Molecular Changes in the Brain of the Wintering Calidris pusilla in the Mangroves of the Amazon River Estuary.

Migrant birds prepare differently to fly north for breeding in the spring and for the flight to lower latitudes during autumn, avoiding the cold and food shortages of the Northern Hemisphere's harsh winter. The molecular events associated with these fundamental stages in the life history of migrants include the differential gene expression in different tissues. Semipalmated sandpipers (Calidris pusilla) are Arctic-breeding shorebirds that migrate to the coast of South America during the non-breeding season. In a previous study, we demonstrated that between the beginning and the end of the wintering period, substantial glial changes and neurogenesis occur in the brain of C. pusilla. These changes follow the epic journey of the autumn migration when a 5-day non-stop transatlantic flight towards the coast of South America and the subsequent preparation for the long-distance flight of the spring migration takes place. Here, we tested the hypothesis that the differential gene expressions observed in the brains of individuals captured in the autumn and spring windows are consistent with the previously described cellular changes. We searched for differential gene expressions in the brain of the semipalmated sandpiper, of recently arrived birds (RA) from the autumnal migration, and that of individuals in the premigratory period (PM) in the spring. All individuals were collected in the tropical coastal of northern Brazil in the mangrove region of the Amazon River estuary. We generated a de novo neurotranscriptome for C. pusilla individuals and compared the gene expressions across libraries. To that end, we mapped an RNA-Seq that reads to the C. pusilla neurotranscriptome in four brain samples of each group and found that the differential gene expressions in newly arrived and premigratory birds were related with neurogenesis, metabolic pathways (ketone body biosynthetic and the catabolic and lipid biosynthetic processes), and glial changes (astrocyte-dopaminergic neuron signaling, astrocyte differentiation, astrocyte cell migration, and astrocyte activation involved in immune response), as well as genes related to the immune response to virus infections (Type I Interferons), inflammatory cytokines (IL-6, IL-1ß, TNF, and NF-κB), NLRP3 inflammasome, anti-inflammatory cytokines (IL-10), and cell death pathways (pyroptosis- and caspase-related changes).

Evaluation of Paralytic Shellfish Toxins in Marine Oyster Farming and Microalgae in the Atlantic Amazon Evidences Safety but Highlights Potential Risks of Shellfish Poisoning.

Marine phycotoxins are organic compounds synthesized by some species of microalgae, which accumulate in the tissues of filter-feeder organisms such as bivalve mollusks. These toxins can cause acute intoxication episodes in humans, a severe threat to aquaculture and fisheries. In the State of Pará, Brazil, oyster farming has community, artisanal and sustainable bases, using mangroves as cultivation environment and seed banks. In small-scale production, there are often no established methods of safeguarding the health of consumers elevating the potential risks of shellfish poisoning outbreaks. Our study evaluated the presence of phycotoxins in oysters cultivated in five municipalities in the region of the Atlantic Amazon (Pará, Brazil) assessing the quality of the final product. We further evaluated the microalgae, water quality, and the spatio-temporal variation of physicochemical factors in the same area. Diatoms dominated the microalgae composition, followed by dinoflagellates, some of which are reported to be potentially toxic and producers of paralytic shellfish toxins. For the first time, we describe the occurrence of the potentially toxic dinoflagellate Ostreopsis sp. in the Amazon region. Furthermore, for the first time, toxins were detected in oyster farming in the northeast of the State of Pará, namely GTX2,3, STX, and dc-STX nevertheless, with nontoxic values. The identified toxins represent a potential threat to shellfish consumers.

Novel insights on aquatic mammal MHC evolution: Evidence from manatee DQB diversity.

The low diversity in marine mammal major histocompatibility complex (MHC) appears to support the hypothesis of reduced pathogen selective pressure in aquatic systems compared to terrestrial environments. However, the lack of characterization of the aquatic and evolutionarily distant Sirenia precludes drawing more generalized conclusions. Therefore, we aimed to characterize the MHC DQB diversity of two manatee species and compare it with those reported for marine mammals. Our results identified 12 and 6 alleles in T. inunguis and T. manatus, respectively. Alleles show high rates of nonsynonymous substitutions, suggesting loci are evolving under positive selection. Among aquatic mammals, Pinnipeda DQB had smaller numbers of alleles, higher synonymous substitution rate, and a dN/dS ratio closer to 1, suggesting it may be evolving under more relaxed selection compared to fully aquatic mammals. This contradicts one of the predictions of the hypothesis that aquatic environments impose reduced pathogen pressure to mammalian immune system. These results suggest that the unique evolutionary trajectories of mammalian MHC may impose challenges in drawing ecoevolutionary conclusions from comparisons across distant vertebrate lineages.

