Genomic characterization of a novel Hepatovirus identified in Maranhão state, Brazil.

Bats are efficient reservoirs of a number of viruses with zoonotic potential, and are involved directly in the transmission cycle of many zoonoses. In the present study, which is part of a larger project that is documenting the viromes of the bat species found in the Mid-North states of Maranhão and Piauí, we analyzed 16 pooled samples obtained from four species of bat of the genus Artibeus-Artibeus obscurus, Artibeus cinereus, Artibeus lituratus and Artibeus planirostris. We describe and identify a Hepatovirus, denominated Hepatovirus H isolate sotense, which was found in a pool of internal organs (liver and lungs) extracted from a specimen of A. planirostris, a frugivorous bat, collected in the Cerrado biome of Maranhão state. This material was analyzed using new generation sequencing, which produced a contig of 7390 nucleotides and presented a degree of identity with a number of existing Hepatovirus sequences available for bats (amino acid identity of 61.5% with Bat hepatovirus C of Miniopterus cf. manavi, 66.6% with Bat hepatovirus G of Coleura afra, 67.4% with Hepatovirus G2 of Rhinolophus landeri, and 75.3% with Hepatovirus H2 of Rhinolophus landeri). The analysis of the functional domains of this contig confirmed a pattern consistent with the characteristics of the genus Hepatovirus (Picornaviridae). In the phylogenetic tree with several other Hepatovirus species, this genome also grouped in a monophyletic clade with Hepatovirus H (HepV-H1; HepV-H2, and HepV-H3) albeit on an external branch, which suggests that it may be a distinct genotype within this species. This is the first isolate of Hepatovirus H identified in bats from South America, and represents an important discovery, given that most studies of viruses associated with bats in the state of Maranhão have focused on the family Rhabdoviridae.

SynGenes: a Python class for standardizing nomenclatures of mitochondrial and chloroplast genes and a web form for enhancing searches for evolutionary analyses.

BACKGROUND: The reconstruction of the evolutionary history of organisms has been greatly influenced by the advent of molecular techniques, leading to a significant increase in studies utilizing genomic data from different species. However, the lack of standardization in gene nomenclature poses a challenge in database searches and evolutionary analyses, impacting the accuracy of results obtained. RESULTS: To address this issue, a Python class for standardizing gene nomenclatures, SynGenes, has been developed. It automatically recognizes and converts different nomenclature variations into a standardized form, facilitating comprehensive and accurate searches. Additionally, SynGenes offers a web form for individual searches using different names associated with the same gene. The SynGenes database contains a total of 545 gene name variations for mitochondrial and 2485 for chloroplasts genes, providing a valuable resource for researchers. CONCLUSIONS: The SynGenes platform offers a solution for standardizing gene nomenclatures of mitochondrial and chloroplast genes and providing a standardized search solution for specific markers in GenBank. Evaluation of SynGenes effectiveness through research conducted on GenBank and PubMedCentral demonstrated its ability to yield a greater number of outcomes compared to conventional searches, ensuring more comprehensive and accurate results. This tool is crucial for accurate database searches, and consequently, evolutionary analyses, addressing the challenges posed by non-standardized gene nomenclature.

Comparative cytogenetics of microsatellite distribution in two tetra fishes Astyanax bimaculatus (Linnaeus, 1758) and Psalidodon scabripinnis (Jenyns, 1842).

Background: The main cytogenetic studies of the Characidae family comprise the genera Astyanax and Psalidodon involving the use of repetitive DNA probes. However, for the microsatellite classes, studies are still scarce and the function of these sequences in the genome of these individuals is still not understood. Thus, we aimed to analyze and compare the distribution of microsatellite sequences in the species Astyanax bimaculatus and Psalidodon scabripinnis. Methods: We collected biopsies from the fins of A. bimaculatus and P. scabripinnis to perform cell culture, followed by chromosome extraction, and mapped the distribution of 14 microsatellites by FISH in both species. Results and Discussion: The diploid number observed for both species was 2n = 50, with an acrocentric B microchromosome in A. bimaculatus and a metacentric B chromosome in P. scabripinnis. Regarding FISH, 11 probes hybridized in the karyotype of A. bimaculatus mainly in centromeric regions, and 13 probes hybridized in P. scabripinnis, mainly in telomeric regions, in addition to a large accumulation of microsatellite hybridization on its B chromosome. Conclusion: Comparative FISH mapping of 14 microsatellite motifs revealed different patterns of distribution both in autosomes and supernumerary chromosomes of A. bimaculatus and P. scabripinnis, suggesting independent evolutionary processes in each of these species, representing excellent data on chromosome rearrangements and cytotaxonomy.

