Eurosta Solidaginis Research Articles

Lysine Methylation and Histone Modifications during Cold Stress of Insects: Freeze-Tolerant Eurosta solidaginis and Freeze-Avoiding Epiblema scudderiana.

Overwintering survival by insects, whether of the freeze-tolerant or freeze-avoiding types, is typically associated with a strong suppression of metabolic rate (e.g., entry into diapause) that involves the differential expression of many genes with regulation at the transcriptional, translational or post-translational levels. Epigenetic modifications have been suggested to play a vital role in regulating cold responses of insects. However, knowledge of the roles of epigenetic mechanisms in modulating gene expression for winter survival of the larvae of two goldenrod gall formers, the freeze-tolerant dipteran Eurosta solidaginis and the freeze-avoiding lepidopteran Epiblema scudderiana, remain unknown. The current study evaluates the role of cold-induced lysine methylation and histone modifications, with enzymes of lysine methylation (SETD8, SETD7, SUV39H1, SMYD2 and ASH2L), as well as relative levels of histone H3 acetylation (H3K9ac, H3K18ac, H3K27ac, H3K56ac) and methylation (H3K4me1, H3K9me3, H3K36me2) examined in two insects. Significant (p < 0.05) reductions were observed in most of the targets of histone methylation/acetylation for decreasing temperatures of Ep. scudderiana larvae, whereas selected histone methylation/acetylation targets were conversely elevated (p < 0.05) in E. solidaginis, particularly under conditions of 5 °C for 4 h. Histone H3 expression was found to be variable without statistical differences in larval goldenrod gall moths and gall flies. These results provide basic information on the patterns of epigenetic regulation involved in insect cold hardiness.

Urban socioeconomic variation influences the ecology and evolution of trophic interactions.

As urbanization expands, it is becoming increasingly important to understand how anthropogenic activity is affecting ecological and evolutionary processes. Few studies have examined how human social patterns within cities can modify eco-evolutionary dynamics. We tested how socioeconomic variation corresponds with changes in trophic interactions and natural selection on prey phenotypes using the classic interaction between goldenrod gall flies (Eurosta solidaginis) and their natural enemies: birds, beetles, and parasitoid wasps. We sampled galls from 84 sites across neighbourhoods with varying socioeconomic levels, and quantified the frequency of predation/parasitism on flies and natural selection by each enemy. We found that bird predation was higher in the highest income neighbourhoods, increasing the strength of selection for smaller galls. Wasp and beetle attack, but not their strength of selection, increased in lower income neighbourhoods. We show that socioeconomic variation in cities can have strong unintended consequences for the ecology and evolution of trophic interactions.

Histone H3 and H4 Modifications Point to Transcriptional Suppression as a Component of Winter Freeze Tolerance in the Gall Fly Eurosta solidaginis.

The goldenrod gall fly (Eurosta solidaginis) is a well-studied model of insect freeze tolerance. In situations of prolonged winter subzero temperatures, larvae of E. solidaginis accept ice penetration throughout extracellular spaces while protecting the intracellular environment by producing extreme amounts of glycerol and sorbitol as cryoprotectants. Hypometabolism (diapause) is implemented, and energy use is reprioritized to essential pathways. Gene transcription is one energy-expensive process likely suppressed over the winter, in part, due to epigenetic controls. The present study profiled the prevalence of 24 histone H3/H4 modifications of E. solidaginis larvae after 3-week acclimations to decreasing environmental temperatures (5 °C, -5 °C and -15 °C). Using immunoblotting, the data show freeze-mediated reductions (p < 0.05) in seven permissive histone modifications (H3K27me1, H4K20me1, H3K9ac, H3K14ac, H3K27ac, H4K8ac, H3R26me2a). Along with the maintenance of various repressive marks, the data are indicative of a suppressed transcriptional state at subzero temperatures. Elevated nuclear levels of histone H4, but not histone H3, were also observed in response to both cold and freeze acclimation. Together, the present study provides evidence for epigenetic-mediated transcriptional suppression in support of the winter diapause state and freeze tolerance of E. solidaginis.

Genomic Assessment of the Contribution of the Wolbachia Endosymbiont of Eurosta solidaginis to Gall Induction.