Glucosidase Inhibitors Screening in Microalgae and Cyanobacteria Isolated from the Amazon and Proteomic Analysis of Inhibitor Producing Synechococcus sp. GFB01.

Microalgae and cyanobacteria are good sources for prospecting metabolites of biotechnological interest, including glucosidase inhibitors. These inhibitors act on enzymes related to various biochemical processes; they are involved in metabolic diseases, such as diabetes and Gaucher disease, tumors and viral infections, thus, they are interesting hubs for the development of new drugs and therapies. In this work, the screening of 63 environmental samples collected in the Brazilian Amazon found activity against ß-glucosidase, of at least 60 min, in 13.85% of the tested extracts, with Synechococcus sp. GFB01 showing inhibitory activity of 90.2% for α-glucosidase and 96.9% against ß-glucosidase. It was found that the nutritional limitation due to a reduction in the concentration of sodium nitrate, despite not being sufficient to cause changes in cell growth and photosynthetic apparatus, resulted in reduced production of α and ß-glucosidase inhibitors and differential protein expression. The proteomic analysis of cyanobacteria isolated from the Amazon is unprecedented, with this being the first work to evaluate the protein expression of Synechococcus sp. GFB01 subjected to nutritional stress. This evaluation helps to better understand the metabolic responses of this organism, especially related to the production of inhibitors, adding knowledge to the industrial potential of these cyanobacterial compounds.

TLR4 and TLR8 variability in Amazonian and West Indian manatee species from Brazil.

Amazonian (Trichechus inunguis) and West Indian (Trichechus manatus) manatees are aquatic mammals vulnerable to extinction found in the Amazon basin and the coastal western Atlantic. Toll-like receptors (TLR) play a key role in recognizing pathogen-associated molecular patterns using leucine-rich repeats (LRRs). We described the diversity of TLR4 and TLR8 genes in these two species of manatee. Amazonian manatee showed seven SNPs in TLR4 and the eight in TLR8, while West Indian manatee shared four and six of those SNPs, respectively. In our analysis, TLR4 showed one non-conservative amino acid replacement substitution in LRR7 and LRR8, on the other hand, TLR8 was less variable and showed only conserved amino acid substitutions. Selection analysis showed that only one TLR4 site was subjected to positive selection and none in TLR8. TLR4 in manatees did not show any evidence of convergent evolution compared to species of the cetacean lineage. Differences in TLR4 and TLR8 polymorphism may be related to distinct selection by pathogens, population reduction of West Indian manatees, or an expected consequence of population expansion in Amazonian manatees. Future studies combining pathogen association and TLR polymorphism may clarify possible roles of these genes and be used for conservation purposes of manatee species.

Characterization and Biotechnological Potential of Intracellular Polyhydroxybutyrate by Stigeoclonium sp. B23 Using Cassava Peel as Carbon Source.

The possibility of utilizing lignocellulosic agro-industrial waste products such as cassava peel hydrolysate (CPH) as carbon sources for polyhydroxybutyrate (PHB) biosynthesis and characterization by Amazonian microalga Stigeoclonium sp. B23. was investigated. Cassava peel was hydrolyzed to reducing sugars to obtain increased glucose content with 2.56 ± 0.07 mmol/L. Prior to obtaining PHB, Stigeoclonium sp. B23 was grown in BG-11 for characterization and Z8 media for evaluation of PHB nanoparticles' cytotoxicity in zebrafish embryos. As results, microalga produced the highest amount of dry weight of PHB with 12.16 ± 1.28 (%) in modified Z8 medium, and PHB nanoparticles exerted some toxicity on zebrafish embryos at concentrations of 6.25-100 µg/mL, increased mortality (<35%) and lethality indicators as lack of somite formation (<25%), non-detachment of tail, and lack of heartbeat (both <15%). Characterization of PHB by scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimeter (DSC), and thermogravimetry (TGA) analysis revealed the polymer obtained from CPH cultivation to be morphologically, thermally, physically, and biologically acceptable and promising for its use as a biomaterial and confirmed the structure of the polymer as PHB. The findings revealed that microalgal PHB from Stigeoclonium sp. B23 was a promising and biologically feasible new option with high commercial value, potential for biomaterial applications, and also suggested the use of cassava peel as an alternative renewable resource of carbon for PHB biosynthesis and the non-use of agro-industrial waste and dumping concerns.