Comparative mitochondrial genome brings insights to slight variation in gene proportion and large intergenic spacer and phylogenetic relationship of mudskipper species.

Fish mitochondrial genome have been largely studied worldwide for evolutionary and other genetic purposes and the structure and gene organization are commonly conservative. However, several studies have demonstrated that this scenario may present variations in some taxa, showing differentiation on the gene rearrangement. In this study, the complete mitogenome of terrestrial fish Boleophthalmus dussumieri was generated and compared with other species of the Exudercidae fishes. The newly complete mitogenome generated is circular and 16,685 bp of length, and it contained 13 protein-coding genes (PCGs), two ribosomal RNA (rRNAs), 22 transfer RNA genes (tRNAs), and one control region (CR), with high conservative structure, like other Mudskippers. Most of the PCG showed similar codon usage bias. The gene length was found to be different specially for the CR, 12S rRNA gene and ND5 gene in some taxon. All the Boleophthalmus species showed a gene duplication in the CR, except for B. dussumieri, and they presented a long intergenic spacer specially on the tRNA-Pro/ OH Tandem duplication/random loss (TDRL) and dimer-mitogenome and nonrandom loss (DMNL) are suitable to explain the mitogenome rearrangement observed in this study. The phylogenetic analysis well supported the monophyly of all mudskipper species and the analysis positioned the Periophthalmus clade as the most basal of the terrestrial fishes. This finding provides basis and brings insights for gene variation, gene rearrangements and replications showing evidence for variety of mitochondrial structure diversity within mudskippers.

DNA barcoding for the assessment of marine and coastal fish diversity from the Coast of Mozambique.

The ichthyological provinces of Mozambique are understudied hotspots of global fish diversity. In this study, we applied DNA barcoding to identify the composition of the fish fauna from the coast of Mozambique. A total of 143 species belonging to 104 genera, 59 families, and 30 orders were identified. The overall K2P distance of the COI sequences within species ranged from 0.00% to 1.51%, while interspecific distances ranged from 3.64% to 24.49%. Moreover, the study revealed 15 threatened species according to the IUCN Red List of Threatened Species, with elasmobranchs being the most represented group. Additionally, the study also uncovered four new species that were not previously recorded in this geographic area, including Boleophthalmus dussumieri, Maculabatis gerrardi, Hippocampus kelloggi, and Lethrinus miniatus. This study represents the first instance of utilizing molecular references to explore the fish fauna along the Mozambican coast. Our results indicate that DNA barcoding is a dependable technique for the identification and delineation of fish species in the waters of Mozambique. The DNA barcoding library established in this research will be an invaluable asset for advancing the understanding of fish diversity and guiding future conservation initiatives.

First neurotranscriptome of adults Tambaquis (Colossoma macropomum) with characterization and differential expression between males and females.

The Tambaqui is one of the most representative Amazon fish species, being highly exploited in fisheries, aquaculture and as a research model. Nonetheless, data about functional genome are still required to evaluate reproductive and nutrition parameters as well as resistance to pathogens. The of next-generation sequencing has allows assessing the transcriptional processes in non-model species by providing comprehensive gene collections to be used as a database in further genomic applications and increased performance of captive populations. In this study, we relied on RNAseq approach to generate the first transcriptome of the telencephalon from adult males and females of Colossoma macropomum, resulting in a reference dataset for future functional studies. We retrieved 896,238 transcripts, including the identification of 267,785 contigs and 203,790 genes. From this total, 91 transcripts were differentially expressed, being 63 and 28 of them positively regulated for females and males, respectively. The functional annotation resulted in a library of 40 candidate genes for females and 20 for males. The functional enrichment classes comprised reproductive processes (GO:0,048,609; GO:0,003,006; GO:0,044,703; GO:0,032,504; GO:0,019,953) being related to sex differentiation (e.g., SAFB) and immune response (e.g., SLC2A6, AHNAK, NLRC3, NLRP3 and IgC MHC I alpha3), thus indicating that the genes in the neurotranscriptome of Tambaqui participate in sex differentiation and homeostasis of captive specimens. These data are useful to design the selection of genes related to sex determination and animal welfare in raising systems of Tambaqui.