We explored the genome of the Wolbachia strain, wEsol, symbiotic with the plant-gall-inducing fly Eurosta solidaginis with the goal of determining if wEsol contributes to gall induction by its insect host. Gall induction by insects has been hypothesized to involve the secretion of the phytohormones cytokinin and auxin and/or proteinaceous effectors to stimulate cell division and growth in the host plant. We sequenced the metagenome of E. solidaginis and wEsol and assembled and annotated the genome of wEsol. The wEsol genome has an assembled length of 1.66 Mbp and contains 1878 protein-coding genes. The wEsol genome is replete with proteins encoded by mobile genetic elements and shows evidence of seven different prophages. We also detected evidence of multiple small insertions of wEsol genes into the genome of the host insect. Our characterization of the genome of wEsol indicates that it is compromised in the synthesis of dimethylallyl pyrophosphate (DMAPP) and S-adenosyl L-methionine (SAM), which are precursors required for the synthesis of cytokinins and methylthiolated cytokinins. wEsol is also incapable of synthesizing tryptophan, and its genome contains no enzymes in any of the known pathways for the synthesis of indole-3-acetic acid (IAA) from tryptophan. wEsol must steal DMAPP and L-methionine from its host and therefore is unlikely to provide cytokinin and auxin to its insect host for use in gall induction. Furthermore, in spite of its large repertoire of predicted Type IV secreted effector proteins, these effectors are more likely to contribute to the acquisition of nutrients and the manipulation of the host's cellular environment to contribute to growth and reproduction of wEsol than to aid E. solidaginis in manipulating its host plant. Combined with earlier work that shows that wEsol is absent from the salivary glands of E. solidaginis, our results suggest that wEsol does not contribute to gall induction by its host.

Abscisic Acid: A Potential Secreted Effector Synthesized by Phytophagous Insects for Host-Plant Manipulation.

Abscisic acid (ABA) is an isoprenoid-derived plant signaling molecule involved in a wide variety of plant processes, including facets of growth and development as well as responses to abiotic and biotic stress. ABA had previously been reported in a wide variety of animals, including insects and humans. We used high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-(ESI)-MS/MS) to examine concentrations of ABA in 17 species of phytophagous insects, including gall- and non-gall-inducing species from all insect orders with species known to induce plant galls: Thysanoptera, Hemiptera, Lepidoptera, Coleoptera, Diptera, and Hymenoptera. We found ABA in insect species in all six orders, in both gall-inducing and non-gall-inducing species, with no tendency for gall-inducing insects to have higher concentrations. The concentrations of ABA in insects often markedly exceeded those typically found in plants, suggesting it is highly improbable that insects obtain all their ABA from their host plant via consumption and sequestration. As a follow-up, we used immunohistochemistry to determine that ABA localizes to the salivary glands in the larvae of the gall-inducing Eurosta solidaginis (Diptera: Tephritidae). The high concentrations of ABA, combined with its localization to salivary glands, suggest that insects are synthesizing and secreting ABA to manipulate their host plants. The pervasiveness of ABA among both gall- and non-gall-inducing insects and our current knowledge of the role of ABA in plant processes suggest that insects are using ABA to manipulate source-sink mechanisms of nutrient allocation or to suppress host-plant defenses. ABA joins the triumvirate of phytohormones, along with cytokinins (CKs) and indole-3-acetic acid (IAA), that are abundant, widespread, and localized to glandular organs in insects and used to manipulate host plants.

A geographic mosaic of coevolution between Eurosta solidaginis (Fitch) and its host plant tall goldenrod Solidago altissima (L.).

A geographic mosaic of coevolution has produced local reciprocal adaptation in tall goldenrod, Solidago altissima (L.), and the goldenrod ball-gall fly, Eurosta solidaginis (Fitch 1855). The fly is selected to induce gall diameters that minimize mortality from natural enemies, and the plant is selected to limit gall growth that reduces plant fitness. We conducted a double reciprocal transplant experiment where S. altissima and E. solidaginis from three sites were grown in gardens at each site to partition the gall morphology variation into fly genotype, plant genotype, and the environment components. The host plant gall diameter induced by each E. solidaginis population was adapted to inhibit local natural enemies from ovipositing on or consuming enclosed larvae. Reciprocally, increasing the gall size induced by the local fly population increased the resistance of the local plant host population to gall growth. Differences among sites in natural enemies produced a mosaic of hotspots of coevolutionary arms races between flies selecting for greater gall diameter and plants for smaller diameters, and coldspots where there is no selection on plant or fly for a change in gall diameter. In contrast, the geographic variations of gall length and gall shape were not due to coevolutionary interactions.

