Chestnut cultivar diversification process in the Iberian Peninsula, Canary Islands, and Azores

This is a large-scale molecular study based on simple sequence repeat (SSR) loci of the diversification process in chestnut cultivars from Portugal and Spain, from the northern Iberian Peninsula to the Canary Islands and the Azores. A total of 593 grafted chestnut trees (Castanea sativa Mill.) were analysed with 10 SSRs: 292 from Portugal and 301 from Spain. Some of the trees studied were more than 300 years old. Accessions were analysed using a model-based Bayesian procedure to assess the geographical structure and to assign individuals to reconstructed populations based on the SSR genotypes. We found 356 different genotypes with a mean value of clonality of 33% owing to grafting. Mutations accounted for 6%, with hybridization being the main diversification process that can explain the great diversity found. Ten main cultivar groups were detected: four in northern Spain, five in the centre of the Iberian Peninsula, and one in southern Spain related to the centre of the Iberian Peninsula. This work demonstrated that cultivar origin and the diversification process was a combination of clonal propagation of selected seedlings, hybridization, and mutations, which allowed high levels of diversity to be maintained with respect to selected clones for fruit production. Furthermore, seedlings and graft sticks facilitated the transport to new destinations in the colonization process, transporting sometimes more than 3000 km if we consider the Azores and the Canary Islands.     

Variability in the settlement of non-indigenous species in benthic communities from an oceanic island

The introduction of non-indigenous species (NIS) in new environments represents a major threat for coastal ecosystems. A good understanding of the mechanisms and magnitude of the impact of NIS colonisation on native ecosystems is becoming increasingly crucial to develop mitigation measures and prevent new invasions. In this present study, we asked if distinct coastal benthic communities from an oceanic island can have different vulnerability to NIS colonisation process. First, PVC settlement plates were deployed for 1 year on the rocky shore of two different locations of Madeira Island (North versus South coast). Then, we implemented a mesocosm experiment where recruited plate communities were maintained under different levels of NIS propagule pressure in order to assess their vulnerability to NIS colonisation process. Results showed that NIS colonisation success was not influenced by the level of propagule pressure, but however, final colonisation patterns varied depending on the origin of the communities. This variability can be attributed to major structural differences between the preponderant species of each community and therefore to the biotic substrate they offer to colonisers. This study highlights how biotic features can alter the NIS colonisation process and importantly, shows that in an urbanisation context, the nature of the resident communities facing invasions risks needs to be closely assessed.     

Field efficacy of triflumuron against Aedes and Culex mosquitoes in temperate Argentina

Aedes aegypti and Culex pipiens s.l. (Linnaeus, 1762 and 1758, respectively) (Diptera: Culicidae) are important vectors of diseases to humans and a growing public health concern. In order to contribute to the control of mosquito vectors by low environmental impact approaches we assessed the susceptibility of natural populations of container-breeding mosquitoes to triflumuron, an insect growth regulator, in temperate Argentina. A field trial was conducted to evaluate the efficacy of two doses (0.5 ppm and 1 ppm) of triflumuron (SC 48%) against natural populations of Ae. aegypti and Culex spp. immatures in flower vases of four cemeteries. The results demonstrated the susceptibility of both target mosquitoes to triflumuron in field conditions. For Ae. aegypti, dose-dependent reductions were achieved in the presence of pupae and the percentage of water-holding containers harbouring L3–4 and/or pupae, whereas the larvae abundance was equally reduced for both doses. For Culex spp., similar levels of reduction of larvae abundance and pupae presence were achieved with both doses. Significant effects on the response variables measured were recorded up to six to eight weeks post-intervention. Bimonthly applying 1 ppm triflumuron in the context of an integrated mosquito management should achieve a lasting control of Ae. aegypti and Culex spp. in small artificial containers with minimal environmental impacts.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

Localisation of Ig heavy chain mRNA positive cells in Atlantic cod (Gadus morhuaL.) tissues; identified byin situhybridisation

Localisation of immunoglobulin heavy chain (IgH) producing cells was determined in sections from head kidney, spleen, thymus, gills, gut, skin, heart and liver from the Atlantic cod. In general, IgH mRNA positive cells were detected in all organs examined and were mainly located to the connective tissue surrounding the vascular system in these organs. In the head kidney and spleen, IgH mRNA positive cells appeared as single distributed cells or as dense clusters, whereas in the thymus only single distributed positive cells were observed. The percentage of Ig heavy chain mRNA positive (plasma) cells in the head kidney, spleen and thymus was estimated at about 1% of the total cell mass. The number of IgH mRNA positive cells was lower than this in all the other organs examined.     

Evidence of aerobic and anaerobic format dehydrogenase and aldehyde dehydrogenase immunological similarities shown by polyclonal antibodies raised against molybdoproteins

Antisera against metal(Mo)-containing dye-linked dehydrogenases from sulphate-reducing bacteria were used to screen for immunological similarities with NAD⁺-linked dehydrogenases detected in aerobic methanol-utilizing bacterial isolates. Out of eleven strains tested, the strains #5, 8, 9 and 11 were shown to have specific formate and aldehyde dehydrogenases displaying antibody cross-reaction against highly purified Mo-containing dye-linked dehydrogenases. The apparent molecular mass of the identified proteins observed during the antibody reaction correlated with the molecular mass of the dehydrogenases obtained after native PAGE electrophoresis. The strains #8 and 11 exhibited one formate dehydrogenase apparently of identical molecular mass 140–145 kDa, whereas strains #5, 9 and 11 synthesized aldehyde dehydrogenases with apparent molecular masses of about 110, 120 and 155 kDa (two forms) and 120 kDa, respectively. All these aerobic enzymes shared antigenic properties with the anaerobic metalloproteins, indicating the existence of structural similarities between those enzymes in spite of having different cofactor moieties.     

Dietary sodium chloride restriction enhances aortic wall lipid storage and raises plasma lipid concentration in LDL receptor knockout mice

This study aimed at measuring the influence of a low salt diet on the development of experimental atherosclerosis in moderately hyperlipidemic mice. Experiments were carried out on LDL receptor (LDLR) knockout (KO) mice, or apolipoprotein E (apoE) KO mice on a low sodium chloride diet (LSD) as compared with a normal salt diet (NSD). On LSD, the rise of the plasma concentrations of TG and nonesterified fatty acid (NEFA) was, respectively, 19% and 34% in LDLR KO mice, and 21% and 35% in apoE KO mice, and that of plasma cholesterol was limited to the LDLR KO group alone (15%). Probably due to the apoE KO severe hypercholesterolemia, the arterial inner-wall fat storage was not influenced by the diet salt content and was far more abundant in the apoE KO than in the LDLR KO mice. However, in the less severe hypercholesterolemia of the LDLR KO mice, lipid deposits on the LSD were greater than on the NSD. Arterial fat storage correlated with NEFA concentrations in the LDLR KO mice alone (n = 14, P = 0.0065). Thus, dietary sodium chloride restriction enhances aortic wall lipid storage in moderately hyperlipidemic mice.     

Molecular Cloning of α-Amylases from Cotton Boll Weevil, Anthonomus grandis and Structural Relations to Plant Inhibitors: An Approach to Insect Resistance

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of α-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two α-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5′ and 3′ RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7 kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several α-amylase inhibitors from plants were assayed against A. grandis α-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and α-AI1 inhibitors on A. grandis α-amylase activity. This work suggests that genetic engineering of cotton to express α-amylase inhibitors may offer a novel route to A. grandis resistance.     

Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

Background

The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.

Methodology/Principal Findings

A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.

Conclusions/Significance

The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.