Optimization of Polyhydroxybutyrate Production by Amazonian Microalga Stigeoclonium sp. B23.

The present work established the optimization and production of biodegradable thermoplastic polyhydroxybutyrate (PHB) from Amazonian microalga Stigeoclonium sp. B23. The optimization was performed in eight different growth media conditions of Stigeoclonium sp. B23, supplemented with sodium acetate and sodium bicarbonate and total deprivation of sodium nitrate. B23 was stained with Nile Red, and PHB was extracted and quantified by correlating the amount of fluorescence and biopolymer concentration through spectrofluorimetry and spectrophotometry, respectively. Our results detected the production of PHB in Stigeoclonium sp. B23 and in all modified media. Treatment with increased acetate and bicarbonate and without nitrate gave the highest concentration of PHB, while the treatment with only acetate gave the lowest among supplemented media. Our results showed a great potential of Stigeoclonium sp. B23, the first Amazonian microalga reported on PHB production. The microalga was isolated from a poorly explored and investigated region and proved to be productive when compared to other cyanobacterial and bacterial species. Additionally, microalga biomass changes due to the nutritional conditions and, reversely, biopolymer is well-synthetized. This great potential could lead to the pursuit of new Amazonian microalgae species in the search for alternative polyesters.

Amazonia Seasons Have an Influence in the Composition of Bacterial Gut Microbiota of Mangrove Oysters (Crassostrea gasar).

The mangrove oysters (Crassostrea gasar) are molluscs native to the Amazonia region and their exploration and farming has increased considerably in recent years. These animals are farmed on beds built in the rivers of the Amazonia estuaries and, therefore, the composition of their microbiome should be directly influenced by environmental conditions. Our work aimed to evaluate the changes in bacterial composition of oyster's microbiota at two different seasons (rainy and dry). For this purpose, we amplified and sequenced the V3-V4 regions of the 16S rRNA gene. Sequencing was performed on the Illumina MiSeq platform. According to the rarefaction curve, the sampling effort was sufficient to describe the bacterial diversity in the samples. Alpha-diversity indexes showed that the bacterial microbiota of oysters is richer during the rainy season. This richness is possibly associated with the diversity at lower taxonomic levels, since the relative abundance of bacterial phyla in the two seasons remained relatively constant. The main phyla found include Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Similar results were found for the species Crassostrea gigas, Crassostrea sikamea, and Crassostrea corteziensis. Beta-diversity analysis showed that the bacterial composition of oyster's gut microbiota was quite different in the two seasons. Our data demonstrate the close relationship between the environment and the microbiome of these molluscs, reinforcing the need for conservation and sustainable management of estuaries in the Amazonia.

The Marine Mammal Class II Major Histocompatibility Complex Organization.

Sirenians share with cetaceans and pinnipeds several convergent traits selected for the aquatic lifestyle. Living in water poses new challenges not only for locomotion and feeding but also for combating new pathogens, which may render the immune system one of the best tools aquatic mammals have for dealing with aquatic microbial threats. So far, only cetaceans have had their class II Major Histocompatibility Complex (MHC) organization characterized, despite the importance of MHC genes for adaptive immune responses. This study aims to characterize the organization of the marine mammal class II MHC using publicly available genomes. We located class II sequences in the genomes of one sirenian, four pinnipeds and eight cetaceans using NCBI-BLAST and reannotated the sequences using local BLAST search with exon and intron libraries. Scaffolds containing class II sequences were compared using dotplot analysis and introns were used for phylogenetic analysis. The manatee class II region shares overall synteny with other mammals, however most DR loci were translocated from the canonical location, past the extended class II region. Detailed analysis of the genomes of closely related taxa revealed that this presumed translocation is shared with all other living afrotherians. Other presumptive chromosome rearrangements in Afrotheria are the deletion of DQ loci in Afrosoricida and deletion of DP in E. telfairi. Pinnipeds share the main features of dog MHC: lack of a functional pair of DPA/DPB genes and inverted DRB locus between DQ and DO subregions. All cetaceans share the Cetartiodactyla inversion separating class II genes into two subregions: class IIa, with DR and DQ genes, and class IIb, with non-classic genes and a DRB pseudogene. These results point to three distinct and unheralded class II MHC structures in marine mammals: one canonical organization but lacking DP genes in pinnipeds; one bearing an inversion separating IIa and IIb subregions lacking DP genes found in cetaceans; and one with a translocation separating the most diverse class II gene from the MHC found in afrotherians and presumptive functional DR, DQ, and DP genes. Future functional research will reveal how these aquatic mammals cope with pathogen pressures with these divergent MHC organizations.