Giant polyploid epidermal cells and male pheromone production in the tephritid fruit fly Eurosta solidaginis (Diptera: Tephritidae)

Eurosta solidaginis males produce large amounts of putative sex pheromone compared to other insect species; however, neither the site of pheromone production nor the release mechanism has been characterized. We compared E. solidaginis males and females, focusing on sexually dimorphic structures that are known to be involved in pheromone production in other tephritid species. Morphological and chemical analyses indicated that the rectum and pleural epidermis are involved in male E. solidaginis pheromone production, storage, or emission. We detected large quantities of pheromone in the enlarged rectum, suggesting that it stores pheromone for subsequent release through the anus. However, pheromone might also discharge through the pleural cuticle with the involvement of unusual pleural attachments of the tergosternal muscles, which, when contracted in males, realign specialized cuticular surface elements and expose less-sclerotized areas of cuticle. In males, pheromone components were also detected in epidermal cells of the pleuron. These cells were 60–100 times larger in mature males than in females and, to our knowledge, are the largest animal epithelial cells ever recorded. Furthermore, because these large cells in males are multinucleated, we presume that they develop through somatic polyploidization by endomitosis. Consequently, the pheromone-associated multinuclear pleural epidermal cells of Eurosta solidaginis may provide an interesting new system for understanding polyploidization.

Regulation of an important glycolytic enzyme, pyruvate kinase, through phosphorylation in the larvae of a species of freeze-tolerant insect, Eurosta solidaginis.

Larvae of the goldenrod gall fly, Eurosta solidaginis, rely on a freeze tolerance strategy to survive the sub-zero temperatures of Canadian winter. Critical to their survival is the accumulation of polyol cryoprotectants and global metabolic rate depression, both of which require the regulation of glycolysis and reorganization of carbohydrate metabolism. This study explored the role that pyruvate kinase (PK) regulation plays in this metabolic reorganization. PK was purified from control (5 °C-acclimated) and frozen (-15 °C-acclimated) larvae and enzyme kinetic properties, structural stability, and post-translational modifications were examined in both enzyme forms. The Km phosphoenolpyruvate (PEP) of frozen PK was 20% higher than that of control PK, whereas the Vmax of frozen PK was up to 50% lower than that of control PK at the lowest assay temperature, suggesting inhibition of the enzyme during the winter. Additionally, the activity and substrate affinity of both forms of PK decreased significantly at low assay temperatures, and both forms were regulated allosterically by a number of metabolites. Pro-Q™ Diamond phosphoprotein staining and immunoblotting experiments demonstrated significantly higher threonine phosphorylation of PK from frozen animals while acetylation and methylation levels remained constant. Together, these results indicate that PK exists in two structurally distinct forms in E. solidaginis. In response to conditions mimicking the transition to winter, PK appears to be regulated to support metabolic rate depression, the accumulation of polyol cryoprotectants, and the need for extended periods of anaerobic carbohydrate metabolism to allow the animal to survive whole-body freezing.

Sensory co‐evolution: The sex attractant of a gall‐making fly primes plant defences, but female flies recognize resulting changes in host‐plant quality

Abstract Tall goldenrod plants Solidago altissima appear to eavesdrop on the communication of a specialist gall‐inducing fly Eurosta solidaginis by detecting the volatile emissions of male flies and priming anti‐herbivore defences. However, the effects of defence priming on fly fitness have not previously been demonstrated, despite some evidence that female flies avoid ovipositing on primed plants in the field. To explore how priming affects female preferences and galling success, we manipulated exposure to the priming cue in multiple genotypes of S. altissima and assessed rates of oviposition and gall development. Priming reduced gall formation overall; however, its effect varied across plant genotypes, as well as by the age of the male flies used for priming, possibly reflecting dosage effects (older flies produce lower emissions). Priming by younger males significantly reduced galling success, while priming by older males slightly improved it. Remarkably, female flies accurately assessed host‐plant quality by avoiding plants primed by young males while preferring those primed by older males. Synthesis. Our results suggest that defence priming in S. altissima in response to the volatile emissions of E. solidaginis males does affect fly fitness; however, the effect of priming varied by plant genotype, suggesting that there may be variation in defensive strategies against the fly. Female flies have, in response to this defence, evolved a sophisticated ability to evaluate the quality of primed plants.

