Induced defences alter the strength and direction of natural selection on reproductive traits in common milkweed.

Evolutionary biologists have long sought to understand the ecological processes that generate plant reproductive diversity. Recent evidence indicates that constitutive antiherbivore defences can alter natural selection on reproductive traits, but it is unclear whether induced defences will have the same effect and whether reduced foliar damage in defended plants is the cause of this pattern. In a factorial field experiment using common milkweed, Asclepias syriaca L., we induced plant defences using jasmonic acid (JA) and imposed foliar damage using scissors. We found that JA-induced plants experienced selection for more inflorescences that were smaller in size (fewer flowers), whereas control plants only experienced a trend towards selection for larger inflorescences (more flowers); all effects were independent of foliar damage. Our results demonstrate that induced defences can alter both the strength and direction of selection on reproductive traits, and suggest that antiherbivore defences may promote the evolution of plant reproductive diversity.

Tolerance to deer herbivory and resistance to insect herbivores in the common evening primrose (Oenothera biennis).

The evolution of plant defence in response to herbivory will depend on the fitness effects of damage, availability of genetic variation and potential ecological and genetic constraints on defence. Here, we examine the potential for evolution of tolerance to deer herbivory in Oenothera biennis while simultaneously considering resistance to natural insect herbivores. We examined (i) the effects of deer damage on fitness, (ii) the presence of genetic variation in tolerance and resistance, (iii) selection on tolerance, (iv) genetic correlations with resistance that could constrain evolution of tolerance and (v) plant traits that might predict defence. In a field experiment, we simulated deer damage occurring early and late in the season, recorded arthropod abundances, flowering phenology and measured growth rate and lifetime reproduction. Our study showed that deer herbivory has a negative effect on fitness, with effects being more pronounced for late-season damage. Selection acted to increase tolerance to deer damage, yet there was low and nonsignificant genetic variation in this trait. In contrast, there was substantial genetic variation in resistance to insect herbivores. Resistance was genetically uncorrelated with tolerance, whereas positive genetic correlations in resistance to insect herbivores suggest there exists diffuse selection on resistance traits. In addition, growth rate and flowering time did not predict variation in tolerance, but flowering phenology was genetically correlated with resistance. Our results suggest that deer damage has the potential to exert selection because browsing reduces plant fitness, but limited standing genetic variation in tolerance is expected to constrain adaptive evolution in O. biennis.

Ambient habitat noise and vibration at the Georgia Aquarium.

Underwater and in-air noise evaluations were completed in performance pool systems at Georgia Aquarium under normal operating conditions and with performance sound tracks playing. Ambient sound pressure levels at in-pool locations, with corresponding vibration measures from life support system (LSS) pumps, were measured in operating configurations, from shut down to full operation. Results indicate noise levels in the low frequency ranges below 100 Hz were the highest produced by the LSS relative to species hearing thresholds. The LSS had an acoustic impact of about 10 dB at frequencies up to 700 Hz, with a 20 dB re 1 µPa impact above 1000 Hz.

Adaptive molecular evolution of a defence gene in sexual but not functionally asexual evening primroses.

Theory predicts that sexual reproduction provides evolutionary advantages over asexual reproduction by reducing mutational load and increasing adaptive potential. Here, we test the latter prediction in the context of plant defences against pathogens because pathogens frequently reduce plant fitness and drive the evolution of plant defences. Specifically, we ask whether sexual evening primrose plant lineages (Onagraceae) have faster rates of adaptive molecular evolution and altered gene expression of a class I chitinase, a gene implicated in defence against pathogens, than functionally asexual evening primrose lineages. We found that the ratio of amino acid to silent substitutions (K(a) /K(s) = 0.19 vs. 0.11 for sexual and asexual lineages, respectively), the number of sites identified to be under positive selection (four vs. zero for sexual and asexual lineages, respectively) and the expression of chitinase were all higher in sexual than in asexual lineages. Our results are congruent with the conclusion that a loss of sexual recombination and segregation in the Onagraceae negatively affects adaptive structural and potentially regulatory evolution of a plant defence protein.

Diversity, ecology and herbivory of hairstreak butterflies (Theclinae) associated with the velvet tree, Miconia calvescens in Costa Rica.

