The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

Bacterial communities associated with sugarcane under different agricultural management exhibit a diversity of plant growth-promoting traits and evidence of synergistic effect.

Plant-associated microbiomes have been a target of interest for the prospection of microorganisms, which may be acting as effectors to increase agricultural productivity. For years, the search for beneficial microorganisms has been carried out from the characterization of functional traits of growth-promotion using tests with a few isolates. However, eventually, the expectations with positive results may not be realized when the evaluation is performed in association with plants. In our study, we accessed the cultivable sugarcane microbiome under two conditions of agronomic management: organic and conventional. From the use of a new customized culture medium, we recovered 944 endophytic and epiphytic bacterial communities derived from plant roots, stalks, leaves, and rhizospheric soil. This could be accomplished by using a large-scale approach, initially performing an in planta (Cynodon dactylon) screening process of inoculation to avoid early incompatibility. The inoculation was performed using the bacterial communities, considering that in this way, they could act synergistically. This process resulted in 38 candidate communities, 17 of which had higher Indole-3-acetic acid (IAA) production and phosphate solubilization activity and, were submitted to a new in planta test using Brachiaria ruziziensis and quantification of functional traits for growth-promotion and physiological tests. Enrichment analysis of selected communities has shown that they derived mainly from epiphytic populations of sugarcane stalks under conventional management. The sequencing of the V3-V4 region of the 16S rRNA gene revealed 34 genera and 24 species distributed among the phylum Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. We also observed a network of genera in these communities where the genus Chryseobacterium stands out with a greater degree of interaction, indicating a possible direct or indirect role as a keystone taxon in communities with plant-growth promotion capacities. From the results achieved, we can conclude that the approach is useful in the recovery of a set of sugarcane bacterial communities and that there is, evidence of synergistic action providing benefits to plants, and that they are compatible with plants of the same family (Poaceae). Thus, we are reporting the beneficial bacterial communities identified as suitable candidates with rated potential to be exploited as bioinoculants for crops.

mCherry fusions enable the subcellular localization of periplasmic and cytoplasmic proteins in Xanthomonas sp.

Fluorescent markers are a powerful tool and have been widely applied in biology for different purposes. The genome sequence of Xanthomonas citri subsp. citri (X. citri) revealed that approximately 30% of the genes encoded hypothetical proteins, some of which could play an important role in the success of plant-pathogen interaction and disease triggering. Therefore, revealing their functions is an important strategy to understand the bacterium pathways and mechanisms involved in plant-host interaction. The elucidation of protein function is not a trivial task, but the identification of the subcellular localization of a protein is key to understanding its function. We have constructed an integrative vector, pMAJIIc, under the control of the arabinose promoter, which allows the inducible expression of red fluorescent protein (mCherry) fusions in X. citri, suitable for subcellular localization of target proteins. Fluorescence microscopy was used to track the localization of VrpA protein, which was visualized surrounding the bacterial outer membrane, and the GyrB protein, which showed a diffused cytoplasmic localization, sometimes with dots accumulated near the cellular poles. The integration of the vector into the amy locus of X. citri did not affect bacterial virulence. The vector could be stably maintained in X. citri, and the disruption of the α-amylase gene provided an ease screening method for the selection of the transformant colonies. The results demonstrate that the mCherry-containing vector here described is a powerful tool for bacterial protein localization in cytoplasmic and periplasmic environments.

Serratia liquefaciens FG3 isolated from a metallophyte plant sheds light on the evolution and mechanisms of adaptive traits in extreme environments.

Serratia liquefaciens strain FG3 (SlFG3), isolated from the flower of Stachytarpheta glabra in the Brazilian ferruginous fields, has distinctive genomic, adaptive, and biotechnological potential. Herein, using a combination of genomics and molecular approaches, we unlocked the evolution of the adaptive traits acquired by S1FG3, which exhibits the second largest chromosome containing the largest conjugative plasmids described for Serratia. Comparative analysis revealed the presence of 18 genomic islands and 311 unique protein families involved in distinct adaptive features. S1FG3 has a diversified repertoire of genes associated with Nonribosomal peptides (NRPs/PKS), a complete and functional cluster related to cellulose synthesis, and an extensive and functional repertoire of oxidative metabolism genes. In addition, S1FG3 possesses a complete pathway related to protocatecuate and chloroaromatic degradation, and a complete repertoire of genes related to DNA repair and protection that includes mechanisms related to UV light tolerance, redox process resistance, and a laterally acquired capacity to protect DNA using phosphorothioation. These findings summarize that SlFG3 is well-adapted to different biotic and abiotic stress situations imposed by extreme conditions associated with ferruginous fields, unlocking the impact of the lateral gene transfer to adjust the genome for extreme environments, and providing insight into the evolution of prokaryotes.

Improved methane production from sugarcane vinasse with filter cake in thermophilic UASB reactors, with predominance of Methanothermobacter and Methanosarcina archaea and Thermotogae bacteria.