Genetic diversity of Mycobacterium tuberculosis from Pará, Brazil, reveals a higher frequency of ancestral strains than previously reported in South America.

There is only scarce information available on genotypic diversity of the Mycobacterium tuberculosis complex (MTBC) clinical isolates circulating in the Northern part of Brazil, a relatively neglected region regarding research on tuberculosis. We therefore characterized 980 MTBC clinical isolates from the state of Pará, by spoligotyping and data was compared with patterns from around the world, besides analyzing drug susceptibility, and collecting sociodemographic data. We also performed 24 loci MIRU-VNTR typing to evaluate phylogenetic inferences among the East-African-Indian (EAI) lineage strains. The Geographic Information System analyses were performed to generate a descriptive visualization of MTBC strain distribution in the region. A total of 249 different spoligopatterns primarily belonging to evolutionary recent Euro-American lineages, as well as Central-Asian, Manu and ancestral EAI lineages, were identified, in addition to strains with reportedly unknown lineage signatures. The most frequent lineages were Latin American Mediterranean, T and Haarlem. Interestingly, EAI lineage strains were found in a significantly higher proportion in comparison with previous studies from South America. Regarding EAI lineage, the absence of spacers 4-9 and 23-24 co-related to 24 loci MIRU-VNTRs may suggest a close evolutionary relationship between such strains in Pará and those prevalent in Mozambique, which might have contributed to the genetic diversity of MTBC strains in this region.

Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA07 Biovar ovis, Isolated from a Sheep Udder in Amazonia.

In this work, we present the draft genome sequence of Corynebacterium pseudotuberculosis strain PA07 biovar ovis, isolated from a caseous secretion from a sheep udder in Pará, Brazil. The genome contains 2,320,235 bp, 52.2% G+C content, 2,191 coding sequences (CDSs), five pseudogenes, 48 tRNAs, and three rRNAs.

Bartolosides E-K from a Marine Coccoid Cyanobacterium.

The glycosylated and halogenated dialkylresorcinol (DAR) compounds bartolosides A-D (1-4) were recently discovered from marine cyanobacteria and represent a novel family of glycolipids, encoded by the brt biosynthetic gene cluster. Here, we report the isolation and NMR- and MS-based structure elucidation of monoglycosylated bartolosides E-K (5-11), obtained from Synechocystis salina LEGE 06099, a strain closely related to the cyanobacterium that produces the diglycosylated 2-4. In addition, a genome region containing orthologues of brt genes was identified in this cyanobacterium. Interestingly, the major bartoloside in S. salina LEGE 06099 was 1 (above 0.5% dry wt), originally isolated from the phylogenetically distant filamentous cyanobacterium Nodosilinea sp. LEGE 06102. Compounds 5-11 are analogues of 1, with different alkyl chain lengths or halogenation patterns. Their structures and the organization of the brt genes suggest that the DAR-forming ketosynthase BrtD can generate structural diversity by accepting fatty acyl-derived substrates of varying length. Compound 9 features a rare midchain gem-dichloro moiety, indicating that the putative halogenase BrtJ is able to act twice on the same midchain carbon.

Draft Genome Sequence of the N2-Fixing Cyanobacterium Nostoc piscinale CENA21, Isolated from the Brazilian Amazon Floodplain.

We announce here the draft genome sequence ofNostoc piscinaleCENA21, a diazotrophic heterocyst-forming cyanobacterium isolated from the Solimões River, Amazon Basin, Brazil. It consists of one circular chromosome scaffold with 11 contigs and total size of 7,094,556 bp. Secondary metabolite annotations indicate a good source for the discovery of novel natural products.

Draft Genome Sequence of the Picocyanobacterium Synechococcus sp. Strain GFB01, Isolated from a Freshwater Lagoon in the Brazilian Amazon.

