Variation in the Presence of Anti-<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Bacteria of Amphibians Across Life Stages and Elevations in Ecuador

Amphibian populations are decreasing worldwide due to a variety of factors. In South America, the chytrid fungus Batrachochytrium dendrobatidis (Bd) is linked to many population declines. The pathogenic effect of Bd on amphibians can be inhibited by specific bacteria present on host skin. This symbiotic association allows some amphibians to resist the development of the disease chytridiomycosis. Here, we aimed (1) to determine for the first time if specific anti-Bd bacteria are present on amphibians in the Andes of Ecuador, (2) to monitor anti-Bd bacteria across developmental stages in a focal amphibian, the Andean marsupial tree frog, Gastrotheca riobambae, that deposits larvae in aquatic habitats, and (3) to compare the Bd presence associated with host assemblages including 10 species at sites ranging in biogeography from Amazonian rainforest (450 masl) to Andes montane rainforest (3200 masl). We sampled and identified skin-associated bacteria of frogs in the field using swabs and a novel methodology of aerobic counting plates, and a combination of morphological, biochemical, and molecular identification techniques. The following anti-Bd bacteria were identified and found to be shared among several hosts at high-elevation sites where Bd was present at a prevalence of 32.5%: Janthinobacterium lividum, Pseudomonas fluorescens, and Serratia sp. Bd were detected in Gastrotheca spp. and not detected in the lowlands (sites below 1000 masl). In G. riobambae, recognized Bd-resistant bacteria start to be present at the metamorphic stage. Overall bacterial abundance was significantly higher post-metamorphosis and on species sampled at lower elevations. Further metagenomic studies are needed to evaluate the roles of host identity, life-history stage, and biogeography of the microbiota and their function in disease resistance.     

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.     

An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Background

Biodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.

Results

A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings.

Conclusions

The results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

     

Factors influencing wolf <Emphasis Type="Italic">Canis lupus</Emphasis> roadkills in Northwest Spain

Roadkill is one of the most prominent causes of wildlife mortality. Much research effort has focussed on collisions with ungulates because of traffic safety. However, studies about large carnivore roadkills are scarce despite vehicles being a main cause of mortality. The absence of studies can be explained in part because of difficulties in obtaining sufficient sample sizes. We collected data from locations of 82 wolf roadkill sites in the Castilla y León Region, northwest Spain. We evaluated different models to characterise collision localities using logistic regressions with corrections for rare events. The best models included traffic and human disturbance parameters. Landscape variables did not improve predictive power. Fencing was a decisive key predictor; roadkill was proportionally higher along fenced highways than on similar major roads that lacked fences. Wolf–vehicle collisions were more common in agricultural areas, although wolf densities were lower in these zones. Both the higher density of important roads and a greater proportion of roaming wolves on the plateau may explain this pattern.     

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac-based transposon vector system using independent vector and transposase helper plasmids. Minimum estimated germ-line transformation frequencies were approximately 13–21% per fertile G₀ individual, similar to previously reported frequencies using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control. The first allows post-integration stabilization of a transposon-vector by deletion of a terminal sequence necessary for mobilization. The complete pB[L1-EGFP-L2-DsRed-R1] vector was integrated into the Chiapas wild type strain with subsequent deletion of the L2-DsRed-R1 sub-vector carrying the piggyBac 3′ terminal sequence. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5–10% in most tests, however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release. The second vector, pB[XL-EGFP, Asß2-tub-DsRed.T3], has the DsRed.T3 fluorescent protein reporter gene regulated by the A. suspensa Asß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm bundles were unambiguously observed in the spermathecae of non-transgenic females mated to transgenic males. One transgenic line apparently had a male-specific Y-chromosome insertion, having potential use for sexing by fluorescent-embryo sorting. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.     

Profile Parsimony (PP): an analysis under Implied Weights (IW)

Faith and Trueman [Syst. Biol. (2001) 331] recently proposed “Profile Parsimony” (PP) as a new form of phylogenetic analysis, but it is equivalent to the Implied Weights (IW) method when certain functions are used. The PP method cannot accommodate missing/inapplicable cells and/or multistate characters. Finally we demonstrate that, as defined, PP scores are not random, and there is no need to generate random trees as proposed by Faith and Trueman.     

Indicators of environmentally sound land use in the humid tropics: The potential roles of expert opinion,knowledge engineering and knowledge discovery

Despite abundant literature on indicators for sustainable resource management, practical tools to help local users to apply its general concepts at a local to regional level are scarce. This means that decisions over land evaluation and land use at a local level are often not based on the formal application of indicators or decision support systems for environmentally sound management but instead on the opinion of local expertise, for instance forest managers, cattle breeders, farmers and/or academics. This is particularly seen to be the case in the tropics where access to modern communication and information technologies is restricted.As the opinions of experts are often based on and influenced by personal experience, intuition, heuristics and bias, their evaluations and decision are often unclear to the non-expert working at a local level. In order to make their reasoning more comprehensible to the non-expert, the ecological condition of 176 plots in the tropical Southeast of Mexico were evaluated by experts on soil fertility, forest management, cattle breeding and agriculture. With the assistance of a knowledge engineer (one who converts expert knowledge and reasoning into a model), these expert opinions and reasoning were then translated into a formal computer model.As an alternative approach we applied a knowledge discovery technique, namely the induction of regression trees and automatically developed models using the expert evaluations as training data. Where knowledge engineering was tedious and time consuming, regression models could be rapidly generated. Moreover, the correspondence between regression trees and expert opinions was considerably higher than the correspondence between expert opinion and their own models. The regression trees used less explicative variables than the models generated by the experts. The minimisation of sampling effort due to variable space reduction means that the application of regression tree induction has a high potential for a rapid development of indicators for narrowly defined ecological assessments, needed for decision making on a local or regional scale.     

Length–weight relationships of 18 fish species from Paraíba do Sul basin,Minas Gerais,Brazil

The present study established the length–weight relationships (LWRs) for 18 species of freshwater fish from Paraíba do Sul River basin, southeastern Brazil. LWR parameters are estimated for the first time for seven of these species. The allometric coefficient b varied from 2.81 to 3.32. These data complement the effort to understand the biology of one of the most threatened Brazilian basins.