Larvae of three species of hairstreak butterflies in the subfamily Theclinae (Lepidoptera: Lycaenidae) were found feeding on developing inflorescences, flower buds, and immature fruits of the velvet tree, Miconia calvescens DC. (Myrtales: Melastomataceae) in Costa Rica. Erora opisena (Druce), Parrhasius polibetes (Cramer), and Temecla paron (Godman and Salvin) were studied in association with M. calvescens, an uncommon tree in its natural range in the neotropics and a target for biocontrol as an invader in Pacific islands. Host plant use by the three theclines was similar, with eggs being laid on inflorescences and cryptic larvae remaining there throughout development. Feeding damage by E. opisena was most abundant in pre-flowering M. calvescens, when 23% of inflorescences showed feeding damage characteristic of this species. Feeding damage by T. paron peaked at flowering, when 30% of inflorescences were affected. At field sites, E. opisena and T. paron damaged an average of 26 and 18% of each attacked inflorescence, respectively. In cage experiments, individual third- and fourth-instar larvae of E. opisena damaged an average of 24 and 21% of an inflorescence before pupating, respectively. This study provides the first host plant record for E. opisena and T. paron, the first record of P. polibetes feeding on Melastomataceae, and the first records of E. opisena and T. paron presence in Costa Rica.

Heritability, covariation and natural selection on 24 traits of common evening primrose (Oenothera biennis) from a field experiment.

This study explored genetic variation and co-variation in multiple functional plant traits. Our goal was to characterize selection, heritabilities and genetic correlations among different types of traits to gain insight into the evolutionary ecology of plant populations and their interactions with insect herbivores. In a field experiment, we detected significant heritable variation for each of 24 traits of Oenothera biennis and extensive genetic covariance among traits. Traits with diverse functions formed several distinct groups that exhibited positive genetic covariation with each other. Genetic variation in life-history traits and secondary chemistry together explained a large proportion of variation in herbivory (r(2) = 0.73). At the same time, selection acted on lifetime biomass, life-history traits and two secondary compounds of O. biennis, explaining over 95% of the variation in relative fitness among genotypes. The combination of genetic covariances and directional selection acting on multiple traits suggests that adaptive evolution of particular traits is constrained, and that correlated evolution of groups of traits will occur, which is expected to drive the evolution of increased herbivore susceptibility. As a whole, our study indicates that an examination of genetic variation and covariation among many different types of traits can provide greater insight into the evolutionary ecology of plant populations and plant-herbivore interactions.

PERMANENT GENETIC RESOURCES: Isolation and characterization of polymorphic microsatellite loci in common evening primrose (Oenothera biennis).

We developed nine polymorphic microsatellite loci for evening primrose (Oenothera biennis). These loci have two to 18 alleles per locus and observed heterozygosities ranging from 0 to 0.879 in a sample of 34 individuals. In a pattern consistent with the functionally asexual reproductive system of this species, 17/36 pairs of loci revealed significant linkage disequilibrium and three loci showed significant deviations from Hardy-Weinberg equilibrium. The loci will be informative in identifying genotypes in multigenerational field studies to assess changes in genotype frequencies.

Genotype-by-environment interactions leads to variable selection on life-history strategy in Common Evening Primrose (Oenothera biennis).

Monocarpic plant species, where reproduction is fatal, frequently exhibit variation in the length of their prereproductive period prior to flowering. If this life-history variation in flowering strategy has a genetic basis, genotype-by-environment interactions (G x E) may maintain phenotypic diversity in flowering strategy. The native monocarpic plant Common Evening Primrose (Oenothera biennis L., Onagraceae) exhibits phenotypic variation for annual vs. biennial flowering strategies. I tested whether there was a genetic basis to variation in flowering strategy in O. biennis, and whether environmental variation causes G x E that imposes variable selection on flowering strategy. In a field experiment, I randomized more than 900 plants from 14 clonal families (genotypes) into five distinct habitats that represented a natural productivity gradient. G x E strongly affected the lifetime fruit production of O. biennis, with the rank-order in relative fitness of genotypes changing substantially between habitats. I detected genetic variation in annual vs. biennial strategies in most habitats, as well as a G x E effect on flowering strategy. This variation in flowering strategy was correlated with genetic variation in relative fitness, and phenotypic and genotypic selection analyses revealed that environmental variation resulted in variable directional selection on annual vs. biennial strategies. Specifically, a biennial strategy was favoured in moderately productive environments, whereas an annual strategy was favoured in low-productivity environments. These results highlight the importance of variable selection for the maintenance of genetic variation in the life-history strategy of a monocarpic plant.

Nontarget effects--the Achilles' heel of biological control? Retrospective analyses to reduce risk associated with biocontrol introductions.