Molossusmelini Montani et al. 2021 (Chiroptera, Molossidae) in Brazil: new insights for distribuition, morphology and genetics.

Background: The species M.melini has been observed in both the Pampa and Spinal ecoregions of Argentina. Researchers have underscored that distinguishing M.melini from other species within the same genus relies primarily on craniometric and molecular analyses. Morphological measurements alone do not offer a clear differentiation between M.melini and other members of this genus.This study aims to document the presence of M.melini within the Brazilian ecoregion, focusing on its morphological, morphometric and genetic characteristics. By undertaking a comprehensive examination, we seek to contribute valuable insights into the distribution and differentiation of M.melini in this region. New information: Molossusmelini specimens exhibited a forearm length ranging from 39.9 to 40.08 mm. The average intraspecific divergence was 1.2%, with specimens from the Argentine Pampas clustering in the same clade with a 98% bootstrap support and a posterior probability of: Regarding dorsal colouration, the specimens displayed fur with two bands-a Snow White base colour and apex colours ranging from Olive Brown, Broccoli Brown, Wood Brown to Yellowish-Brown. This marks the first record of M.melini in Brazil, expanding its distribution 1,300 km northeastwards into the Curitiba, Paraná, Atlantic Forest Ecoregion. The findings contribute valuable information on the distribution, morphology, morphometrics and genetics of this species.

Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

Sequencing and Analysis of the Mitochondrial Genome of Aedes aegypti (Diptera: Culicidae) from the Brazilian Amazon Region.

Aedes aegypti is a mosquito native to the African continent, which is now widespread in the tropical and subtropical regions of the world. In many regions, it represents a major challenge to public health, given its role in the cycle of transmission of important arboviruses, such as Dengue, Zika, and Chikungunya. Considering the epidemiological importance of Ae. aegypti, the present study sequenced the partial mitochondrial genome of a sample collected in the municipality of Balsas, in the Brazilian state of Maranhão, followed by High Throughput Sequencing and phylogenetic analyses. The mitochondrial sequence obtained here was 15,863 bp long, and contained 37 functional subunits (thirteen PCGs, twenty-two tRNAs and two rRNAs) in addition to a partial final portion rich in A+T. The data obtained here contribute to the enrichment of our knowledge of the taxonomy and evolutionary biology of this prominent disease vector. These findings represent an important advancement in the understanding of the characteristics of the populations of northeastern Brazil and provide valuable insights into the taxonomy and evolutionary biology of this prominent disease vector.

Molecular delimitation methods validate morphologically similar species of red snappers (Perciformes: Lutjanidae).

In this study, we tested the taxonomic validation of red snappers species (Southern red snapper Lutjanus purpureus; Silk snapper L. vivanus; Blackfin snapper L. buccanella; and Pacific red snapper L. peru) based on comparative analysis, using four methods for species delimitation. These methods were based on either genetic similarity or phylogenetic trees inferred from two mitochondrial (Cytochrome b and D-loop) and two nuclear (Myostatin and S7 introns) markers. On one hand, the genetic results corroborated the presence of four red snapper species, confirming their taxonomic validation despite their remarkable morphological similarity. On the other hand, few incongruencies in the species delimitation methods were observed according to the phylogenetic reconstruction method (maximum likelihood or Bayesian inference) when using. Based on the phylogenetic results, L. buccanella should represent a more ancient lineage in relation to the clade that encompasses L. purpureus, L. peru and L. vivanus. The single-locus phylogenetic analysis based on Cytb recovered each the red snapper species as a well-supported clade. Overall, this study provided a DNA-based validation of the traditional morphological taxonomy of red snappers.

Species delimitation by DNA barcoding reveals undescribed diversity in Stelliferinae (Sciaenidae).

