Identification of point mutations in a putative carboxylesterase and their association with acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Chemical control based on the use of pyrethroid and organophosphate compounds has selected resistant genotypes in populations of Rhipicephalus (Boophilus) microplus. Point mutations in esterase-encoding genes represent one of the main resistance mechanisms in this species. In this study, the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique was used to investigate the presence of mutations in a fragment of a putative carboxylesterase in a population of ticks with a history of resistance. The digestion of a fragment of 372 pb with EcoRI revealed three genotypes: W, H and M, observed in different frequencies. The homozygous wild-type genotype (W) was detected only in sensitive strains, with high frequency. The heterozygous genotype (H) was observed in all the strains, albeit with higher frequency in the strains with a moderate resistance, while the homozygous mutant genotype (M) was found only in the moderate resistant strain and resistant strains, with higher frequency in the resistant strains. A comparison of the sequences indicated the presence of other mutations, besides EcoRI polymorphism in the moderate resistant and resistant strains. Also found was the presence of stop codons generating truncated proteins in the sensitive and moderate resistant strains. A domain analysis revealed the presence of additional domains in the resistant strain. These findings suggest that different point mutations, as well as the influence of post-translational modification mechanisms, are altering the activity of the translated proteins and may be associated with resistance.     

Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and Malassezia pachydermatis strains from dogs

Yeasts of the genera Candida and Malassezia can be found as commensal microorganisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans (n=5), C. tropicalis (n=3) and M. pachydermatis (n=32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (ITC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64microg/mL and all were resistant to KTC and ITC (MIC>16microg/mL). The MIC for two strains of C. tropicalis were >16microg/mL for KTC and ITC, and >64microg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25-1.0microg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) 0.0075microg/mL. The MIC for 29 strains was the same (MFC0.0075microg/mL) for KTC. The MFCs for FLC varied from 1 to 16microg/mL, and for AMB, the MFC interval was 0.125-8microg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and M. pachydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents.     

An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge

Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA.     

Thermal Structure of Jupiter's Upper Atmosphere Derived from the Galileo Probe

Temperatures in Jupiter's atmosphere derived from Galileo Probe deceleration data increase from 109 kelvin at the 175-millibar level to 900 ± 40 kelvin at 1 nanobar, consistent with Voyager remote sensing data. Wavelike oscillations are present at all levels. Vertical wavelengths are 10 to 25 kilometers in the deep isothermal layer, which extends from 12 to 0.003 millibars. Above the 0.003-millibar level, only 90- to 270- kilometer vertical wavelengths survive, suggesting dissipation of wave energy as the probable source of upper atmosphere heating.     

A metabarcoding tool to detect predation of the honeybee Apis mellifera and other wild insects by the invasive Vespa velutina

The invasive Vespa velutina has been widely referred as an effective predator of honeybees. Despite the potential risk to pollination services provision and honey production, there is no accurate quantification and assessment of its real consequences for honeybees. To date, the identification of the honeybee and other insects in the diet of V. velutina has been investigated by direct observation of adult foraging or examination of food pellets. To overcome these limitations, in this study we used a DNA metabarcoding approach to evaluate the usefulness of different types of sample (jaws and stomachs collected from workers and larval faecal pellets taken from the hornet comb) to investigate the predation of V. velutina upon honeybees, and potentially on other insects. Honeybee DNA was identified in all types of samples, but larval faecal pellets retrieved the higher number of reads of honeybee DNA and the largest diversity at all taxonomic levels. Over all samples we could identify 4 orders, 9 families, 6 genera and 1 species of prey. We estimate that collecting 6 workers is sufficient to identify honeybee predation by a colony using worker’s jaws. Stomachs were the least useful sample type to detect honeybee DNA. The presence of honeybee DNA in all analysed colonies irrespective of collection site, and the variety of insect orders detected in the diet support current concerns over the acknowledged negative impact of V. velutina on managed honeybees and its potential threat to pollination services provision.

