Exploring the symbiont diversity of ancient western redcedars: arbuscular mycorrhizal fungi of long‐lived hosts

Arbuscular mycorrhizal fungi (AMF) are globally distributed, monophyletic root symbionts with ancient origins. Their contribution to carbon cycling and nutrient dynamics is ecologically important, given their obligate association with over 70% of vascular plant species. Current understanding of AMF species richness and community structure is based primarily on studies of grasses, herbs and agricultural crops, typically in disturbed environments. Few studies have considered AMF interactions with long‐lived woody perennial species in undisturbed ecosystems. Here we examined AMF communities associated with roots and soils of young, mature and old western redcedar (Thuja plicata) at two sites in the old‐growth temperate rainforests of British Columbia. Due to the unique biology of AMF, community richness and structure were assessed using a conservative, clade‐based approach. We found 91 AMF OTUs across all samples, with significantly greater AMF richness in the southern site, but no differences in richness along the host chronosequence at either site. All host age classes harboured AMF communities that were overdispersed (more different to each other than expected by chance), with young tree communities most resembling old tree communities. A comparison with similar clade richness data obtained from the literature indicates that western redcedar AMF communities are as rich as those of grasses, tropical trees and palms. Our examination of undisturbed temperate old‐growth rainforests suggests that priority effects, rather than succession, are an important aspect of AMF community assembly in this ecosystem.     

C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind l-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N^G-nitro-l-arginine, but not its inactive d-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.     

Landscape analysis of adult codling moth (Lepidoptera: Tortricidae) distribution and dispersal within typical agroecosystems dominated by apple production in central Chile

We analyzed the spatial distribution and dispersal of codling moth, Cydia pomonella (L.), adults within two heterogeneous agroecosystems typical of central Chile: commercial apple, Malus domestica Borkhausen, orchards surrounded by various unmanaged host plants. Both a geostatistical analysis of catches of adult males with a grid of sex pheromone-baited traps and an immunological self-marking technique combined with traps baited with a male and female attractant were used. The spatial analyses identified the key sources of moths within these diverse landscapes. Codling moth catches in traps were spatially associated within distances of ≈ 150-300 m. Similarly, the mean distance from the immunological self-marking plots within the commercial apple orchard to the traps that captured marked adults was 282 m. In contrast, the mean distance in the capture of marked moths from unmanaged self-marking plots to a commercial orchard was 828 m. These data suggest that the success of any future area-wide management programs for codling moth in Chilean pome fruit must include a component for managing or removing noncommercial hosts that surround orchards. This analysis also suggests that the selection pressure for resistance imposed by insecticide sprays within managed orchards is likely dampened by the influx of susceptible moths from unmanaged sites common in central Chile.     

Landscape composition modulates population genetic structure of Eriosoma lanigerum (Hausmann) on Malus domestica Borkh in central Chile

Landscape genetics have been particularly relevant when assessing the influence of landscape characteristics on the genetic variability and the identification of barriers to gene flow. Linking current practices of area-wide pest management information on pest population genetics and geographical barriers would increase the efficiency of these programs. The woolly apple aphid, Eriosoma lanigerum (Hausmann), an important pest of apple orchards worldwide, was collected on apple trees (Malus domestica Borkh) from different locations in a 400 km north-south transect trough central Chile. In order to determine if there was population structure, diversity and flow were assessed. A total of 215 individuals from these locations were analysed using Inter Simple Sequence Repeat (ISSR) markers. Four ISSR primers generated a total of 114 polymorphic loci. The percentage of molecular variation among locations was 18%. As the algorithm used by structure may be poorly suited for inferring the number of genetic clusters in a data set that has an IBD relationship, the number of genetic clusters in the samples was also analyzed using a Bayesian clustering method implemented in software Baps version 4.14. We inferred the presence of four genetic clusters in the study region. Clustering of individuals followed a pattern explained by some geographical barriers. Using partial Mantel tests, we detected barriers to gene flow other than distance, created by a combination of main rivers and mountains. Although landscape genetics are rarely used in pest management, our results suggest that these tools may be suitable for the design of area-wide pest management programs.     

On the variability of the chaetognath Sagitta friderici Ritter-Zàhony at Cananéia Lagoon Estuarine system, São Paulo, Brazil

Diel and monthly variations in abundance, stage composition, biomass, production and feeding rates of the chaetognath Sagitta friderici were studied in the Cananéia Lagoon Estuarine System, São Paulo, Brazil. Plankton samples were collected at intervals of 4 h over several 24-h periods from February 1995 to January 1996. Sagitta friderici occurred throughout the year. Abundance of S. friderici showed considerable diel and monthly variations, ranging from 2 to 373 ind. m^?3. During the low-salinity period of February and March, higher abundance was found when salinity was higher, but from May to January abundance did not show consistent pattern in relation to time of day and tidal cycle. Seasonal variation in total body length was related to temperature variations. Mean biomass ranged from 10.593 to 57.533 mgDW m^?3. The daily production rate of S. friderici ranged from 0.009 to 4.488 mgC m^?3 d^?1. Sagitta friderici preyed mainly upon copepods (63.2%) such as Acartia (8.3%), Oncaea (7.4%) and Corycaeus (6.7%), among other prey. The daily mean feeding rate was 1.33 prey d^?1 and it was related to the water temperature.     

An alternative approach for gene transfer in trees using wild-typeAgrobacterium strains

Micropropagated shoots of three forest tree species, poplar (Populus tremula × P.alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra × J. regia), were inoculated each with six different wild-typeAgrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with anAgrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carryingneo (kanamycin resistance) anduidA (-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to expressneo anduidA genes. These results suggest that wild-typeAgrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.This work is dedicated to the late Marie-France Michel who initiated the poplar biotechnology project at INRA.     

Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we determine if rare fungal species can be reliably detected by T-RFLP analysis. Spores from three arbuscular mycorrhizal fungal species were each mixed at a range of concentrations (1%, 10%, 50%, and 100%) with Glomus irregulare to establish a minimum detection threshold. T-RFLP analysis was capable of detecting diagnostic peaks of rare taxa at concentrations as low as 1%. The relative proportion of the target taxa in the sample and DNA concentration influenced peak detection reliability. However, low concentrations produced small, inconsistent electropherogram peaks contributing to difficulty in differentiating true peaks from signal noise. The results of this experiment suggest T-RFLP is a reproducible and high fidelity procedure, which requires careful data interpretation in order to accurately characterize sample diversity.     

Significance of uncoupling protein 3 in mitochondrial function upon mid- and long-term dietary high-fat exposure

Uncoupling protein 3 (UCP3) may reduce mitochondrial ROS production, and thereby protect against mitochondrial dysfunction in skeletal muscle. UCP3 has been suggested to specifically fulfill this role under high-fat conditions. Here we show that UCP3 knockout mice indeed have elevated mitochondrial ROS production after short-term (8 weeks) high-fat feeding. After 26 weeks of high-fat feeding, UCP3 knockout mice exhibited reduced mitochondrial function as measured ex vivo in isolated mitochondria. In conclusion, these data suggest that UCP3 may have a role in the protection of mitochondria against lipid-induced mitochondrial dysfunction, but only after long-term exposure to high-fat.