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.     

Cuticular lipids and desiccation resistance in overwintering larvae of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae)

Within their gall, larvae of the goldenrod gall fly (Eurosta solidaginis) experience severe desiccating conditions as well as highly variable thermal conditions and extreme cold during winter. Through the autumn and early winter, field-collected larvae acquired markedly enhanced resistance to desiccation and freezing. At the same time, they increased their cuticular surface hydrocarbons. Hydrocarbons were the major lipid class extracted by hexane or chloroform from the cuticular surface of overwintering gall fly larvae. The major hydrocarbon classes were the 2-methylalkanes which consisted mainly of 2-methyltriacontane. 2-Methyltriacontane comprised 48-68% of the total hydrocarbons during the larval stages. Total hydrocarbons increased from 122 ng/larva in early third instar larvae collected in September to 4900 ng/larva in those collected in January. Although washing of the cuticular surface with chloroform or chloroform:methanol (2:1, v:v) caused marked increases in rates of water loss, treatment with hexane and methanol had little effect on water loss rates.     

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 (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees 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 degrees 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 > or = -6 degrees 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 degrees 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.     

Evidence for a novel cryoprotective protein from freeze-tolerant larvae of the goldenrod gall fly Eurosta solidaginis

Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) from populations in northern North America transition from freeze-susceptible to freeze-tolerant just prior to the onset of winter. While studies have documented the accumulation of carbohydrate cryoprotectants during this transition, protein cryoprotectants common to other freeze-tolerant species have not been reported in the gall fly. Using larvae collected from a population in Madison County, NY, which changes from freeze-susceptible to freeze-tolerant in early October, we assayed for the presence of factors that could preserve the catalytic activity of the cold-labile enzyme, rabbit muscle lactate dehydrogenase. Freezing this enzyme with a heat-stable, hydrophilic fraction derived from homogenates of both freeze-tolerant larvae and those in the process of becoming freeze-tolerant preserved between 70% and 80% of this enzyme's activity. Neither a comparable solution of bovine serum albumin nor the naturally-occurring carbohydrates (glycerol, sorbitol, or trehalose) conferred this level of cryoprotection. The putative cryoprotective protein from gall fly larvae did not bind to a weak anion exchanger, implying that its character may be cationic.     

Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes

This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at -80 degrees C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY-14) and propidium iodide.Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20-30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.     

The distribution of risk and density-dependent mortality in the galls of Eurosta solidaginis, the goldenrod gall fly

ABSTRACT.

• 1 This paper explores the net effect of a suite of mortality factors on a sedentary prey, the larvae of the goldenrod gall fly, Eurosta solidaginis Fitch (Diptera: Tephritidae).
• 2 Mortality is caused by unknown factors early in larval development, two species of parasitoid wasp (Hymenoptera: Eurytomidae), an inquiline beetle larva (Coleoptera: Mordellidae), and during the winter months downy woodpeckers Picoides pubescens (L.).
• 3 Distribution of mortality among galls relative to prey (gall) distribution was measured and discussed with respect to the distribution of relative risk of predation.
• 4 Galls are by and large contagiously distributed among quadrats, and mortality is distributed in a comparable pattern to that of galls.
• 5 The pattern of mortality on Eurosta larvae is neither density-dependent nor aggregated independently of gall distribution. Persistence in the system is probably a result of a combination of other factors such as adult mortality and early larval death which may have intergenerational density-dependent effects, and the linkage of locally unstable sub-populations via migration.

     

31P-NMR studies of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis

31P NMR was applied to an examination of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from -30 degrees to +25 degrees C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about -8 degrees C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15 degrees and -20 degrees C, the beta peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1-6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15 degrees C to pH 7.3 at -12 degrees C with a change, delta pH/delta 0, of -0.0185 degrees C consistent with alphastat regulation.     

Freezing and cellular metabolism in the gall fly larva,Eurosta solidaginis

Summary The effects of extracellular freezing on intracellular metabolism were monitored over both a short (9 h) and long (12 weeks) time course using the freeze tolerant larvae of the gall fly,Eurosta solidaginis.The process of freezing, monitored over the short time course, had no effect upon cellular energy levels (adenylates, arginine phosphate) but initiated a rise in glucose-6-P and lactate levels. This suggests that freezing initiates a shift towards glycolysis as the predominant mode of energy production. The process of thawing at 3°C (after 24 h at –16°C) also had no effect, even transient, on cellular energy levels demonstrating that thawing and the rapid redistribution of water and solutes which must accompany it does not disrupt cellular metabolism. During thawing accumulated lactate was quickly cleared with a t 1/2 of 20–30 min.Long term freezing at –16°C had dramatic effects on energy metabolism. Freezing for up to 1 week had minimal effects with only a small drop in arginine phosphate reserves and an increase in lactate content noted. Between 1 and 2 weeks of freezing, however, larvae showed strong signs of energy stress. The arginine phosphate pool fell from 75% to 30% of control levels, ATP content dropped by 50% and energy charge dropped to 0.75. This state, with continued lactate accumulation, was maintained through 4 weeks of freezing. Between 6 and 12 weeks of freezing energy stress became even greater. Phosphagen and ATP contents dropped to 5 and 25% of control values and energy charge decreased to about 0.50. Despite this stress, however, 94% of larvae survived 12 weeks of freezing with an 86% hatch rate of adults. The data demonstrate that the larvae can survive prolonged periods of winter freezing drawing upon glycolysis and phosphagen reserves to supply the continued basal energy demands of the cell.     