     

Case studies on breeding systems and its consequences for germplasm conservation

Summary This study gives the results of allozyme diversity within and among 20 wild Lima bean populations uniformly distributed throughout the Central Valley of Costa Rica. The electrophoretic analysis of seven enzyme systems show five monomorphic loci and a relatively low level of polymorphism for the other loci. This moderate level of polymorphism is unexpected for a species for which a fair amount of allogamy rates has been reported, at least among the cultivated forms. The genetic parameters (mainly percentage of polymorphic loci, mean genetic diversity, percentage of heterozygotes and fixation index) indicate a tendency for a predominantly selfing breeding system in the wild Lima beans although some values range between selfing and mixed-animal breeding systems. Very low within-population diversity is observed while a good diversity is found among populations. Results also show a departure from Hardy-Weinberg equilibrium on most analyzed populations in the target site This might be due to populations divided into subpopulations among which no natural crosses occur randomly, to weeding practices or to overlap of generations within some populations. All the results obtained in this work are discussed in view of further studies for the planning of in situ conservation in a quickly evolving tropical environment.     

Case studies on breeding systems and its consequences for germplasm conservation: 3. Electrophoretic mobility of phaseolins in wild populations of Lima beans (Phaseolus lunatus L.) in the Central Valley of Costa Rica

We studied the variation in the electrophoretic mobility of phaseolins of 202 individual seeds from thirty-eight wild populations of Lima beans (Phaseolus lunatus) in the Central Valley of Costa Rica. We found four main polypeptides or banding regions with molecular weights which ranged from 21.5 to 31 kDaltons. Two of these peptides were polymorphic, and their combination resulted in four distinct genotypes. The frequency of the different genotypes varies among populations, but two of them, namely B¹¹C¹¹, B²²C²², were found in most of the populations studied (37 out of 38 populations examined). Overall, there was a lack of heterozygotes among the individuals examined, which strongly suggests that there is gametic disequilibrium for the encoding regions involved in the synthesis of these polypeptides. When we arranged the populations into eight groups according to their proximity and phenology, we found that the distribution of the peptides was also variable among them. The possible association between the distribution of the patterns of electrophoretic mobility observed and climatic factors, such as mean annual rainfall, mean annual temperature and mean annual relative humidity is discussed.     

The relationship between vesicular-arbuscular mycorrhiza and lime: Associated effects on the growth and nutrition of brachiaria grass (Brachiaria decumbens)

Summary We studied the effects of limining on growth and nutrient concentrations of Brachiaria decumbens inoculated with five vesicular-arbuscular mycorrhizal (VAM) fungal assemblages which orginated from soils with different acidity. Liming increased plant growth when applied at rates up to 3 g kg^-1 soil and depressed growth at higher rates. Mycorrhizal plants grew better than non-mycorrhizal ones in unlimed soil and also liming rates of 4.5 and 6.0 g kg^-1 soil. The growth amelioration effects of VAM in highly acid or over-limed soils were related to nutrient uptake. VAM fungi isolated from an acidic soil exhibited a high symbiotic effectiveness and were better adapted to unlimed soil than those that originated from non-acidic soils. VAM root colonization, 90 days after planting, was little affected by liming. Fungal spore production and species compositions were highly affected by liming. A mixture of Glomus diaphanum and Glomus occultum predominated in unlimed soils inoculated with VAM assemblages isolated from non-acidic soils. In these fungal assemblages, an increased liming rate favored Glomus etunicatum over the other VAM fungi. Gigaspora margarita sporulated abundantly when introduced into unlimed soils, but rarely in limed soils. VAM appear to be crucial for the establishment of brachiaria pastures in the nutrient-deficient acidic soils of Central Brazil. It is suggested that liming may cause striking shifts in VAM populations which may, in turn, have a long-term impact on agricultural productivity in the tropics.