Divergence in toxin antigenicity and venom enzymes in Tityus melici, a medically important scorpion, despite transcriptomic and phylogenetic affinities with problematic Brazilian species.

The Brazilian scorpion Tityus melici, native to Minas Gerais and Bahia, is morphologically related to Tityus serrulatus, the most medically significant species in Brazil. Despite inhabiting scorpion-envenomation endemic regions, T. melici venom remains unexplored. This work evaluates T. melici venom composition and function using transcriptomics, enzymatic activities, and in vivo and in vitro immunological analyses. Next-Generation Sequencing unveiled 86 components putatively involved in venom toxicity: 39 toxins, 28 metalloproteases, seven disulfide isomerases, six hyaluronidases, three phospholipases and three amidating enzymes. T. serrulatus showed the highest number of toxin matches with 80-100 % sequence similarity. T. melici is of medical importance as it has a venom LD50 of 0.85 mg/kg in mice. We demonstrated venom phospholipase A2 activity, and elevated hyaluronidase and metalloprotease activities compared to T. serrulatus, paralleling our transcriptomic findings. Comparison of transcriptional levels for T. serrulatus and T. melici venom metalloenzymes suggests species-specific expression patterns in Tityus. Despite close phylogenetic association with T. serrulatus inferred from COI sequences and toxin similarities, partial neutralization of T. melici venom toxicity was achieved when using the anti-T. serrulatus antivenom, implying antigenic divergence among their toxins. We suggest that the Brazilian therapeutic scorpion antivenom could be improved to effectively neutralize T. melici venom.

De novo assembly of the complete mitochondrial genome of Mycetophylax simplex Emery, 1888 through organelle targeting revels no substantial expansion of gene spacers, but rather some slightly shorter genes.

Mitochondria play a key role in cell biology and have their own genome, residing in a highly oxidative environment that induces faster changes than the nuclear genome. Because of this, mitochondrial markers have been exploited to reconstruct phylogenetic and phylogeographic relationships in studies of adaptation and molecular evolution. In this study, we determined the complete mitogenome of the fungus-farming ant Mycetophylax simplex (Hymenoptera, Formicidae) and conducted a comparative analysis among 29 myrmicine ant mitogenomes. Mycetophylax simplex is an endemic ant that inhabits sand dunes along the southern Atlantic coast. Specifically, the species occur in the ecosystem known as "restinga", within the Atlantic Forest biome. Due to habitat degradation, land use and decline of restinga habitats, the species is considered locally extinct in extremely urban beaches and is listed as vulnerable on the Brazilian Red List (ICMBio). We employed a mitochondrion-targeting approach to obtain the complete mitogenome through high-throughput DNA sequencing technology. This method allowed us to determine the mitogenome with high performance, coverage and low cost. The circular mitogenome has a length of 16,367 base pairs enclosing 37 genes (13 protein-coding genes, 22 tRNAs and 2 rRNAs) along with one control region (CR). All the protein-coding genes begin with a typical ATN codon and end with the canonical stop codons. All tRNAs formed the fully paired acceptor stems and fold into the typical cloverleaf-shaped secondary structures. The gene order is consistent with the shared Myrmicinae structure, and the A + T content of the majority strand is 81.51%. Long intergenic spacers were not found but some gene are slightly shorter. The phylogenetic relationships based on concatenated nucleotide and amino acid sequences of the 13 protein-coding genes, using Maximum Likelihood and Bayesian Inference methods, indicated that mitogenome sequences were useful in resolving higher-level relationship within Formicidae.

Genetic diversity and relatedness in captive collared peccaries Dicotyles tajacu (Linnaeus, 1758) (Cetartiodactyla: Tayassuidae) estimated by microsatellite genotyping using high-throughput sequencing: Implications for their conservation and reintroduction.