A small‐tiled geographic mosaic of coevolution between Eurosta solidaginis and its natural enemies and host plant

AbstractWe identified a geographic mosaic of Eurosta solidaginis fly traits produced by coevolution of the stem gall‐forming fly with both its natural enemies and its host plants at small geographic scales in the presence of gene flow. These tritrophic interactions between the fly with its natural enemies and with its host‐plant Solidago altissima produced what has been termed a small‐tiled geographic mosaic of coevolution. Selection on gall diameter and length varies between prairie and forest habitats due to differences in host plants and natural‐enemy communities. At the prairie–forest ecotone where prairie and forest habitats are intermixed, we found that geographic selection mosaics on gall diameter and length varied on a scale of a few kilometers. Gall diameter variation among sites correlated with selection on gall diameters, indicating local adaptation. In contrast, gall lengths did not correlate with selection, indicating that gene flow may have prevented local adaptation of this trait. Eastern (forest) and western (prairie) subspecies of E. solidaginis have been proposed based on fly wing patterns, and these had intermediate forms in the ecotone indicating gene flow between these subspecies. Variation in wing patterns correlated with gall diameter, indicating that gene flow between prairie and forest fly populations may influence the distribution of gall traits. The ratio of forest to prairie vegetation increases with latitude, but there was no indication of latitudinal clines in gall or wing traits. Our results indicate that selection for differentiation in coevolved traits is strong enough to overcome gene flow in small tiles of habitat. The result is that ecological forces produce a dynamic mosaic of genetically differentiated locally adapted populations. It also indicates that prairie and forest host races of E. solidaginis form a mosaic hybrid zone in this region.

Salivary Origins of Exogenous Cytokinins

Cytokinins are a class of phytohormones which play an important role in a variety of plant processes, such as cell growth and differentiation, formation of roots and shoots, and the reallocation of nutrients to form mobilizing sinks. Exogenous cytokinins found in plant‐manipulating insects are known to contribute to many effects on host plants. Perhaps the most apparent effect is that cytokinins, in the presence of auxin, leads to cell division and the proliferation of plant tissue; consequently, plant galls or tumors are formed. Cytokinins have also been implicated in the reallocation of sugars and nutrients for the oviposition site to act as a mobilizing sink, where the intention may be gall induction or sustenance for the organism. There are two known pathways for the biosynthesis of isoprenoid cytokinins. The first is the tRNA‐ipt pathway, in which an adenine residue is modified by the addition of an isoprenoid chain by the tRNA‐ipt gene. In plant plastids and bacteria, the methyl erythritol (MEP) pathway is the main source of the prenyl group, but in the cytosol of plants and insects, the prenyl group is synthesized via the mevalonate (MVA) pathway. Cytokinins are produced upon the degradation of the prenylated tRNA. To further investigate the role of cytokinins in gall induction by insects, I performed gene expression experiments using the gall‐inducing Eurosta solidaginis and its bacterial symbiont Wolbachia sp. I also performed targeted gene expression analyses in order to examine the expression of genes in the MEP, MVA, and tRNA‐ipt pathways. Up‐regulation of these pathways in the salivary gland implies that cytokinins are being synthesized in the salivary glands by either the insect and/or its bacterial symbiont. Along with the known widespread distribution of cytokinins in insects, these further imply that cytokinin production and secretion has been amplified and is used by the insects to manipulate their host plants. However, the genes involved in cytokinin production have also been found in other insect groups, so this plant manipulation may not just be exclusive to gall‐inducing insects, suggesting widespread hostplant manipulation among plant‐eating insects.Support or Funding InformationNIH MBRS‐RISE: R25‐GM059298

Overexpression of diacylglycerol acetyltransferase from Euonymus europaeus in Yarrowia lipolytica leads to the production of single-cell oil enriched with 3-acetyl-1,2-diacylglycerols.

