Goldenrod Gall Fly Eurosta Solidaginis Research Articles

Repeated freezing induces a trade-off between cryoprotection and egg production in the goldenrod gall fly, Eurosta solidaginis.

Internal ice formation leads to wholesale changes in ionic, osmotic and pH homeostasis, energy metabolism, and mechanical damage, across a small range of temperatures, and is thus an abiotic stressor that acts at a distinct, physiologically relevant, threshold. Insects that experience repeated freeze-thaw cycles over winter will cross this stressor threshold many times over their lifespan. Here, we examined the effect of repeatedly crossing the freezing threshold on short-term physiological parameters (metabolic reserves and cryoprotectant concentration) as well as long-term fitness-related performance (survival and egg production) in the freeze-tolerant goldenrod gall fly, Eurosta solidaginis We exposed overwintering prepupae to a series of low temperatures (-10, -15 or -20°C) with increasing numbers of freezing events (3, 6 or 10) with differing recovery periods between events (1, 5 or 10 days). Repeated freezing increased sorbitol concentration by about 50% relative to a single freezing episode, and prompted prepupae to modify long-chain triacylglycerols to acetylated triacylglycerols. Long-term, repeated freezing did not significantly reduce survival but did reduce egg production by 9.8% relative to a single freezing event. Exposure temperature did not affect any of these measures, suggesting that threshold crossing events may be more important to fitness than the intensity of stress in overwintering E. solidaginis.

Characterization of cold-associated microRNAs in the freeze-tolerant gall fly Eurosta solidaginis using high-throughput sequencing

Significant physiological and biochemical changes are observed in freeze-tolerant insects when confronted with cold temperatures. These insects have adapted to winter by retreating into a hypometabolic state of diapause and implementing cryoprotective mechanisms that allow them to survive whole body freezing. MicroRNAs (miRNAs), a family of short ribonucleic acids, are emerging as likely molecular players underlying the process of cold adaptation. Unfortunately, the data is sparse concerning the signature of miRNAs that are modulated following cold exposure in the freeze-tolerant goldenrod gall fly Eurosta solidaginis. Leveraging for the first time a next-generation sequencing approach, differentially expressed miRNAs were evaluated in 5°C and −15°C-exposed E. solidaginis larvae. Next-generation sequencing expression data was subsequently validated by qRT-PCR for selected miRNA targets. Results demonstrate 24 differentially expressed freeze-responsive miRNAs. Notable, miR-1-3p, a miRNA modulated at low temperature in another cold-hardy insect, and miR-14-3p, a miRNA associated with stress response in the fruit fly, were shown to be significantly up-regulated in −15°C-exposed larvae. Overall, this work identifies, for the first time in a high-throughput manner, differentially expressed miRNAs in cold-exposed E. solidaginis larvae and further clarifies an emerging signature of miRNAs modulated at low temperatures in cold-hardy insects.

The limits of drought-induced rapid cold-hardening: Extremely brief, mild desiccation triggers enhanced freeze-tolerance in Eurosta solidaginis larvae

Rapid cold-hardening (RCH) is a highly conserved response in insects that induces physiological changes within minutes to hours of exposure to low temperature and provides protection from chilling injury. Recently, a similar response, termed drought-induced RCH, was described following as little as 6h of desiccation, producing a loss of less than 10% of fresh mass. In this study, we investigated the limits and mechanisms of this response in larvae of the goldenrod gall fly Eurosta solidaginis (Diptera, Tephritidae). The cold-hardiness of larvae increased markedly after as few as 2h of desiccation and a loss of less than 1% fresh mass, as organismal survival increased from 8% to 41% following exposure to −18°C. Tissue-level effects of desiccation were observed within 1h, as 87% of midgut cells from desiccated larvae remained viable following freezing compared to 57% of controls. We also demonstrated that drought-induced RCH occurs independently of neuroendocrine input, as midgut tissue desiccated ex vivo displayed improved freeze-tolerance relative to control tissue (78–11% survival, respectively). Finally, though there was an increase in hemolymph osmolality beyond the expected effects of the osmo-concentration of solutes during dehydration, we determined that this increase was not due to the synthesis of glycerol, glucose, sorbitol, or trehalose. Our results indicate that E. solidaginis larvae are extremely sensitive to desiccation, which is a triggering mechanism for one or more physiological pathways that confer enhanced freeze-tolerance.

Repeated freezing induces oxidative stress and reduces survival in the freeze-tolerant goldenrod gall fly, Eurosta solidaginis