Stelliferinae is the third most speciose subfamily of Sciaenidae, with 51 recognized species arranged in five genera. Phylogenies derived from both morphological and molecular data support the monophyly of this subfamily, although there is no general consensus on the intergeneric relationships or the species diversity of this group. We used the barcoding region of the cytochrome oxidase C subunit I (COI) gene to verify the delimitation of Stelliferinae species based on the Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescence (GMYC), and Bayesian Poisson Tree Process (bPTP) methods. In general, the results of these different approaches were congruent, delimiting 30-32 molecular operational taxonomic units (MOTUs), most of which coincided with valid species. Specimens of Stellifer menezesi and Stellifer gomezi were attributed to a single species, which disagrees with the most recent review of this genus. The evidence also indicated that Odontoscion xanthops and Corvula macrops belong to a single MOTU. In contrast, evidence also indicates presence of distinct lineages in both Odontoscion dentex and Bairdiella chrysoura. Such results are compatible with the existence of cryptic species, which is supported by the genetic divergence and haplotype genealogy. Therefore, the results of the present study indicate the existence of undescribed diversity in the Stelliferinae, which reinforces the need for an ample taxonomic review of the fish in this subfamily.

DNA barcode reveals occurrence of threatened species and hidden diversity on Teleost fish trade in the Coastal Amazon.

This study aimed to identify the teleost fish species sold in Bragança, a major fishing hub on the north coast of Brazil. The COI gene analysis was performed for the identification of fish species. The local market uses common names that are not accurate and do not reflect the diversity of the species. 204 sequences were obtained, with 119 haplotypes. 83 species were identified by comparing with public databases and constructing phylogenetic trees, with Carangidae being the most prevalent family. The study also found Haemulon atlanticus, Menticirrhus cuiaranensis and Hoplias misioneira, a newly described species from the Amazon basin, among the samples. Additionally, 73 commercial names were recorded, including 10 categories, and the illegal trade of Epinephelus itajara was detected. The DNA Barcode method proved to be effective for discriminating the species. The study highlights that common and commercial names are vague and underestimate the fish diversity, and that Brazil needs to revise its regulations for commercial and scientific names.

In silico prospecting of the mtDNA of Macrobrachium amazonicum from transcriptome data.

BACKGROUND: Macrobrachium amazonicum is a freshwater prawn widely distributed in South America that is undergoing speciation, so the denomination "M. amazonicum complex" is used for it. The mitochondrial cytochrome c oxidase subunit I (COI) gene has been used to elucidate this speciation, but heteroplasmies and pseudogenes have been recorded, making separation difficult. Obtaining genes from cDNA (RNA) rather than genomic DNA is an effective tool to mitigate those two types of occurrences. The aim of this study was to assemble in silico the mitochondrial DNA (mtDNA) of the Amazonian coastal population of M. amazonicum inhabiting the state of Pará. RESULTS: Sequences were obtained from the prawn's transcriptome using the de novo approach. Six libraries of cDNA from the androgen gland, hepatopancreas, and muscle tissue were used. The mtDNA of M. amazonicum was 14,960 bp in length. It contained 13 protein-coding genes, 21 complete transfer RNAs, and the 12S and 16S subunits of ribosomal RNA. All regions were found on the light strand except tRNAGln, which was on the heavy strand. The control region (D-loop) was not recovered, making for a gap of 793 bp. The cladogram showed the formation of the well-defined Macrobrachium clade, with high support value in the established branches (91-100). The three-dimensional spatial conformation of the mtDNA-encoded proteins showed that most of them were mainly composed of major α-helices that typically shows in those proteins inserted in the membrane (mitochondrial). CONCLUSIONS: It was possible to assemble a large part of the mitochondrial genome of M. amazonicum in silico using data from other genomes deposited in GenBank and to validate it through the similarities between its COI and 16S genes and those from animals of the same region deposited in GenBank. Depositing the M. amazonicum mtDNA sequences in GenBank may help solve the taxonomic problems recorded for the species, in addition to providing complete sequences of candidate coding genes for use as biomarkers in ecological studies.

Genetic Diversity in Natural Populations of the Near-Threatened Species Lignobrycon myersi (Characiformes, Triportheidae): Implications for Species Conservation.

