Genetic architecture of resistance to aphids and mites in a willow hybrid system.

Hybrid plants often differ in resistance to arthropods compared to the parental species from which they are derived. To better understand the relative contribution of genetic effects in influencing plant resistance to arthropods, we examined the genetic architecture of resistance in a willow hybrid system, Salix eriocephala, S. sericea, and their interspecific hybrids. Resistance to two arthropods, a willow leaf aphid (Chaitophorus sp.: Aphididae) and an eriophyoid mite (Aculops tetanothrix: Eriophyidae), were compared because resistance to different herbivores may be controlled by different traits and influenced by different genetic effects. We found additive and nonadditive genetic effects to be important in explaining the difference between willow species in resistance to aphids and mites. F2 hybrids exhibited low resistance to aphids, suggesting breakdown of favourable epistatic interactions that confer resistance. F2 hybrids, however, exhibited high resistance to mites, suggesting either the breakdown of interactions that affect traits used by mites in host location or the creation of favourable epistatic interactions. This study demonstrates the potential role of herbivores in affecting plant genetic structure, such that selection by herbivores can potentially lead to the creation of gene interactions that influence host resistance traits or host recognition traits used by the herbivore.

Genetic architecture of susceptibility to herbivores in hybrid willows.

We performed a common garden experiment using parental, F1, F2, and backcross willow hybrids to test the hypothesis that hybrid willows experience breakdown of resistance to herbivores. After exposing plants to herbivores in the field, we measured the densities/damage caused by 13 insect herbivores and one herbivorous mite. Using joint-scaling tests, we determined the contribution of additive, dominance, and epistasis to variation in susceptibility to herbivores (measured either as density or damage level) among the six genetic classes. We found the genetic architecture of susceptibility/resistance in the parental species to be complex, involving additive, dominance, and epistasis for each herbivore species. Although genic interactions altered plant susceptibility for each of the 14 herbivores, three distinct patterns of response of herbivores to hybrids were expressed. One pattern, observed in four herbivore species, supported the hypothesis of breakdown of resistance genes in recombinant hybrids. A second pattern, shown by six other herbivore species, supported the hypothesis of hybrid breakdown of host recognition genes. In other words, epistatic interactions for host recognition traits (probably oviposition/feeding stimulants or attractants) appeared to be important in determining herbivore abundance for those six species. The final patterns supported a structure of dominance, either for host recognition traits (in the case of three herbivore species) or for host resistance traits (for one herbivore species). The combination of differing responses of herbivore species, including members of the same genus and tribe, and the ubiquitous importance of epistasis suggests that many genes affect herbivore resistance in this hybrid willow system.

Symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses.

In experimental human influenza infection initiated by nasal inoculation, the magnitude of viral replication, fever, and symptoms correlate with nasopharyngeal lavage fluid levels of various cytokines. Our aim was to assess these relationships in patients with naturally occurring acute influenza. Patients with culture-positive influenza illness of less than 36 hr of duration were studied. Nasopharyngeal washing were collected at enrollment and on Day 2, 4, 6 and 8 for quantitative virus isolation and IL-6, TNF-alpha, INF-alpha, INF-gamma and IL-10 determinations. Blood samples collected at entry and on Day 2 and 6 were processed to assess plasma cytokines and circulating influenza RNA. Patients received either oseltamivir or placebo for 5 days. We assessed the correlation between nasopharyngeal lavage fluid or blood levels of cytokines before treatment and viral titers, symptom severity and fever. Sixteen adult subjects (median age of 22 years) were studied. In this small group of patients no significant differences between placebo and oseltamivir patients were found in viral replication or measures of cytokines. Thus the data for all 16 subjects were pooled for analysis. At entry, influenza A viruses were cultured from nasopharyngeal washes at a median titer of 4.8 log(10)TCID(50)/ml of wash. Viral titers correlated positively with symptom score (P = 0.006) and temperature values (P < 0.001). Viral titers, fever and symptoms were highest at enrollment and fell in parallel during the subsequent days. RT-PCR assays failed to detect influenza RNA in the white blood cells from any patient. We observed a significant release, in both nasopharyngeal lavage fluid and in plasma, of IL-6, TNF-alpha, INF-alpha, INF-gamma and IL-10. At entry high IL-6 levels were detected in the nasopharyngeal lavage fluid (median 10.3 pg/ml) and plasma (median 5.1 pg/ml) of all patients. We found a positive correlation between plasma IL-6 levels and both symptom scores and temperature values (P < 0.05), as well as a positive correlation between nasopharyngeal lavage fluid levels of IL-6 and TNF-alpha and temperature (P < 0.05). We did not find significant associations between symptoms, fever and levels of INF-alpha, INF-gamma or IL-10. The magnitude of early decrease in viral titers correlated with initial levels of INF-gamma in nasopharyngeal lavage fluid (P < 0.05). Significant production of IL-6, TNF-alpha, INF-alpha, INF-gamma and IL-10 occurs in response to community acquired influenza A illness. As in experimental influenza, symptoms and fever in natural acute influenza correlate with the release of IL-6.