Controversy exists over ecological risks in classical biological control. We reviewed 10 projects with quantitative data on nontarget effects. Ten patterns emerged: (a) Relatives of the pest are most likely to be attacked; (b) host-specificity testing defines physiological host range, but not ecological range; (c) prediction of ecological consequences requires population data; (d) level of impact varied, often in relation to environmental conditions; (e) information on magnitude of nontarget impact is sparse; (f) attack on rare native species can accelerate their decline; (g) nontarget effects can be indirect; (h) agents disperse from agroecosystems; (i) whole assemblages of species can be perturbed; and (j) no evidence on adaptation is available in these cases. The review leads to six recommendations: Avoid using generalists or adventive species; expand host-specificity testing; incorporate more ecological information; consider ecological risk in target selection; prioritize agents; and pursue genetic data on adaptation. We conclude that retrospective analyses suggest clear ways to further increase future safety of biocontrol.

Effect of ultraviolet B radiation and 100 Hz electromagnetic fields on proliferation and DNA synthesis of Jurkat cells.

The use of ultraviolet B light (UVB) has been proven to be highly effective for treatment of various inflammatory skin diseases, but UVB phototherapy is limited by its carcinogenic side effects. It is necessary to uncover effectors that augment UVB so that similar or improved efficacy can be obtained with lower UVB doses. We found that low frequency, low intensity electromagnetic fields (EMFs) can act as such an effector and synergistically inhibit T lymphocyte proliferation. We first characterized the effects of UVB on Jurkat cells, a model for cutaneous T lymphocytes, and determined UVB's dose dependent inhibition of cell proliferation and induction of apoptosis. Cells exposed to a sublethal UVB dose retained their sensitivity to UVB, but repetitive irradiation seemed to cause accumulation of delayed DNA damage. We then exposed cells to combinations of UVB plus EMFs and found that 100 Hz, 1 mT EMFs decrease DNA synthesis of UVB-activated Jurkat cells by 34 +/- 13% compared to UVB alone. The decrease is, however, most effective when relatively high UVB doses are employed. Since EMFs alone had only a very weak inhibitory effect (10 +/- 2%), the data suggest that EMFs augment the cell killing effects of UVB in a synergistic way. These findings could provide the basis for development of new and improved clinical phototherapy protocols.

Central processes for the multiparametric control of arm movements in primates.

Recent single-unit recording studies have clarified how multiple parameters of movement are signaled by individual cortical and cerebellar neurons, and also that multiple coordinate frames are utilized. Cognitive processes also modulate the firing of these neurons. The various signals and coordinate systems vary in time and evolve throughout a behavioral sequence, consistent with the demands of the task and the required sensorimotor transformations.

Inactivation of the murine pyruvate dehydrogenase (Pdha1) gene and its effect on early embryonic development.

A deficiency of pyruvate dehydrogenase complex (PDC) in humans results in lactic acidosis and neurological dysfunction that frequently results in death during infancy. Using gene targeting technology, a silent mutation was introduced into the murine X-linked Pdha1 gene that encodes the alpha subunit of the pyruvate dehydrogenase or E1 component of the complex. Two loxP sequences were introduced into intronic sequences flanking exon 8 to generate the Pdha1(flox8) allele. In vitro studies in embryonic stem cells demonstrated that deletion of exon 8 ablated PDC activity. Homozygous Pdha1(flox8) females were bred with male mice carrying a wild-type Pdha1 allele and a transgene that ubiquitously expresses the Cre recombinase to produce progeny with a deletion in exon 8, Pdha1(Deltaex8). The majority of progeny were found to be mosaic with the presence of both the flox and deleted alleles, and there were no apparent phenotypic effects associated with the null allele. The mosaic mice were interbred to increase the degree of mosaicism for the Pdha1(Deltaex8) allele in the subsequent generation, resulting in a significantly smaller litter size (54% reduction). Embryos carrying predominantly the Pdha1(Deltaex8) allele were found to be globally delayed in development by 9.5 days postcoitus, with resorption occurring over the following several days. These findings demonstrate an essential role for oxidative metabolism of glucose during the early postimplantation period of prenatal development.

Electromagnetic field effects: changes in protein phosphorylation in the Jurkat E6.1 cell line.