Freeze tolerant insects must not only survive extracellular ice formation but also the generation of reactive oxygen species (ROS) during oxygen reperfusion upon thawing. Furthermore, diurnal fluctuations in temperature place temperate insects at risk of being exposed to multiple freeze–thaw cycles, yet few studies have examined metrics of survival and oxidative stress in freeze-tolerant insects subjected to successive freezing events. To address this, we assessed survival in larvae of the goldenrod gall fly Eurosta solidaginis, after being subjected to 0, 5, 10, 20, or 30 diurnally repeated cold exposures (RCE) to −18°C or a single freeze to −18°C for 20days. In addition, we measured indicators of oxidative stress, levels of cryoprotectants, and total aqueous antioxidant capacity in animals exposed to the above treatments at 8, 32, or 80h after their final thaw. Repeated freezing and thawing, rather than time spent frozen, reduced survival as only 30% of larvae subjected to 20 or 30 RCE successfully pupated, compared to those subjected to fewer RCE or a single 20d freeze, of which 82% pupated. RCE had little effect on the concentration of the cryoprotectant glycerol (4.26±0.66μgglycerol·ngprotein−1 for all treatments and time points) or sorbitol (18.8±2.9μgsorbitol·mgprotein−1 for all treatments and time points); however, sorbitol concentrations were more than twofold higher than controls (16.3±2.2μgsorbitol·mgprotein−1) initially after a thaw in larvae subjected to a single extended freeze, but levels returned to values similar to controls at 80h after thaw. Thawing likely produced ROS as total aqueous antioxidant capacities peaked at 1.8-fold higher than controls (14.7±1.6mmoltrolox·ngprotein−1) in animals exposed to 5, 10, or 20 RCE. By contrast, aqueous antioxidant capacities were similar to controls in larvae subjected to 30 RCE or the single 20d freeze regardless of time post final thaw, indicating these animals may have had an impaired ability to produce primary antioxidants. Larvae lacking an antioxidant response also had elevated levels of oxidized proteins, nearly twice that of controls (21.8±3.2mmolchloramine-T·mgprotein−1). Repeated freezing also lead to substantial oxidative damage to lipids that was independent of aqueous antioxidant capacity; peroxides were, on average, 5.6-fold higher in larvae subjected to 10, 20 or 30 RCE compared to controls (29.1±7.3mmolTMOP·μgprotein−1). These data suggest that oxidative stress due to repeated freeze–thaw cycles reduces the capacity of E. solidaginis larvae to survive freezing.

Seasonal Changes in Phospholipid Class and Class‐Specific Fatty Acid Composition Associated with the Onset of Freeze Tolerance in Third‐Instar Larvae ofEurosta solidaginis

Abstract Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) are freeze tolerant in winter. During freezing, cell membranes must compensate for both low temperature and partial dehydration. Documented adaptations to low temperature include increased fatty acid unsaturation and enrichment of cone-shaped phosphatides, both of which inhibit formation of gel phase lipid domains. These changes appear inconsistent with adaptations known to prevent formation of the hexagonal II phospholipid phase at low water activities, namely, increased fatty acid saturation and increased proportions of cylindrical phosphatides. To address these inconsistencies, changes in E. solidaginis phospholipid composition and class-specific fatty acid composition were studied from August to November 2002. Cylindrical phosphatides, mostly phosphatidylcholine (PC), increased transiently and significantly, from 35% of the total to nearly 50%, during the transition from freeze susceptible to freeze tolerant. Monoenes in both PC and phosphatidylethanolamine (PE) represented 35% of total fatty acids in freeze-susceptible larvae but accumulated in PC to 48% and in PE to 42% in freeze-tolerant larvae. Moreover, PC accumulated the most unsaturated acid in this species, 18:3(n-3), to a significantly greater degree than PE. This combination of changes may represent a finely tailored response to both low temperatures and freeze-induced dehydration.

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.

Plant senescence cues entry into diapause in the gall flyEurosta solidaginis: resulting metabolic depression is critical for water conservation

Mechanisms and possible cues for seasonal increases in desiccation resistance in larvae of the goldenrod gall fly Eurosta solidaginis, were examined before and after natural and premature plant senescence, or after being removed from their gall and placed in either 100, 95 or 75% relative humidity (RH). Rates of water loss were 8.6-fold lower, averaging 0.7+/-0.2 microg mm(-2) h(-1), in larvae from senescent gall tissue and after all RH treatments than in control larvae from pre-senescent plants. Enhanced desiccation resistance occurred quickly, within 3 days of removal from their gall. Contrary to most previous reports, a large majority of the increased desiccation resistance (approximately 85%) was due to reduced respiratory transpiration with the remainder being the result of a lowered cuticular permeability. Rates of cuticular water loss were reduced by the presence of a vapor pressure gradient between the larval hemolymph and environmental water vapor and were probably due to increases in cuticular lipids and/or production of the cryoprotectant glycerol. Metabolic rate was reduced by over fourfold, averaging 0.07+/-0.01 microl CO2 g(-1) h(-1), in larvae from senescent gall tissue and all RH treatments compared to larvae from pre-senescent plants. The magnitude of the reduction in metabolic rates indicated that these larvae had entered diapause. In addition, larvae entered diapause in response to removal from, or degeneration of, the gall tissue they feed, on rather than seasonal changes in temperature or photoperiod. The low metabolic rates of the diapausing larvae probably allowed them to dramatically reduce their respiratory transpiration and total rate of water loss compared with non-diapausing controls. Thus, diapause, with its associated lowered metabolic rate, may be essential for conserving water in overwintering temperate insects, which may be dormant for six or more months of the year.

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.

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.

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.

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.

Metabolic response to temperature pulsing in the goldenrod gall fly Eurosta solidaginis: Cryoprotectant synthesis and catabolism

Metabolic response to temperature pulsing in the goldenrod gall fly Eurosta solidaginis: Cryoprotectant synthesis and catabolism