Morphological and molecular evidence for hybridization and introgression in a willow (Salix) hybrid zone.

Hybrid zones provide biologists with the opportunity to examine genetic and ecological interactions between differentiated populations. Accurate identification of hybrid genealogies is considered a necessary prerequisite to understanding observed patterns of hybridization-related phenomena. We analysed molecular and morphological data from individuals in a hybrid zone between two species of willows (Salix sericea Marshall and S. eriocephala Michaux) and report the use of randomly amplified polymorphic DNA (RAPD), chloroplast DNA (cpDNA), and ribosomal DNA (rDNA) markers, as well as vegetative morphology and foliar chemistry data to identify individuals in terms of hybrid genealogy and to infer the direction and extent of backcrossing and introgression within the hybrid zone. A novel version of a maximum likelihood estimate approach (developed for this study) was used to calculate hybrid index scores from RAPD marker data; this method produced results similar to those obtained using traditional arithmetic methods. Distribution of rDNA, cpDNA, and chemistry data were examined within the graphical context of RAPD-based hybrid index score histograms and principal component analyses (PCA) on RAPD and morphology data. Seven of the 21 plants classified as S. eriocephala in the field were possible introgressants. Another plant presented an unequivocal example of backcrossed S. sericea chemistry and RAPD markers. Inter- and intraspecific chloroplast diversity found within the hybrid zone suggests both historic introgression (perhaps in a glacial refugium), and contemporary hybridization. Patterns of inheritance and expression within the hybrid zone suggest that morphological characters are often not expressed in a simple additive fashion, and problems associated with both morphological and molecular data are considered.

Use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment.

CONTEXT: Influenza virus neuraminidase is thought to be essential for virus replication in humans; however, to date, available neuraminidase inhibitors are limited to zanamivir, which is topically administered. OBJECTIVE: To determine the safety, tolerability, and antiviral activity of oral neuraminidase inhibitor oseltamivir (GS4104/Ro64-0796) for prevention and the early treatment of influenza in experimentally infected humans. DESIGN: Two randomized, double-blind, placebo-controlled trials conducted between June and July 1997. SETTING: Individual hotel rooms; 2 large US university medical schools. PARTICIPANTS: A total of 117 healthy adult volunteers (aged 18-40 years; median age, 21 years) who were susceptible (hemagglutination-inhibition antibody titer < or =1:8). INTERVENTIONS: All subjects were inoculated intranasally with influenza A/Texas/36/91 (H1N1) virus. For the prophylaxis study, oral oseltamivir (100 mg once daily [n = 12], 100 mg twice daily [n = 12], or matching placebo [n = 13], starting 26 hours before virus inoculation) was administered. For the treatment study, the same drug was given (20 mg, 100 mg, or 200 mg twice daily, 200 mg once daily, or matching placebo [n = 16], in each group starting 28 hours after inoculation). All regimens were continued for 5 days. MAIN OUTCOME MEASURES: Comparing placebo groups with pooled treatment groups, for prophylaxis, outcomes included frequency of infection and viral shedding; for treatment, viral shedding in titers. RESULTS: In the prophylaxis study, 8 (67%) of 12 placebo and 8 (38%) of 21 oseltamivir recipients became infected (P = .16; efficacy, 61%); 6 (50%) placebo compared with 0 oseltamivir recipients shed virus (P<.001; efficacy, 100%), and 33% of placebo but no oseltamivir recipient had infection-related respiratory illness (P<.01). Among infected subjects in the treatment study (n = 69), the viral titer area under the curve of the combined oseltamivir groups (n = 56) was lower (median [interquartile range [IQR]], 80 [23-151] vs 273 [79-306] log10 tissue culture-infective doses50 per milliliter x hour; P = .02) than the placebo group (n = 13), and the median (IQR) duration of viral shedding with therapy was reduced from 107 (83-131) to 58 (35-59) hours (P = .003). Oseltamivir treatment also reduced symptom scores (median [IQR] score-hours, 225 [97-349] vs 400 [189-645]; P = .05), and nasal proinflammatory cytokine levels. Transient mild to moderate nausea after dosing was observed in 15 (17%) of 88 oseltamivir and 2 (7%) of 29 placebo recipients (95% confidence interval for difference, -11% to 68%), which was largely prevented by ingestion with food. CONCLUSIONS: In these trials, prophylaxis and early treatment with oral oseltamivir were both associated with significant antiviral and clinical effects in experimental human influenza.