This study is aimed at expanding the role of electromagnetic field (EMF) therapy for treatment of inflammatory diseases and obtaining new information on the biophysical mechanism of action of weak EMFs. The mechanism of action of EMFs on biological systems is a question that has yet to be answered. Several models have been proposed to explain the coupling of low frequency fields to biological systems, although no consensus has been reached as to which most adequately portrays the true mechanism. Protein phosphorylation is a major cellular metabolic regulator. As such, it has the potential to be a valuable indicator of the impact of EMFs on cellular metabolism. Using a well-controlled EMF exposure system, we examined the regulatory role of EMFs on low molecular weight protein phosphorylation in Jurkat E6.1 cells, a transformed human leukemic T cell line. Jurkat cells were grown to mid-log phase, preloaded with 32P and exposed to EMF (0.1 mT, 60 Hz) or sham for 30 minutes. Cell proteins were separated by SDS-polyacrylamide gel electrophoresis and incorporated radioactivity of low molecular weight proteins (18-23 kDa) was quantified by AMBIS data analysis. Three of five experiments showed no difference in protein phosphorylation in EMF exposed samples compared to controls, while two experiments revealed an EMF effect. We identified stathmin, an important T cell signaling phosphoprotein, as one of the low molecular weight proteins present in our Jurkat cell system. Stathmin expression as well as its phosphorylation was decreased in samples that were exposed to EMFs compared to controls. These data indicate that phosphorylation of individual proteins might be masked by the presence of numerous other proteins in whole cell lysate experiments. Further studies testing other low molecular weight T cell signaling molecules may validate this hypothesis.

Effect of a wound healing electromagnetic field on inflammatory cytokine gene expression in rats.

In earlier studies, we have shown that pulsed electromagnetic fields (PEMFs) induce programmed cell death in cultured T cells and that rats exposed in vivo to PEMFs have decreased T-cell proliferative capacity. These data led us to hypothesize that PEMFs might be used to control proliferation of inflammatory lymphocytes and therefore beneficially affect inflammatory diseases. Tendinitis is characterized by painful inflammation of the tendon. Inflammation is characterized by massive infiltration of T lymphocytes, neutrophils and macrophages into the damaged tissue. These inflammatory cells produce a variety of cytokines, which are the cellular regulators of inflammation. The current study tests whether in vivo PEMF effects are mediated via systemic cytokine production in rat tendinitis. Inflammation was chemically induced in female Harlan Sprague Dawley rats Achilles' tendons and a wound healing PEMF (Electrobiology, Inc.) was applied for 4 hours immediately following injury. Spleens from control and experimental animals were harvested 24 hours later and total RNA was extracted from the tissues. Gene expression was analyzed by reverse transcription of mRNA, and polymerase chain reaction amplification (RT-PCR) using primers specific for the cytokines IFN-gamma, IL-1 beta, IL-6, TNF-alpha, and TGF-beta, as well as for the control beta-actin. RT-PCR products were separated on 1.5% agarose gels and band intensities were normalized to beta-actin gene expression of the same sample. TGF-beta was the only cytokine produced at high levels in rats with tendinitis in comparison to the other cytokines. PEMFs did not show an effect on any cytokine expression in the spleens, 24 hours after induction of tendinitis. Further studies need to test if cumulative exposures of PEMFs are able to regulate inflammatory cytokine expression either at the site of inflammation or at the local lymph nodes.

Electromagnetic fields used clinically to improve bone healing also impact lymphocyte proliferation in vitro.

An important aspect of medical device development is the need to understand how a device produces a specific biological effect. The focus can then be on optimizing that effect by device modification and repeated testing. Several reports from this lab have targeted programmed cell death, or apoptosis, as a cellular pathway that is induced by exposure of transformed leukemic T-cells in culture to specific frequency and intensity electromagnetic fields (EMFs). An EMF delivery device capable of selectively inducing T-cell apoptosis in human tissues could be used to enhance healing by limiting the production of molecules that promote inflammatory disorders such as psoriasis and tendonitis. In the present study, we examined the normal T-cell response to EMF exposure in vitro. In the peripheral blood, 70-80% of the lymphocytes are T-cells, and thus is a rich source of normal cells that match the transformed T-cells used in other experiments (Jurkat cells). We isolated lymphocytes from the peripheral blood of humans and rats, cultured them in nutritive medium and exposed them to either a complex 1.8 mT pulsed EMF (Electrobiology, Inc.), a 0.1 mT, 60 Hz power frequency EMF or a 0.2 mT, 100 Hz sinusoidal EMF. Control lymphocytes were cultured similarly, without field exposure. Lymphocytes were then treated with T-cell mitogens and evaluated for proliferative capacity after an additional 72 hours culture. Results indicate that T-cell proliferation is modulated by in vitro exposure to defined EMFs. The potential use of an EMF delivery device capable of selectively inducing such T-cell effects is discussed.

