936-577X; Online ISSN: 1616-1572 Copyright © 1995 Inter-Research.

Temperature acclimation and seasonal responses by enzymes in cold‐hardy gall insects

AbstractChanges in the activity of over 20 enzymes of intermediary metabolism in 15°C or −4°C acclimated goldenrod gall moth (Epiblema scudderiana) and gall fly (Eurosta solidaginis) larvae were measured. Increased activities of glyco‐genolytic and hexose monophosphate shunt enzymes in cold‐acclimated Epiblema scudderiana suggest a role for coarse control in the conversion of glycogen reserves into glycerol cryoprotectant synthesis. In Eurosta solidaginis, high glycogen phosphorylase activity with decreased activities of glycolytic enzymes may account in part for the temperature‐dependent switch from glycerol to sorbitol synthesis in these larvae upon cold acclimation. Isoelectric focusing analyses of five enzymes in overwintering Epiblema scudderiana revealed transient mid‐winter changes in the isoelectric points of phosphofructokinase and pyruvate kinase, suggesting seasonal changes in the phosphorylation state of these enzymes. A distinct developmental pattern of aldolase isozymes suggests a role for a new isozyme during overwintering or upon spring emergence. Regulation of metabolism by changes in enzyme activities is indicated for both larvae. © 1995 Wiley‐Liss, Inc.

Comparative ultrastructure of fat body cells of freeze-susceptible and freeze-tolerant Eurosta solidaginis larvae after chemical fixation and high pressure freezing

In response to low environmental temperatures third instar larvae of the goldenrod gall fly, Eurosta solidaginis, undergo the process of cold-hardening in which they acquire freeze-tolerance. Cold-hardening includes the elevation of the supercooling point, the temperature at which body water spontaneously freezes, and the accumulation of the low-molecular-mass cryoprotectants, glycerol, sorbitol and trehalose. Although it is generally believed that freezing survival is only possible if the ice lattice is restricted to the extracellular space, the larval fat body cells survive intracellular ice formation. Fat body cells of freeze-susceptible (September-collected) and freeze-tolerant (January-collected) third instar larvae were prepared for transmission electron microscopic analysis using both conventional chemical fixation and high pressure freezing with freeze substitution. High pressure cryofixation procedures were superior to conventional fixation in both freeze-susceptible and freeze-tolerant cells. Endogenous cryoprotectants in freeze-tolerant larvae contributed to the superior ultrastructure of cytoplasmic organelles and nucleoplasm in high-pressure frozen fat body cells, while freeze-susceptible larvae, which lack high levels of cryoprotectants, contained fat body cells which sustained ice crystal damage following cryofixation. Mitochondria in fat body cells prepared using high pressure freezing/freeze substitution were characterized by smooth outer membranes and uniformly electron dense mitochondrial matrix. Conventionally prepared samples exhibited crenated cytoplasmic organelles with undulating membrane profiles; smooth profiles characterized high-pressure frozen structures. Lipid droplets were abundant in all fat body cells, however glycogen, the energy source of cryoprotectants, was variable in both freeze-susceptible and freeze-tolerant cells. Coalescence of lipid droplets, induced by intracellular freezing at progressively lower temperatures, caused displacement of interior cytoplasmic organelles including the nucleus to the cell cortex. This shift in cytoplasm had no affect on survival of the cells.

Occurrence of Goldenrod Galls: Study of Insect Ovipositing Behavior

Goldenrod galls are a valuable resource for use in biology labs (Peard 1992). Goldenrods and the associated gall-forming insects are often abundant in early stages of old-field succession. In addition, these old-field habitats can be accessible to groups of students. The laboratory exercise described here takes advantage of this resource to explore a question concerning the behavior of the female gall-forming insects as they lay eggs on the goldenrod plant. The most common gall-makers on goldenrods are Eurosta solidaginis (Diptera: Tephritidae), Gnorimoschema gallaesolidaginis (Lepidoptera: Gelechiidae), and Rhopalomyia solidaginis (Diptera: Cecidomyiidae). The goldenrod gall fly, Eurosta solidaginis, produces a spherical swelling (ball gall) on the upper stem of goldenrods, about 2.5 cm in diameter and containing a single fly larva (Milne 1940). The larva pupates in early spring, emerging from the gall in April or May. Adult flies mate, and females begin laying eggs by late May (Miller 1959). Oviposition, the laying of an egg by the female, was described in detail by Abrahamson et al. (1989):

Loss of Ice-Nucleating Activity and Avoidance of Inoculative Freezing with Puparium Formation Induced by 20-Hydroxyecdysone in Eurosta solidaginis(Diptera:Tephritidae)

Overwintering larvae of the goldenrod gall fly Eurosta solidaginis begin to freeze at temperatures above -10°C. The initiation of freezing at relatively high subzero temperatures may involve multiple ice nucleation mechanisms to protect the insect from lethal intracellular freezing. In spring, the insect changes the mode of its cold-hariness from freeze-tolerant to freeze-susceptible type, with the metamorphosis from larva to pupa. The change is accompanied by a lowering of the supercooling points (spontaneous freezing points) and an increase in the resistance to inoculative freezing. The present study demonstrated that the change in cold-hardiness accompanied by puparium formation is artificially induced by 20-hydroxyecdysone treatment. In the beginning of the puparium formation, the insects lowered their supercooling points and lost their freeze tolerance, becoming resistant to inoculative freezing. At 25°C, the serial changes were completed within a week of the hormonal treatment.