The 3-acetyl-1,2-diacylglycerols (acTAGs) are the molecules that are structurally similar to triacylglycerols (TAGs). They are naturally produced by plants of the family Celastraceae and animals such as Cervus nippon and Eurosta solidaginis. The presence of acetate in the sn-3 position of the glycerol backbone confers advantages to these compounds, for example, lower viscosity and calorific value compared to classical TAGs. In this work, the gene EeDAcT, which encodes diacylglycerol acetyltransferase in a species of bush (Euonymus europaeus), was overexpressed in strains Po1d (capable of accumulating storage lipids) and JMY1877 (incapable of accumulating storage lipids) of Yarrowia lipolytica, to test the activity of the gene EeDAcT and the production of acTAGs in oleaginous and nonoleaginous genetic backgrounds. It was observed that both the strains containing the gene EeDAcT (YL33 and YL35 for Po1d and JMY1877 strains, respectively) produced acTAGs. The strain YL33 accumulated up to 20% intracellular lipids, 20% of which was acTAGs, and 40% was TAGs. On the other hand, the strain YL35, which showed interrupted TAGs accumulation, produced up to 10% acTAGs as the only storage lipid. Unfortunately, the quantity of acTAGs produced in YL35 was insignificant, as the overall lipid accumulated in the strain was not more than 4% of the biomass. The fatty acid profile of acTAGs produced by the YL33 strain was remarkably similar to TAGs, and both of these structures were rich in oleic (45%) and palmitic (25%) acids.

Glucose-6-phosphate dehydrogenase is posttranslationally regulated in the larvae of the freeze-tolerant gall fly, Eurosta solidaginis, in response to freezing.

The freeze-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) undergo substantial alterations to their molecular physiology during the winter including the production of elevated quantities of glycerol and sorbitol, which function as cryoprotectants to survive whole body freezing. Production of these cryoprotectants depends on cytosolic pools of nicotinamide adenine dinucleotide phosphate H (NADPH), a major source being the pentose phosphate pathway (PPP). Glucose-6-phosphate dehydrogenase (G6PDH) mediates the rate-limiting and committed step of the PPP and therefore its molecular properties were explored in larvae sampled from control versus frozen states. G6PDH was purified from control (5°C) and frozen (-15°C) E. solidaginis larvae by a single-step chromatography method utilizing 2',5'-ADP agarose and analyzed to determine its enzymatic parameters. Studies revealed a decrease in Km for G6P in the frozen animals (to 50% of control values) suggesting an increased flux through the PPP. Immunoblotting of the purified enzyme showed differences in the relative extent of several posttranslational modifications, notably ubiquitination (95% decrease in frozen larvae), cysteine nitrosylation (61% decrease), threonine (4.1 fold increase), and serine phosphorylation (59% decrease). Together these data suggested that the increased flux through the PPP needed to generate NADPH for cryoprotectants synthesis is regulated, at least in part, through posttranslational alterations of G6PDH.

Distribution of the Specialist Aphid Uroleucon nigrotuberculatum (Homoptera: Aphididae) in Response to Host Plant Semiochemical Induction by the Gall Fly Eurosta solidaginis (Diptera: Tephritidae).

Many plants use terpenoids and other volatile compounds as semiochemicals. Reception of plant volatiles by conspecifics may trigger a defensive phytochemical response. These same compounds can also function as host recognition signals for phytophagous insects. In this experiment, we find that when the specialist gall-forming fly Eurosta solidaginis (Fitch; Diptera: Tephritidae) attacks its tall goldenrod (Solidago altissima (L.; Asterales: Asteraceae)) host plant, the fly indirectly induces a phytochemical response in nearby tall goldenrod plants. This phytochemical response may, in turn, act as a positive signal attracting the goldenrod specialist aphid Uroleucon nigrotuberculatum (Olive; Hemiptera: Aphididae). Laboratory-based experiments exposing ungalled tall goldenrod plants to the volatiles released by E. solidaginis galls demonstrated a consistent increase in foliar terpenoid concentrations in ungalled plants. Analysis of tall goldenrod stem and gall tissue chemistry revealed induction of terpenoids in gall tissue, with a simultaneous decrease in green leaf volatile concentrations. Field experiments demonstrated a consistent spatial relationship in tall goldenrod foliar terpenoid concentrations with distance from an E. solidaginis gall. Both laboratory and field experiments establish consistent induction of the terpene β-farnesene, and that this compound is a strong positive predictor of U. nigrotuberculatum aphid presence on goldenrod plants along with plant biomass and several other foliar terpenoids. These findings suggest E. solidaginis induced phytochemistry, especially β-farnesene, may be acting as a kairomone, driving aphid distribution in the field.