Nasal cytokine and chemokine responses in experimental influenza A virus infection: results of a placebo-controlled trial of intravenous zanamivir treatment.

The local immune response to influenza virus infection was characterized by determining cytokine and chemokine levels in serial nasal lavage fluid samples from 15 volunteers experimentally infected with influenza A/Texas/36/91 (H1N1). The study was part of a randomized double-blind placebo-controlled trial to determine the prophylactic effect of intravenous zanamivir (600 mg 2x/day for 5 days), a highly selective inhibitor of influenza A and B virus neuraminidases, on the clinical symptoms of influenza infection. Nasal lavage fluid levels of interleukin (IL)-6, tumor necrosis factor-alpha, interferon-gamma, IL-10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1alpha and -1beta increased in response to influenza virus infection and correlated statistically with the magnitude and time course of the symptoms. Treatment with zanamivir prevented the infection and abrogated the local cytokine and chemokine responses. These results reveal a complex interplay of cytokines and chemokines in the development of symptoms and resolution of influenza.

Secondary chemistry of hybrid and parental willows: Phenolic glycosides and condensed tannins inSalix sericea, S. eriocephala, and their hybrids.

Salix sericea andS. eriocephala differ markedly in secondary chemistry.S. sericea produces phenolic glycosides, salicortin and 2'-cinnamoylsalicortin, and low concentrations of condensed tannin. In contrast,S. eriocephala produces no phenolic glycosides, but high concentrations of condensed tannins. Hybrid chemistry is intermediate for both types of chemicals, suggesting predominantly additive inheritance of these two defensive chemical systems from the parental species. However, there is extensive variation among hybrids. This variation may be due to genetic variation among parental genotypes, which genes were passed on, or to subsequent back-crossing. The differences in chemistry are likely to exert a strong effect on the relative susceptibility of hybrid and parental willows to herbivores.

Interspecific hybridization of plants and resistance to herbivores: hypotheses, genetics, and variable responses in a diverse herbivore community.

We studied the morphology, molecular genetics, and hebivory of two species of willows (Salix sericea and S. eriocephala) and their interspecific hybrids to test four alternative hypotheses concerning the effects of hybridization on plant resistance. Individually marked plants were identified using morphological traits in the field and measurements of stipule and leaf pubescence were made and compared using Canonical Discriminant Function Analysis. DNA was extracted from the leaves of a sample of the marked plants and RAPD-PCR analysis was performed to establish the genetic status of parental and hybrid plants. RAPD band analysis generally verified the genetic status of parental plants. Hybrid plants were usually correctly identified in the field with a few exceptions. However, the hybrid plants were a heterogeneous group of plants made up of most plants that appear to be F1s and a few plants that appear to be backcrosses to S. sericea. Morphological variables were useful for distinguishing S. sericea from S. eriocephala and hybrids, but were not as dependable in distinguishing between S. eriocephala and hybrids. We compared the densities of 11 herbivore species and the infection by a leaf rust pathogen (Melampsora sp.) on the leaves and stems of two parents and the hybrids in the field. We found support for the Additive hypothesis (3 species), the Dominance hypothesis (2 species) and the Hybrid Susceptibility hypothesis (7 species, 6 herbivores and the Melampsora rust). We found no evidence for the Hybrid Resistance hypothesis. Guild membership was not a good predictor of similar responses of species to hybrid versus parental plants. A Canonical Discriminant Function Analysis showed discrete separation of the taxa based on herbivore densities, illustrating different community structures on hybrid and parental plants. This study demonstrates the diversity of responses of phytophages in response to interspecific hybridization.

The ecology and evolution of host-plant resistance to insects.

Genetic techniques have yielded new insights into plant-herbivore coevolution. Quantitative genetic tests of herbivory theory reveal that in some cases insect herbivores impose selection on resistance traits. Also, some resistance traits are costly while others appear not to be, and genetic models can explain these results. Genetic variation in plant resistance influences insect community structure by modifying interactions of herbivores with competitors and natural enemies. Therefore, models of multispecies coevolution are more realistic than pairwise coevolutionary models. Ecological genetics will facilitate further theoretical and empirical exploration of multispecies coevolution of plants and herbivores.

Nutritional regulation of hepatic glucose-6-phosphate dehydrogenase. Transient activation of transcription.

Hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is subject to nutritional and hormonal regulation. Previous work has shown that increased amounts of mRNA encoding G6PDH can account for the increase in enzyme activity. The results of this study demonstrate that transcription of the G6PDH gene is transiently elevated after ingestion of a high-carbohydrate diet. However, the increased rate of transcription cannot totally account for the increased G6PDH mRNA. The half-life of the G6PDH mRNA appears to be about 4-5-fold higher during ingestion of a high-carbohydrate diet. Thus increased transcription as well as mRNA stability are each partially responsible for the nutritional regulation of G6PDH mRNA.

Regulation of glucose-6-phosphate dehydrogenase by diet and thyroid hormone.

The regulation of hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) RNA by thyroid hormone and high carbohydrate (sucrose) diet was studied. Previous studies from several laboratories have demonstrated that thyroid hormone modulates G6PDH activity. However, the point at which thyroid hormone exerts this regulation has not been adequately addressed. In order to assess the role of thyroid hormone in this regulation, levels of G6PDH mRNA were determined in hypothyroid rats maintained on normal or high carbohydrate diets with or without thyroid hormone (triiodothyronine; T3) supplementation. A dot-blot hybridization procedure with nick-translated cDNA probes was used to directly assess the relative concentrations of G6PDH mRNA. Enzyme activity increased when the animals were treated with T3 and/or placed on a high carbohydrate diet. However, there was no effect of T3 and diet, alone or in combination, on G6PDH mRNA levels in hypothyroid rats. The data suggests that thyroid hormone and high carbohydrate diet are acting at a translational level to increase G6PDH enzyme activity in these animals.

Patterns of intra- and interspecific association of gall-forming sawflies in relation to shoot size on their willow host plant.

Four species of gall-forming sawflies were each frequently found to have clumped distributions among shoots within their willow host plant across four sites and three years. When all species were considered together by clone, year, and site, species showed independence of distribution among shoots two thirds of the time and showed positive covariance one third of the time. When pairs of species were considered separately, but clones were combined within sites and years, 60% of the chi-square tests of association were significant. All but one of the significant tests showed positive associations between pairs of species. The stem galler was positively associated with the leaf folder at all sites in all years, and the petiole galler was positively associated with the stem galler and leaf folder for most year by site combinations. When species paris co-occurred on shoots they were usually found at the same or higher density as when found alone on shoots. Only 2 of 100 tests showed a depressed density of a species when co-occurring on shoots with heterospecifics.All sawfly species were found on shoots that were significantly larger (mean node number) than on shoots without sawflies, and species responded to shoot size variation similarly. Sizes of shoots occupied by heterospecific species combinations were usually significantly larger than shoots with only conspecifics, for all species. These data supported the hypothesis that similar species' responses to within-plant variation would lead to positive rather than negative or random species associations. The data do not support the hypothesis that interspecific competition was important in determining shoot choice or species density.

Variation in herbivore density among host plants and its consequences for community structure : Field studies on willow sawflies.

The densities of four species of gall-forming sawflies were found to vary significantly among willow host plant clones. Two of the speices varied among host plants at four sites in each of three years. The other two species varied in density among host plants at most of the sites in two of the three years. Total sawfly density also varied significantly among clones. Individual species densities on willow clones were significantly positively correlated between years when all sites were combined and frequently when sites were considered separately. Most pairwise species combinations were independent in density between years, but some negative correlations existed between the stem galler and the leaf galler. Gall-former densities also were largely independent among clones within years with all sites combined and with sites considered separately. The significant correlations were nearly all positive. At all four sites the combination of significant variation in sawfly densities among willow clones in the field and independence of species densities among clones resulted in significantly different communities (relative abundance of species) among willow clones in three years. Although sawfly abundances differed substantially among the four sites, this remained true. It is argued that the pattern of community structure among clones is the result of variation in host plant quality of clones. We propose an hypothesis to account for patterns of herbivore species associations based on intrapopulation host plant variation.

Glucose-6-phosphate dehydrogenase mRNA sequence abundance in primary cultures of rat hepatocytes. Effect of insulin and dexamethasone.

Hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is subject to nutritional regulation. To assess the possible role of hormones in this regulation, the amounts of G6PDH mRNA were studied in primary cultures of rat hepatocytes treated with insulin and dexamethasone, alone or in combination. Relative concentrations of G6PDH mRNA were directly assessed by a dot-blot hybridization procedure with nick-translated cDNA probes. G6PDH sequence abundance increased when the cultures were treated with insulin or dexamethasone, but the G6PDH mRNA induced by dexamethasone was not expressed at the protein level as active enzyme. In cultures treated with insulin and dexamethasone in combination, enzyme activity and G6PDH sequence abundance were greater than those induced by insulin alone. Our results directly demonstrate that G6PDH mRNA amounts are modulated in liver by these two classes of hormones and can partially account for the dietary induction of the enzyme observed in vivo.