The global population of Dicotyles tajacu (Linnaeus, 1758) (Cetartiodactyla: Tayassuidae), commonly known as the collared peccary and distributed in the Neotropics, is currently in decline due to anthropogenic pressures. In this study, five microsatellite loci were used to genetically characterize a group of 20 captive-born collared peccaries intended for reintroduction. This study aimed to evaluate the genetic diversity and relatedness of captive individuals using microsatellite markers. The genetic data generated were used to evaluate the viability of the reintroduction and to propose measures for the management and conservation of this species. In this study, we found relatively high genetic diversity indices, indicating that the group was genetically diverse. Inbreeding coefficients with negative values were observed, indicating an excess of alleles in heterozygosis and an absence of inbreeding. One locus showed deviation from Hardy-Weinberg equilibrium, which may have been caused by the mixing of individuals from different origins. Relatedness analysis indicated that some individuals were highly related, with coefficients indicating they may be first-degree relatives. Our findings indicate that the studied group has enough genetic diversity to be released into nature, but the high individual relatedness found would require the adoption of strategies after the release of animals in the wild to ensure their persistence.

Eco-evolutionary factors that influence its demographic oscillations in Prochilodus costatus (Actinopterygii: Characiformes) populations evidenced through a genetic spatial-temporal evaluation.

The human activity impact on wild animal populations is indicated by eco-evolutionary and demographic processes, along with their survival and capacity to evolve; consequently, such data can contribute toward enhancing genetic-based conservation programs. In this context, knowledge on the life-history and the eco-evolutionary processes is required to understand extant patterns of population structure in Prochilodus costatus a Neotropical migratory fish that has been threatened due to loss and fragmentation of its natural habitat since 1960s promoted by the expansion of hydroelectric power plant construction programs. This study evaluated the eco-evolutionary parameters that cause oscillations in the demography and structure of P. costatus populations. An integrated approach was used, including temporal and spatial sampling, next-generation sequencing of eight microsatellite loci, multivariate genetic analysis, and demographic life-history reconstruction. The results provided evidence of the complex interplay of ecological-evolutionary and human-interference events on the life history of this species in the upper basin. In particular, spawning wave behavior might have ecological triggers resulting in an overlapping of distinct genetic generations, and arising distinct migratory and nonmigratory genetic patterns living in the same area. An abrupt decrease in the effective population size of the P. costatus populations in the recent past (1960-80) was likely driven by environment fragmentation promoted by the construction of the Três Marias hydropower dam. The low allelic diversity that resulted from this event is still detected today; thus, active stocking programs are not effective at expanding the genetic diversity of this species in the river basin. Finally, this study highlights the importance of using mixed methods to understand spatial and temporal variation in genetic structure for effective mitigation and conservation programs for threatened species that are directly affected by human actions.

The complete mitochondrial genome of Sporophila nigricollis (Aves, Passeriformes).

BACKGROUND: Sporophila nigricollis, popularly known as Yellow-bellied Seedeater, is a commonly trafficked bird in Brazil. This study describes the complete mitogenome of the species and its phylogenetic position. METHODS AND RESULTS: DNA sample was sequenced on MiSeq (Illumina®) sequencer. Reads were assembled to reference using Geneious. A total of 11,740 paired sequence reads were used to produce the complete mitochondrial DNA sequence with average coverage of 176x. The mitogenome was found to be circular in shape and consisted of 16,777 base pairs. The mitochondrial genome was annotated with MITOS and verified with ExPASy. Its structure contains two ribosomal RNA (rRNA) genes, 22 transporter RNA (tRNA) genes, 13 protein-coding genes (PCGs) and one control region. Twelve of the 13 PCGs have ATG as start codon. Seven of the 13 PCGs contain a TAA stop codon. Most of the tRNA genes and PCGs are encoded on the heavy strand. Phylogenetic analyses were conducted with MEGA using the maximum likelihood method. Sporophila nigricollis grouped together with other Thraupidae species. CONCLUSION: This study presents the first complete mitogenome of Sporophila nigricollis and can be useful for research on evolution, ecology and conservation of this species.

An overview of Tityus cisandinus scorpion venom: Transcriptome and mass fingerprinting reveal conserved toxin homologs across the Amazon region and novel lipolytic components.