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.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight     

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

Extracellular freezing and dehydration concentrate hemolymph solutes, which can lead to cellular injury due to excessive water loss. Freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, may experience extreme cold and desiccation in winter. To determine whether larvae employ protective mechanisms against excessive cellular water loss we examined the effect of extracellular freezing and dehydration on hemolymph volume, and cryoprotectant and ion levels in the hemolymph. Dehydrated larvae or ones that had been frozen at −5 or −20 °C had a significantly smaller proportion of their body water as hemolymph (26.0-27.4%) compared to controls (30.5%). Even with this reduction in water content, hemolymph osmolality was similar or only slightly higher in frozen or dehydrated individuals than controls (908 mOsm kg⁻¹), indicating these stresses led to a reduction in hemolymph solutes. Hemolymph and intracellular content of ions remained largely unchanged between treatment groups; although levels of Mg⁺⁺ in the hemolymph were lower in larvae subjected to freezing (0.21 ± 0.01 μg mg⁻¹ dry mass) compared to controls (0.29 ± 0.01 μg mg⁻¹ dry mass), while intracellular levels of K⁺ were lower in groups exposed to low temperature (8.31 ± 0.21 μg mg⁻¹ dry mass). Whole body glycerol and sorbitol content was similar among all treatment groups, averaging 432 ± 25 mOsm kg⁻¹ and 549 ± 78 mOsm kg⁻¹ respectively. However, larvae subjected to dehydration and freezing at −20 °C had a much lower relative amount of cryoprotectants in their hemolymph (∼35%) compared to controls (54%) suggesting these solutes moved into intracellular compartments during these stresses. The correlation between reduced hemolymph volume (i.e. increased cellular water content) and intracellular movement of cryoprotectants may represent a link between tolerance of dehydration and cold in this species.     

Mitochondrial DNA variation among host races of Eurosta solidaginis Fitch (Diptera: Tephritidae)

Eurosta solidaginis Fitch (Diptera: Tephritidae) induces galls on two species of goldenrod, Solidago (Compositae), in the northern regions of the United States. Recent studies have demonstrated that E. solidaginis is comprised of two host races that differ in adult emergence times, mate preference, and host preference. However, it is not known how much genetic variation, if any, exists among E. solidaginis host-associated populations west of Minnesota where the two host races occur in sympatry. Sequencing analysis was used to characterize two mitochondrial gene fragments: (1) NADH1 dehydrogenase (ND1: 539 bp) and (2) cytochrome oxidase II + tRNA(Lys) + tRNA(Asp) (CO2KD: 396 bp) from sympatric, host-associated populations of E. solidaginis in North Dakota. Our results indicated that two genetically distinct lineages exist among E. solidaginis in North Dakota that correspond with host-plant association.     

The effects of temperature and season on the O2 consumption of third-instar larvae of the goldenrod gall fly (Eurosta solidaginis)

Abstract. 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.     

Ultrastructural effects of lethal freezing on brain,muscle and Malpighian tubules from freeze-tolerant larvae of the gall fly,Eurosta solidaginis

In preparation for winter low temperatures, larvae of the gall fly, Eurosta solidaginis, accumulate the cryoprotectants glycerol, sorbitol, and trehalose. The fat body cells of these freeze-tolerant larvae can survive intracellular freezing to -80 degrees C for 48 h even though no whole larvae survive this treatment. We hypothesized that some other tissue was more susceptible to freezing and therefore may be responsible for larval death. This paper compares the ultrastructure of brain, muscle, and Malpighian tubules between non-lethally frozen and lethally frozen freeze-tolerant larvae. The nuclei of cortical brain cells from lethally frozen larvae exhibited clumped chromatin and nuclear membranes with occasional expansions or 'blebs' of the intermembranous space, while the cytoplasm contained swollen spheres of endoplasmic reticulum. In contrast, non-lethally frozen brain contained nuclei with evenly dispersed chromatin, smooth nuclear membranes and a cytoplasm free of swollen endoplasmic reticulum. Muscle tissue of lethally frozen larvae contained disrupted myofilaments surrounding the Z-line in comparison to non-lethally frozen muscle which had myofilaments extending all the way to the Z-line. Alterations of Malpighian tubule cells from lethally frozen larvae included an extracted cytoplasm with swollen and rounded mitochondria. In contrast, Malpighian tubule cells from non-lethally frozen larvae had a more concentrated cytoplasm with many rod-shaped mitochondria. Results show alterations to all three tissue types due to lethal freezing. The brain tissue contained the most observable alterations and therefore may be the most susceptible to lethal freeze damage.     

Sequential radiation of unrelated organisms: the gall fly Eurosta solidaginis and the tumbling flower beetle Mordellistena convicta

Host shifts and the formation of insect-host races are likely common processes in the speciation of herbivorous insects. The interactions of goldenrods Solidago (Compositae), the gall fly Eurosta solidaginis (Diptera: Tephritidae) and the beetle Mordellistena convicta (Coleoptera: Mordellidae) provide behavioural, ecological and genetic evidence of host races that may represent incipient species forming via sympatric speciation. We summarize evidence for Eurosta host races and show that M. convicta has radiated from goldenrod stems to Eurosta galls to form host-part races and, having exploited the galler's host shift, has begun to differentiate into host races within galls. Thus, host-race formation has occurred in two interacting, but unrelated organisms representing two trophic levels, resulting in 'sequential radiation' (escalation of biodiversity up the trophic system). Distributions of host races and their behavioural isolating mechanisms suggest sympatric differentiation. Such differentiation suggests host-race formation and subsequent speciation may be an important source of biodiversity.