Biogas production from sugarcane vinasse has enormous economic, energy, and environmental management potential. However, methane production stability and biodigested vinasse quality remain key issues, requiring better nutrient and alkalinity availability, operational strategies, and knowledge of reactor microbiota. This study demonstrates increased methane production from vinasse through the use of sugarcane filter cake and improved effluent recirculation, with elevated organic loading rates (OLR) and good reactor stability. We used UASB reactors in a two-stage configuration, with OLRs up to 45gCODL-1d-1, and obtained methane production as high as 3LL-1d-1. Quantitative PCR indicated balanced amounts of bacteria and archaea in the sludge (109-1010copiesg-1VS), and of the predominant archaea orders, Methanobacteriales and Methanosarcinales (106-108copiesg-1VS). 16S rDNA sequencing also indicated the thermophilic Thermotogae as the most abundant class of bacteria in the sludge.

A balanced microbiota efficiently produces methane in a novel high-rate horizontal anaerobic reactor for the treatment of swine wastewater.

A novel combination of structurally simple, high-rate horizontal anaerobic reactors installed in series was used to treat swine wastewater. The reactors maintained stable pH, alkalinity, and volatile acid levels. Removed chemical oxygen demand (COD) represented 68% of the total, and the average specific methane production was 0.30L CH4 (g removed CODtot)(-1). In addition, next-generation sequencing and quantitative real-time PCR analyses were used to explore the methane-producing Archaea and microbial diversity. At least 94% of the sludge diversity belong to the Bacteria and Archaea, indicating a good balance of microorganisms. Among the Bacteria the Proteobacteria, Bacteroidetes and Firmicutes were the most prevalent phyla. Interestingly, up to 12% of the sludge diversity belongs to methane-producing orders, such as Methanosarcinales, Methanobacteriales and Methanomicrobiales. In summary, this system can efficiently produce methane and this is the first time that horizontal anaerobic reactors have been evaluated for the treatment of swine wastewater.

Type IV Secretion System Is Not Involved in Infection Process in Citrus.

The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Xanthomonas citri subsp. citri contains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS in Xcc, we analyzed, in vitro and in planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, only hrpA was downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated on Citrus leaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress.

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.

Polymorphism analysis of the hsp70 stress gene in Broiler chickens (Gallus gallus) of different breeds

The promoter region and the beginning of the coding region of the hsp70 stress gene were analysed in broiler chickens of a commercial breed (Hubbard-Pettersen), a breed selected for weight gain (PP1) and a non-selected breed (naked-neck Label Rouge). The naked neck gene (Naked neck, Na), which reduces feathering in birds and is thus related to heat resistance, was present in both PP1 and Label Rouge breeds. Genomic DNA was restricted with PstI and Southern blotting analysis of the samples revealed the absence of polymorphic sites for that enzyme in the promoter region and beginning of the coding region of the hsp70 gene of studied birds. PCR-SSCP analysis of these regions, however, indicated the presence of polymorphisms in the beginning of the coding region and the sequencing of the PCR products confirmed and identified two polymorphic sites in this region: a transition A ® G in position +258 and a transversion C ® G in position +276. Both mutations were considered to be silent, since they did not modify the aminoacid sequence of the protein Hsp70. The promoter region of the hsp70 gene was identical in all studied birds, indicating that the regulation pattern of this gene must be the same in all birds at the promoter region. Three different alleles (hsp70-1, hsp70-2 and hsp70-3) were identified for the hsp70 gene from the observed mutations. The allele hsp70-3 was detected in only two breeds, Hubbard-Pettersen and PP1, but at a low frequency (0,016 and 0,006, respectively)

Hepatic mRNA expression and plasma levels of insulin-like growth factor-I (IGF-I) in broiler chickens selected for different growth rates

The hepatic expression and plasma concentrations of IGF-I were investigated in three broiler chicken strains selected for different growth rates (HP-Hubbard-Pettersen, a fast growing strain; NN-Naked-neck, a strain with an intermediate growth rate and a heterozygous genotype, and C-Caipira, a slow growing crossbred strain). The chickens were studied at 1, 21 and 42 days of age and had free access to food throughout the study. Hepatic IGF-I mRNA expression was assessed by dot blot analysis using a randomly labeled chicken IGF-I cDNA as the probe and plasma IGF-I concentrations were assayed by radioimmunoassay. The hepatic levels of IGF-I mRNA increased from 1 to 21 days of age in all strains, with NN chickens showing a higher (p < 0.05) IGF-I expression than the other strains. Plasma IGF-I concentrations increased (p < 0.05) with broiler chicken age, but there were no significant differences among the strains. These results indicate that despite differences in the growth rates among the strains, the changes in the expression of IGF-I mRNA in liver and in the plasma levels of IGF-I were independent of broiler chicken strain, but varied with chicken age.