Tityus cisandinus, a neglected medically important scorpion in Ecuadorian and Peruvian Amazonia, belongs to a complex of species related to the eastern Amazon endemic Tityus obscurus, spanning a distribution of ca. 4000 km. Despite high morbidity and mortality rates, no effective scorpion antivenom is currently available in the Amazon region. Knowledge of the structural/functional relationships between T. cisandinus venom components and those from related Amazonian species is crucial for designing region-specific therapeutic antivenoms. In this work, we carried out the first venom gland transcriptomic study of an Amazonian scorpion outside Brazil, T. cisandinus. We also fingerprinted its total venom through MALDI-TOF MS, which supported our transcriptomic findings. We identified and calculated the expression level of 94 components: 60 toxins, 25 metalloproteases, five disulfide isomerases, three amidating enzymes, one hyaluronidase, and also uncovered transcripts encoding novel lipolytic beta subunits produced by New World buthid scorpions. This study demonstrates the high similarity between T. cisandinus and T. obscurus venoms, reinforcing the existence of a neglected complex of genetically and toxinologically related Amazonian scorpions of medical importance. Finally, we demonstrated the low recognition of currently available therapeutic sera against T. cisandinus and T. obscurus venoms, and concluded that these should be improved to protect against envenomation by Amazonian Tityus spp.

Analysis of NGS data from Peruvian Loxosceles laeta spider venom gland reveals toxin diversity.

Accidents involving spiders from the genus Loxosceles cause medical emergencies in several countries of South America. The species Loxosceles laeta is ubiquitously present in Peru and is responsible for severe accidents in this country. To further characterize L. laeta venom components and to unveil possible variations in the Peruvian population, we provide an overview of the toxins-related transcripts present in the venom gland of Peruvian L. laeta. A dataset from a cDNA library previously sequenced by MiSeq sequencer (Illumina) was re-analyzed and the obtained data was compared with available sequences from Loxosceles toxins. Phospholipase-D represent the majority (69,28 %) of the transcripts related to venom toxins, followed by metalloproteases (20,72 %), sicaritoxins (6,03 %), serine-proteases (2,28 %), hyaluronidases (1,80 %) and Translationally Controlled Tumor Protein (TCTP) (0,56 %). New sequences of phospholipases D,sicaritoxins, hyaluronidase, TCTP and serine proteinases were described. Differences between the here-described toxin sequences and others, previously identified in venom glands from other spiders, were visualized upon sequence alignments. In addition, an in vitro hyaluronidase activity assay was also performed to complement comparisons between Peruvian and Brazilian L. laeta venom enzymatic activities, revealing a superior activity in the venom from Brazilian specimens. These new data provide a molecular basis that can help to explain the difference in toxicity among L. laeta venoms from different countries in South America.

A prokaryote system optimization for rMEPLox expression: A promising non-toxic antigen for Loxosceles antivenom production.

Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD600nm = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment.

The complete mitochondrial genome of Abramites hypselonotus (Günther, 1868) (Ostariophysi: Characiformes: Anostomidae): an aquarium headstander.

Abramites hypselonotus, commonly known as marbled headstander, is an important freshwater aquarium fish from Brazil, found in the Orinoco, Amazon, Paraguay and lower Parana River basins. This genus has only two species and only this species occurs in Brazil. The complete mitochondrial genome of Abramites hypselonotus is 16,685 bp in length and it includes 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNAs genes and a control region with 1,028 bp. It has two PCGs with GTG start codon and the others with ATG start codon. Four of the 13 PCGs appear TAA stop codon, three incomplete TA_ stop codon, four incomplete T_ _ stop codon, one contain AGG stop codon and one TAG stop codon. Phylogenetic analysis showed that Abramites hypselonotus formed a sister group of Leporinus affinis (AP011994.1), thus maintaining the Family Anostomidae as a clade.

Endophytic fungus diversity in soybean plants submitted to conditions of elevated atmospheric CO2 and temperature.

Global climatic changes can have drastic impacts on plant species, including severe consequences for the agricultural species productivity. Many of these species present important mutualisms with endophytic fungi that positively influence their performance. The present study evaluated whether the increases in CO2 and temperature predicted for the year 2100 may cause changes in foliar carbon (C) and nitrogen (N) concentrations in soybean (Glycine max) and, consequently, the interactions with its endophytic fungi. The effects of elevated CO2 and temperature were evaluated in four treatments in open-top chambers: (i) control, (ii) increased temperature, (iii) increased CO2, and (iv) increased CO2 and temperature. Increased atmospheric CO2 resulted in decreased foliar N concentration, while increased temperature increased it. A total of 16 taxa of endophytic fungi were identified based on sequencing internal transcribed spacer regions of rRNA subunits. Increased atmospheric CO2 and temperature were observed to potentially modify the endophytic mycobiota of soybean plants. The results suggest that the fungi species substitution is a consequence of changes in foliar N concentration and C/N ratio. Predicted climatic changes shall affect the relationships between plant and endophytes, which in turn, will affect the performance and resistance of soybean, one of the most important crops in the world.