Costs of plant defense priming: exposure to volatile cues from a specialist herbivore increases short-term growth but reduces rhizome production in tall goldenrod (Solidago altissima)

BackgroundBy sensing environmental cues indicative of pathogens or herbivores, plants can “prime” appropriate defenses and deploy faster, stronger responses to subsequent attack. Such priming presumably entails costs—else the primed state should be constitutively expressed—yet those costs remain poorly documented, in part due to a lack of studies conducted under realistic ecological conditions. We explored how defence priming in goldenrod (Solidago altissima) influenced growth and reproduction under semi-natural field conditions by manipulating exposure to priming cues (volatile emissions of a specialist herbivore, Eurosta solidaginis), competition between neighbouring plants, and herbivory (via insecticide application).ResultsWe found that primed plants grew faster than unprimed plants, but produced fewer rhizomes, suggesting reduced capacity for clonal reproduction. Unexpectedly, this effect was apparent only in the absence of insecticide, prompting a follow-up experiment that revealed direct effects of the pesticide esfenvalerate on plant growth (contrary to previous reports from goldenrod). Meanwhile, even in the absence of pesticide, priming had little effect on herbivore damage levels, likely because herbivores susceptible to the primed defences were rare or absent due to seasonality.ConclusionsReduced clonal reproduction in primed plants suggest that priming can entail significant costs for plants. These costs, however, may only become apparent when priming cues fail to provide accurate information about prevailing threats, as was the case in this study. Additionally, our insecticide data indicate that pesticides or their carrier compounds can subtly, but significantly, affect plant physiology and may interact with plant defences.

Are Eurosta solidaginis on Solidago rugosa a divergent host-associated race?

The ball-gall fly Eurosta solidaginis is considered a classic example of host-race formation in herbivorous insects, with host-associated races evolving at least twice, including the well-known pair on Solidago altissima and S. gigantea. Yet E. solidaginis has been occasionally observed galling other Solidago species. Here, we explore the origins of E. solidaginis on Solidago rugosa. We hypothesize that flies associated with S. rugosa are derived from the S. altissima host-race, and ask whether S. rugosa-associated flies have initiated host-race formation. We compared genetic variation among seventeen E. solidaginis populations collected from S. rugosa, S. altissima, and S. gigantea, in an adjacent COI/COII region of the mitochondrial genome. Across the study area, E. solidaginis flies from S. rugosa were as diverged from S. altissima-flies as they were from S. gigantea-flies (pairwise ΦPT 0.075 and 0.78 respectively) but S. altissima and S. gigantea-flies appeared considerably less diverged (0.002). This pattern was driven by the majority of flies, regardless of host-plant, sharing the same haplotype across the study area. However, we detected several site/region-specific haplotypes, not shared among host species. At the local site scale we were able to distinguish S. gigantea-associated fly haplotypes from either S. altissima or S. rugosa haplotypes, but the majority of S. altissima and S. rugosa-flies shared the same haplotype locally. These patterns of haplotype diversity support existing evidence of host-associated divergence in S. altissima- and S. gigantea-associated flies, and suggest that S. rugosa flies are either the same host-race as, or are recently derived from, S. altissima flies. Successful development on S. rugosa and the use of S. rugosa in the absence of use on sympatric S. altissima suggests that E. solidaginis either has a single oligophagous race using both S. altissima and S. rugosa or is in the earliest stages of host-associated differentiation.