We present the draft genome of the cyanobacterium strain Synechococcus sp. GFB01, the first genome sequencing of this genus isolated from South America. This draft genome consists of 125 contigs with a total size of 2,339,812 bp. Automatic annotation identified several genes involved with heavy metal resistance and natural transformation.

Polymorphic color vision in captive Uta Hick's cuxiús, or bearded sakis (Chiropotes utahickae).

The pitheciines (Chiropotes, Pithecia, and Cacajao) are frugivorous Neotropical primates that specialize on the predation of seeds from unripe fruits, usually cryptic against the foliage. However, little is known about the color vision distribution within this taxon, and even less about the abilities shared by these animals regarding discrimination of chromatic targets. The aim of this study was to evaluate the color vision perception of captive Uta Hick's cuxiús, or bearded sakis (Chiropotes utahickae) through a behavioral paradigm of color visual discrimination, as well as to estimate, by genetic studies, the number and kinds of medium to long wavelength cone photopigment (opsins) encoded by this species. Among 12 cuxiús (7 males and 5 females) studied only 1 female was diagnosed as a trichromat. Results from genotyping were in line with our behavioral data and showed that cuxiús carried one (dichromat) or two (trichromat) medium to long wavelength pigments alleles, demonstrating a color vision polymorphism in C. utahickae similar to the majority of Neotropical Primates.

Whole-genome analyses resolve early branches in the tree of life of modern birds.

To better determine the history of modern birds, we performed a genome-scale phylogenetic analysis of 48 species representing all orders of Neoaves using phylogenomic methods created to handle genome-scale data. We recovered a highly resolved tree that confirms previously controversial sister or close relationships. We identified the first divergence in Neoaves, two groups we named Passerea and Columbea, representing independent lineages of diverse and convergently evolved land and water bird species. Among Passerea, we infer the common ancestor of core landbirds to have been an apex predator and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence and high levels of incomplete lineage sorting that occurred during a rapid radiation after the Cretaceous-Paleogene mass extinction event about 66 million years ago.

Chromobacterium violaceum: important insights for virulence and biotechnological potential by exoproteomic studies.

Chromobacterium violaceum is a beta-proteobacterium with high biotechnological potential, found in tropical environments. This bacterium causes opportunistic infections in both humans and animals, that can spread throughout several tissues, quickly leading to the death of the host. Genomic studies identified potential mechanisms of pathogenicity but no further studies were done to confirm the expression of these systems. In this study 36 unique protein entries were identified in databank from a two-dimensional profile of C. violaceum secreted proteins. Chromobacterium violaceum exoproteomic preliminary studies confirmed the production of proteins identified as virulence factors (such as a collagenase, flagellum proteins, metallopeptidases, and toxins), allowing us to better understand its pathogenicity mechanisms. Biotechnologically interesting proteins (such as chitinase and chitosanase) were also identified among the secreted proteins, as well as proteins involved in the transport and capture of amino acids, carbohydrates, and oxidative stress protection. Overall, the secreted proteins identified provide us important insights on pathogenicity mechanisms, biotechnological potential, and environment adaptation of C. violaceum.

Genome sequence of the Corynebacterium pseudotuberculosis Cp316 strain, isolated from the abscess of a Californian horse.

The bacterium Corynebacterium pseudotuberculosis is of major veterinary importance because it affects livestock, particularly sheep, goats, and horses, in several countries, including Australia, Brazil, the United States, and Canada, resulting in significant economic losses. In the present study, we describe the complete genome of the Corynebacterium pseudotuberculosis Cp316 strain, biovar equi, isolated from the abscess of a North American horse.

Genome sequence of Exiguobacterium antarcticum B7, isolated from a biofilm in Ginger Lake, King George Island, Antarctica.

Exiguobacterium antarcticum is a psychotropic bacterium isolated for the first time from microbial mats of Lake Fryxell in Antarctica. Many organisms of the genus Exiguobacterium are extremophiles and have properties of biotechnological interest, e.g., the capacity to adapt to cold, which make this genus a target for discovering new enzymes, such as lipases and proteases, in addition to improving our understanding of the mechanisms of adaptation and survival at low temperatures. This study presents the genome of E. antarcticum B7, isolated from a biofilm sample of Ginger Lake on King George Island, Antarctic peninsula.

Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia.

Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the α subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95% similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified - the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean Mcrα observed in the present study varied little, and presented approximately 70% identity in comparison with the Mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.