Molecular tracking of pathogens in central venous catheter.

BACKGROUND: Central venous catheter-related bloodstream infection is an important adverse event in health care. Molecular methods are not yet substitutive of microbiological in the detection of the pathogens responsible for the infection, but they can help in the epidemiological characterization. AIM: To detect bacteria by polymerase chain reaction, from material extracted from the tip of central catheters of patients suspected of infection at the intensive care unit. METHODS: Catheters (n = 34) of patients suspected of central venous catheter-related infection were analyzed by polymerase chain reaction. The findings were compared with culture of catheter tip and blood cultures performed by the hospital. FINDINGS: The prevalence of bacteria was Staphylococcus aureus (50%), Enterococcus faecalis (41.2%), Klebsiella pneumoniae (32.4), Pseudomonas aeruginosa (20.6%), Acinetobacter baumannii (38.2%), Escherichia coli (2.9%), and Enterobacter cloacae (0%). No blood culture showed bacterial growth, the culture of catheter tip revealed bacteria in 21 (61.8%) and the polymerase chain reaction had positivity in 31 (91.2%) of the catheters. The mean central venous catheter time was 11 days, and the jugular vein was the site of insertion. CONCLUSION: The molecular method identified more bacteria than microbiological methods and revealed colonization of the catheters. The most commonly found bacteria are in the environment and in the microbiota of the skin, which suggests contamination by the hands of health professionals and points out the need for more efforts in preventive strategies.

Exploring antibiotic resistance in environmental integron-cassettes through intI-attC amplicons deep sequencing.

INTRODUCTION: Freshwater ecosystems provide propitious conditions for the acquisition and spread of antibiotic resistance genes (ARGs), and integrons play an important role in this process. MATERIAL AND METHODS: In the present study, the diversity of putative environmental integron-cassettes, as well as their potential bacterial hosts in the Velhas River (Brazil), was explored through intI-attC and 16S rRNA amplicons deep sequencing. RESULTS AND DISCUSSION: ORFs related to different biological processes were observed, from DNA integration to oxidation-reduction. ARGs-cassettes were mainly associated with class 1 mobile integrons carried by pathogenic Gammaproteobacteria, and possibly sedentary chromosomal integrons hosted by Proteobacteria and Actinobacteria. Two putative novel ARG-cassettes homologs to fosB3 and novA were detected. Regarding 16SrRNA gene analysis, taxonomic and functional profiles unveiled Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria as dominant phyla. Betaproteobacteria, Alphaproteobacteria, and Actinobacteria classes were the main contributors for KEGG orthologs associated with resistance. CONCLUSIONS: Overall, these results provide new information about environmental integrons as a source of resistance determinants outside clinical settings and the bacterial community in the Velhas River.

Novel components of Tityus serrulatus venom: A transcriptomic approach.

Several research groups have studied the components produced by the venom gland of the scorpion Tityus serrulatus, which has one of the most lethal venoms in the world. Various methodologies have been employed to clarify the complex mechanisms of action of these components, especially neurotoxins and enzymes. Transcriptomes and proteomes have provided important information for pharmacological, biochemical, and immunological research. Next-generation sequencing (NGS) has allowed the description of new transcripts and completion of partial sequence descriptions for peptides, especially those with low expression levels. In the present work, after NGS sequencing, we searched for new putative venom components. We present a total of nine new transcripts with neurotoxic potential (Ts33-41) and describe the sequences of one hyaluronidase (TsHyal_4); three enzymes involved in amidation (peptidyl-glycine alpha-amidating monooxygenase A, peptidyl-alpha-hydroxyglycine alpha-amidating lyase, and peptidylglycine alpha-hydroxylating monooxygenase), which increases the lethal potential of neurotoxins; and also the enzyme Ts_Chitinase1, which may be involved in the venom's digestive action. In addition, we determined the level of transcription of five groups: toxins, metalloproteases, hyaluronidases, chitinases and amidation enzymes, including new components found in this study. Toxins are the predominant group with an expression level of 91.945%, followed by metalloproteases with only 7.790% and other groups representing 0.265%.