Repeated freezing costs flies offspring

In the search for inhabitable planets beyond our solar system, water is top of the list of requirements for life. However, when temperatures fall below 0°C, water becomes our enemy, shattering delicate cell structures and destroying life. Brent Sinclair, from the University of Western Ontario, Canada, explains that overwintering insects in Canada frequently experience temperatures well below −40°C as maggots, yet emerge unscathed as adults in spring; and the survivors have usually endured multiple icy encounters. He suspects that goldenrod gall flies (Eurosta solidaginis) – which spend the winter entombed in a dead plant gall – can freeze as many as 20 times before spring. ‘But most of the experiments have been done by simply exposing animals to one cycle of cold’, says Sinclair. Wondering how the robust maggots respond as the mercury repeatedly plunges below zero, Sinclair and Katie Marshall designed an extensive series of simulated winters to test the insects’ survival strategies.Fortunately, 2009 was a bumper year for the flies, with more than half of the 5000 goldenrod galls harvested by Marshall and an army of undergraduates yielding juicy fat maggots. Marshall then repeatedly froze the undeveloped insects to temperatures of −10, −15 or −20°C for 12 h intervals, interspersed with recovery periods at 0.2°C of 1, 5 and 10 days, before repeating the chilly experience 2, 5 or 9 more times. However, 3 days in, disaster stuck. Marshall seriously fractured her leg slipping on ice. Fortunately, the lab rallied round. ‘Katie knew this was going to be an epic experiment, so had made a detailed protocol for what treatments needed to happen and when’, recalls Sinclair, adding, ‘Even so, Katie did a lot of this from a wheelchair’.Eventually, after months of patiently collecting maggots at the end of their personal chilling protocols, Marshall meticulously measured the amount of antifreeze in their bodies – including sweet-tasting sorbitol – in addition to how the types of lipids varied and recording how many maggots survived, going on to develop into adults.Impressively, the maggots survived repeated freezing remarkably well, with the insects that had the longest recovery period (10 days) recording a survival rate of 80% at the lowest temperature (−20°C). Also, the maggots that had experienced ten 12 h long freezing sessions contained ∼50% more sorbitol than the maggots that had been frozen for a single 120 h period. In addition, Marshall discovered that the repeatedly frozen insects had converted more of their high-energy lipids into acetylated triacylglycerols, which are less energy rich but remain fluid at low temperatures.Most intriguing was the effect that repeated freezing had on the amount of energy that each female could invest in her own reproduction. Knowing that the adult female flies emerge complete with a lifetime's supply of eggs ready for their brief existence, Marshall wondered how repeated freezing might impact the number of eggs carried by the new adults. After allowing some of the maggots to develop, she counted the eggs carried by the female adults; the winter experience had taken a toll, reducing egg production by almost 10%.‘Repeated freezing matters’, says Sinclair, adding that extrapolations based on single freezing events could significantly underestimate what happens in the real world. He also hopes to continue his long-term field study of overwintering maggots in order to identify factors that will allow him to predict how well insects will tolerate future winters. ‘We've been going for a decade … maybe another 15 years or so will do the trick’, he smiles.

A New Class of Acetyl‐TAG Present in Seed Oils of Polygala Species

A New Class of Acetyl‐TAG Present in Seed Oils of <i>Polygala</i> Species

Consumer-resource interactions along urbanization gradients drive natural selection.

Urbanization is an important component of global change. Urbanization affects species interactions, but the evolutionary implications are rarely studied. We investigate the evolutionary consequences of a common pattern: the loss of high trophic-level species in urban areas. Using a gall-forming fly, Eurosta solidaginis, and its natural enemies that select for opposite gall sizes, we test for patterns of enemy loss, selection, and local adaptation along five urbanization gradients. Eurosta declined in urban areas, as did predation by birds, which preferentially consume gallmakers that induce large galls. These declines were linked to changes in habitat availability, namely reduced forest cover in urban areas. Conversely, a parasitoid that attacks gallmakers that induce small galls was unaffected by urbanization. Changes in patterns of attack by birds and parasitoids resulted in stronger directional selection, but loss of stabilizing selection in urban areas, a pattern which we suggest may be general. Despite divergent selective regimes, gall size did not very systematically with urbanization, suggesting but not conclusively demonstrating that environmental differences, gene flow, or drift, may have prevented the adaptive divergence of phenotypes. We argue that the evolutionary effects of urbanization will have predictable consequences for patterns of species interactions and natural selection.

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.