A system for simultaneous ultraviolet light and electromagnetic field exposure in in vitro experiments.

Ultraviolet light (UV) is a common treatment for skin diseases such as psoriasis, but bears the risk of carcinogenic side effects. We have biological evidence that electromagnetic fields (EMFs) can act additively with UV so that new therapeutic protocols combining UV and EMF might be developed to improve psoriasis phototherapy. In this study we report on a system that allows in vitro experiments testing this hypothesis. For simultaneous exposure of cell cultures to UVB and EMF, we built Merritt coils with an integrated UV exposure system. The coils can be operated in a sham or experimental mode (up to 1.5 mT and 20,000 Hz). Two UV bulbs were fitted inside the coils for UVB doses between 100-1000 J/m2/nm. In the exposure area the EMF is uniform within 0.0038%. For exposure, the cells are cultured in standard culture plates and placed in a specifically designed box. The box holds two plates in a top chamber covered with a Saran Wrap lid (91% UV transmission) so that cells are exposed to UVB and EMFs. The bottom chamber holds two plates, where cells are screened from UVB and only exposed to EMFs. Temperature control is maintained (+/- 1 degree C) by airflow vents on the side of the box and a fan placed 25 cm away from the cell culture box. To maintain sterility within the box the vents are covered with a bacterial filter. The box lid has additional ventilation through two air direction changes to create an additional bacterial barrier similar to that in culture plate lids.

Randomized trial of progressive resistance training to counteract the myopathy of chronic heart failure.

Chronic heart failure (CHF) is characterized by a skeletal muscle myopathy not optimally addressed by current treatment paradigms or aerobic exercise. Sixteen older women with CHF were compared with 80 age-matched peers without CHF and randomized to progressive resistance training or control stretching exercises for 10 wk. Women with CHF had significantly lower muscle strength (P < 0.0001) but comparable aerobic capacity to women without CHF. Exercise training was well tolerated and resulted in no changes in resting cardiac indexes in CHF patients. Strength improved by an average of 43.4 +/- 8.8% in resistance trainers vs. -1.7 +/- 2.8% in controls (P = 0.001), muscle endurance by 299 +/- 66% vs. 1 +/- 3% (P = 0.001), and 6-min walk distance by 49 +/- 14 m (13%) vs. -3 +/- 19 m (-3%) (P = 0.03). Increases in type I fiber area (9.5 +/- 16%) and citrate synthase activity (35 +/- 21%) in skeletal muscle were independently predictive of improved 6-min walk distance (r2 = 0.78; P = 0.0024). High-intensity progressive resistance training improves impaired skeletal muscle characteristics and overall exercise performance in older women with CHF. These gains are largely explained by skeletal muscle and not resting cardiac adaptations.

Actinide interactions with microbial chelators: the dioxobis[pyridine-2,6-bis(monothiocarboxylato)]uranium(VI) ion.

The title complex, bis(tetraphenylphosphonium) dioxobis(pyridine-2,6-dicarbothioato-O,N,O')uranium(VI), (C(24)H(20)P)(2)[UO(2)(C(7)H(3)NO(2)S(2))(2)], was prepared by reacting two equivalents of pyridine-2,6-bis(monothiocarboxylate) (pdtc) with uranyl nitrate. The geometry of the eight-coordinate U atom is hexagonal bipyramidal, with the uranyl O atoms in apical positions. This is the first reported complex in which this ligand binds a metal through the O and not the S atoms. Principal bond lengths include uranyl lengths of 1.774 (2) A, U--O distances of 2.434 (2) and 2.447 (3) A, and two U--N distances of 2.647 (3) A. The anion lies on an inversion centre.

Population code for tracking velocity based on cerebellar Purkinje cell simple spike firing in monkeys.

Velocity is an important determinant of the simple spike discharge of cerebellar Purkinje cells. In a previous study, Purkinje cells in the intermediate and lateral cerebellum recorded during manual tracking were found to be tuned to a combination of direction and speed, (i.e. preferred velocity). In this study a population analysis of this simple spike discharge was used to determine whether the velocity of tracking could be predicted. For the majority (30/32) of direction-speed combinations, the population response accurately specified the target velocity. A temporal analysis showed how the population response gradually converged to the required velocity 200 ms prior to the onset of tracking. Therefore, the simple spike discharge of a Purkinje cell ensemble contains sufficient information to reconstruct target velocity, providing support for the hypothesis that the cerebellum controls or signals movement velocity.