Complete mitochondrial genome sequence of the high altitude Brazilian treefrog Pithecopus megacephalus (Anura, Phyllomedusidae).

The first complete mitochondrial genome (mtDNA) for the family Phyllomedusidae (genus Pithecopus) is presented. It is a circular molecule with 17713 pb including 13 protein coding genes, 22 tRNA genes, two rRNA genes, and a control region (D-loop). Pithecopus megacephalus was close to the only other phyllomedusid whose complete mtDNA sequence is available, but had the cytb gene 147 pb smaller.

Genetic and toxinological divergence among populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones: Buthidae) inhabiting Paraguay and Argentina.

Envenoming by scorpions in genus Tityus is a public health problem in Tropical America. One of the most medically significant species is Tityus trivittatus, which is known to occur from southwest Brazil to central-northern and eastern Argentina. In this work, we studied the lethality, composition, antigenicity, and enzymatic activity of venom from a T. trivittatus population found further north in urban areas of eastern Paraguay, where it has caused serious envenomation of children. Our results indicate that the population is of medical importance as it produces a potently toxic venom with an LD50 around 1.19 mg/kg. Venom neutralization in preliminary mouse bioassays was complete when using Brazilian anti-T. serrulatus antivenom but only partial when using Argentinean anti-T. trivittatus antivenom. Venom competitive solid-phase enzyme immunoassays and immunoblotting from Argentinean and Paraguayan T. trivittatus populations indicated that antigenic differences exist across the species range. SDS-PAGE showed variations in type and relative amounts of venom proteins between T. trivitattus samples from Argentina and Paraguay. MALDI-TOF mass spectrometry indicated that while some sodium channel toxins are shared, including ß-toxin Tt1g, others are population-specific. Proteolytic activity by zymography and peptide identification through nESI-MS/MS also point out that population-specific proteases may exist in T. trivitattus, which are postulated to be involved in the envenoming process. A time-calibrated molecular phylogeny of mitochondrial COI sequences revealed a significant (8.14%) genetic differentiation between the Argentinean and Paraguayan populations, which appeared to have diverged between the mid Miocene and early Pliocene. Altogether, toxinological and genetic evidence indicate that T. trivitattus populations from Paraguay and Argentina correspond to distinct, unique cryptic species, and suggest that further venom and taxonomic diversity exists in synanthropic southern South American Tityus than previously thought.

Genetic evaluation of migratory fish: Implications for conservation and stocking programs.

Fish stocking programs have been implemented to mitigate the blockage of original riverbeds by the construction of hydropower dams, which affects the natural migration of fish populations. However, this method raises concerns regarding the genetic rescue of the original populations of migratory fish species. We investigated the spatial distribution of genetic properties, such as genetic diversity, population structure, and gene flow (migration), of the Neotropical migratory fish Prochilodus costatus in the Três Marias dam in the São Francisco River basin, Brazil, and examined the possible effects of fish stocking programs on P. costatus populations in this region. In total, 1,017 specimens were sampled from 12 natural sites and a fish stocking program, and genotyped for high-throughput sequencing at 8 microsatellite loci. The populations presented low genetic variability, with evidence of inbreeding and the presence of only four genetic pools; three pools were observed throughout the study region, and the fourth was exclusive to one area in the Paraopeba River. Additionally, we identified high unidirectional gene flow between regions, and a preferred migratory route between the Pará River and the upper portion of the São Francisco River. The fish stocking program succeeded in transposing the genetic pools from downstream to upstream of the Três Marias dam, but, regrettably, promoted genetic homogenization in the upper São Francisco River basin. Moreover, the data show the fragility of this species at the genetic level. This monitoring strategy could be a model for the development of conservation and management measures for migratory fish populations that are consumed by humans.

Molecular cloning and functional characterization of recombinant Loxtox from Loxosceles similis venom.

