Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion.     

Association of Pro-Inflammatory Cytokines and Iron Regulatory Protein 2 (IRP2) with Leishmania Burden in Canine Visceral Leishmaniasis

Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2) were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.     

Computer-aided comparison of protein electrophoretic patterns for grouping and identification of heterotrophic bacteria from mineral water

The microflora of a natural mineral water was studied immediately after bottling (T0) and after 7 d storage (T7) during 6 months, and isolates were clustered by SDS-PAGE of wholecell protein profiles. Isolates from each cluster were further characterized by API 20NE, fatty acid composition and quinone profiles. The numerical analysis of the electrophoregrams of all bacteria isolated from the mineral water formed 15 clusters and five unclustered strains. Except for five minor clusters, all clusters were composed of strains isolated over several months. The numerical analysis of the electrophoregrams of bacteria isolated immediately after bottling formed 15 clusters while after 7 d storage only four of these populations could be isolated, indicating that populations present in the mineral water were stable and that changes occurring after bottling probably resulted from a selection process. Only one unclustered strain was identified simultaneously by all the systems, as Sphingomonas paucimobilis. The monitoring of the aquifer and the bottling system, and the construction of a large database with bacteria of the autochthonous flora allows the detection of alterations in the aquifer by changes in the microflora.     

Attraction of Fruit-Eating Bats with Essential Oils of Fruits: A Potential Tool for Forest Restoration

Previous tests with essential oils from ripe chiropterochoric fruits suggested they can be used to attract and capture fruit-eating bats inside forest remnants. Here we evaluated the efficiency of these oils to attract frugivorous bats to open areas. We performed field tests with artificial fruits impregnated with essential oils of the genera Piper or Ficus that were attached to two groups of mist-nets set 50 m outside the border of a forest remnant. One group of artificial fruits received the corresponding oil isolated through hydrodistillation and the other received water only. Fruits with oils attracted significantly more fruit-eating bats, especially Artibeus lituratus that regularly crosses open habitats to reach other forest remnants. The highly significant attraction of A. lituratus by the oil of Piper was unexpected, since this bat is a specialist on Ficus fruits. We hypothesize that in habitats with no fruit available it is possible to attract frugivorous bats with the odor of several ripe fruit species. Furthermore, we verified that almost half of the individuals captured defecated seeds, indicating that the oils also attract recently fed bats, even when their preferred food is available nearby. This technique potentially may increase seed rain at specific locations, being particularly promising to restoration projects.     

Quantitative analysis of race-specific resistance to Colletotrichum lindemuthianum in common bean

Molecular genetic maps continue to play a major role in breeding of crop species. The common bean genetic map of the recombinant inbred line population IAC-UNA × CAL 143 (UC) has been used to detect loci controlling important agronomic traits in common bean. In the current study, new microsatellite markers were added to the UC map and the linkage analysis was refined using current genomic resources of common bean, in order to identify quantitative resistance loci (QRL) associated with different races of the anthracnose pathogen. A single race inoculation was conducted in greenhouse using four plants per plot. Both race-specific and joint-adjusted disease severity means, obtained from linear-mixed model, were used to perform multiple interval mapping (MIM) and multi-trait MIM (MTMIM). In total, 13 and 11 QRL were identified by MIM and MTMIM analyses, respectively; with nine being observed in both analyses. ANT02.1^UC and ANT07.1^UC showed major effects on resistance both for MIM and MTMIM. Common major QRL for resistance to the three anthracnose races were expected, since high genetic pairwise-correlation was observed between the race-specific and joint-adjusted disease severity means. Therewith, both ANT02.1 and ANT07.1 can be regarded as valuable targets for marker-assisted selection; and so, putative genes potentially involved in the resistance response were identified in these QRL regions. Minor effect QRL were also observed, showing differential affects either on race-specific or multi-trait analyses and may play a role on durable horizontal resistance. These results contribute to a better understanding of the host-pathogen interaction and to breeding for enhancing resistance to Colletotrichum lindemuthianum in common bean.     

Functional Feeds Reduce Heart Inflammation and Pathology in Atlantic Salmon (Salmo salar L.) following Experimental Challenge with Atlantic Salmon Reovirus (ASRV)

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.     

Classification of chili powders by thin-layer chromatography and principal component analysis

Silica gel, aluminium oxide, diatomaceous earth, polyamide, cyano, diol and amino plates have been tested for their capacity to separate the color pigments of six chili powders of different origin by both adsorption and reversed-phase thin-layer chromatography. The plates were evaluated at 340 and 440 nm wavelengths. Best separation of color pigments was obtained on impregnated diatomaceous earth layer using acetone-water 17:3 v/v eluent. It was found that the pigment composition of chili powders showed marked differences. Principal component analysis employed for the classification of the chili powders according to their pigment composition indicated that these differences can be used for the determination of the similarity or dissimilarity of the chili powders.     

Human exploitation shapes productivity–biomass relationships on coral reefs

Coral reef fisheries support the livelihoods of millions of people in tropical countries, despite large‐scale depletion of fish biomass. While human adaptability can help to explain the resistance of fisheries to biomass depletion, compensatory ecological mechanisms may also be involved. If this is the case, high productivity should coexist with low biomass under relatively high exploitation. Here we integrate large spatial scale empirical data analysis and a theory‐driven modelling approach to unveil the effects of human exploitation on reef fish productivity–biomass relationships. We show that differences in how productivity and biomass respond to overexploitation can decouple their relationship. As size‐selective exploitation depletes fish biomass, it triggers increased production per unit biomass, averting immediate productivity collapse in both the modelling and the empirical systems. This ‘buffering productivity’ exposes the danger of assuming resource production–biomass equivalence, but may help to explain why some biomass‐depleted fish assemblages still provide ecosystem goods under continued global fishing exploitation.