Loxoscelism is a recognized public health problem in Brazil, but the venom from Loxosceles similis, which is widespread in Brazil due to its adaptability to the urban environment, remains poorly characterized. Loxtox is a family of phospholipase D enzymes (PLDs), which are the major components of Loxosceles venom and are responsible for the clinical effects of loxoscelism. Loxtox toxins correspond to 15% of L. similis venom gland transcripts, but the Loxtox family of L. similis has yet to be fully described. In this study, we cloned and functionally characterized recLoxtox s1A and recLoxtox s11A. These recombinant toxins exhibited different in vitro activities depending on pH, and recLoxtox s1A had more intense effects on rabbit skin than did recLoxtox s11A in vivo. Both recombinant toxins were used in immunization protocols, and mapping of their epitopes revealed different immunological reactions for the produced immune serums. Additionally, polyclonal antibodies raised against recLoxtox s1A had greater capacity to significantly reduce the in vitro and in vivo effects of L. similis venom. In summary, we obtained and characterized two novel Loxtox isoforms from L. similis venom, which may be valuable biotechnological and immunological tools against loxoscelism.

Engineered antigen containing epitopes from Loxosceles spp. spider toxins induces a monoclonal antibody (Lox-mAb3) against astacin-like metalloproteases.

Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 (184ENNTRTIGPFDYDSIMLYGAY205) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism.

Use of complete mitochondrial genome sequences to identify barcoding markers for groups with low genetic distance.

Complete mitochondrial sequences can be rapidly obtained and are widely available, providing a great source of species information and allowing for the discovery of new specific molecular markers. However, for some taxonomic groups, traditional approaches for species delimitation are impaired by the low genetic distance values. In these cases, other species-level markers are used. For Prochilodus, which includes important neotropical fish species, species-level delimitation usually results in poor phylogenetic resolution when using mitochondrial COI/cytB genes as barcoding markers because of low genetic variability and low species-level resolution. Thus, in this study, we developed an approach to design and validate new barcoding markers with high species-level resolution obtained from the D-loop region, using Prochilodus spp. as a model. For the new barcoding marker validation, the amplicon region was used to infer the phylogenetic relationships of Prochilodus spp. through three distinct methods: Bayesian inference (BI), Neighbor-Joining method (NJ), and Maximum Likelihood method (ML). The phylogenetic relationships of Prochilodus spp. revealed high resolution at species-level, nonoverlapping clades, and high branch support. The genetic distance results allied to two different clustering methods (Bayesian Poisson tree processes and automatic barcode gap discovery) revealed the existence of a barcoding gap, thus, validating the use of the barcoding markers designed in this study. The approach proposed here may, therefore, be expanded to other taxa to access and validate new barcoding markers with higher resolution at the species level.

New set of microsatellite markers for the Great-billed Seed-finch (Sporophila maximiliani - Passeriformes: Thraupidae): tools for inspection and conservation.

The Thraupidae family is one of the most wanted by bird breeders in Brazil because it is represented by its diverse, colorful and melodious singers. The Great-billed Seed-finch, Sporophila maximiliani, is the only representative of the genus Sporophila considered critically endangered in Brazil. Due to the demands of environmental agencies and of conservation programs, there is a need to increase the number of molecular markers available for the genus and specially for S. maximiliani. Therefore, this work aimed to provide a new set of microsatellite markers for S. maximiliani in order to help bird breeders and environmental agencies on fulfilling its demands as well as contributing with extra genetics tools for conservation programs of the S. maximiliani. Of the 30 markers developed, 25 successfully amplified, and 22 were polymorphic. Annealing temperature varied from 52 to 64 °C, number of alleles from 2 to 13, and the medium allele richness was 7.25 and medium expected, observed heterozygosity and PIC were, respectively, 0.812, 0.661 and 0.752. The probability of identity estimate was 8.54 × 10-27 and all the other probabilities of non-exclusion (sib-identity, parent pair and first-parent) were < 0.001, indicating that this set of microsatellite markers have high genetic variability and high power of individual genetic differentiation for S. maximiliani. Therefore, this work increases the options of molecular markers to be used on inspection for environmental agencies and for conservation programs on analyzing genetic variability and population studies for S. maximiliani.