The river basins of Brazil contain a highly diverse ichthyofauna of remarkable endemism, including several threatened species. Accordingly, Lignobrycon myersi is a fish species distributed only in a few rivers from the state of Bahia, northeastern Brazil. Since this species is classified as Near Threatened and is poorly studied, efforts to understand the genetic structure of populations and putative cryptic forms should help define efficient strategies of management and conservation. Herein, the molecular identification and the population genetic diversity of specimens of L. myersi across their range (Almada, Contas, and Cachoeira river basins) were assessed using mitochondrial markers (16S rDNA and D-Loop, respectively). The inferences based on phylogenetics, genetic distance, and species delimitation methods invariably identified all samples as L. myersi. In addition, sequencing of D-loop fragments revealed significant haplotype diversity and a considerable level of population genetic structure. Despite their geographic isolation, these data suggested that populations from Almada and Contas rivers represent a single evolutionary lineage that could be managed as a whole. In contrast, the population from Cachoeira River was highly differentiated from the others and should be managed separately as a unique and endemic unit, particularly focused on the conservation of native habitats.

Genetic connectivity and population expansion inferred from multilocus analysis in Lutjanus alexandrei (Lutjanidae-Perciformes), an endemic snapper from Northeastern Brazilian coast.

Previous studies about the genetic diversity, connectivity and demographic history in Lutjanidae fishes have reported a common pattern of genetic homogeneity and expansion in populations from Western South Atlantic. In the present work, we inferred the population structure, the levels of genetic diversity and the demographic history of the Brazilian snapper Lutjanus alexandrei, a recently described and endemic species from Northeastern coast of Brazil. Five different fragments, including mitochondrial DNA (Control Region, Cyt b and ND4) and nuclear DNA (Myostatin and S7) regions were analyzed in 120 specimens of L. alexandrei from four localities in Northeastern Brazil, representing the first study of population genetics in this species. High levels of genetic diversity were observed following a panmictic pattern, probably related to the larval dispersal by the current tides along the Brazilian coast. In addition, both demographic history and neutrality tests indicated that L. alexandrei has undergone population expansion during Pleistocene. In this sense, the sea level variation from this period could have increased the available resources and suitable habitats for the Brazilian snapper.

Genetic diversity analysis in the Brazilian Amazon reveals a new evolutionary lineage and new karyotype for the genus Mesomys (Rodentia, Echimyidae, Eumysopinae).

Morphological, molecular and chromosomal studies in the genera Lonchothrix and Mesomys have contributed to a better understanding of taxonomic design, phylogenetic relationships and karyotypic patterns. Recent molecular investigations have shown a yet undescribed diversity, suggesting that these taxa are even more diverse than previously assumed. Furthermore, some authors have questioned the limits of geographic distribution in the Amazon region for the species M. hispidus and M. stimulax. In this sense, the current study sought to understand the karyotypic evolution and geographic limits of the genus Mesomys, based on classical (G- and C-banding) and molecular cytogenetic analysis (FISH using rDNA 18S and telomeric probes) and through the sequencing of mitochondrial genes Cytochrome b (Cytb) and Cytochrome Oxidase-Subunit I (CO using phylogeny, species delimitation and time of divergence, from samples of different locations in the Brazilian Amazon. The species M. stimulax and Mesomys sp. presented 2n = 60/FN = 110, while M. hispidus presented 2n = 60/FN = 112, hitherto unpublished. Molecular dating showed that Mesomys diversification occurred during the Plio-Pleistocene period, with M. occultus diverging at around 5.1 Ma, followed by Mesomys sp. (4.1 Ma) and, more recently, the separation between M. hispidus and M. stimulax (3.5 Ma). The ABGD and ASAP species delimiters support the formation of 7 and 8 potential species of the genus Mesomys, respectively. Furthermore, in both analyzes Mesomys sp. was recovered as a valid species. Our multidisciplinary approach involving karyotypic, molecular and biogeographic analysis is the first performed in Mesomys, with the description of a new karyotype for M. hispidus, a new independent lineage for the genus and new distribution data for M. hispidus and M. stimulax.

Molecular characterization of juvenile fish from the Amazon estuary using DNA barcoding approach.

The efficiency of the DNA barcoding relies on sequencing fragment of the Cytochrome C Subunit I (COI) gene, which has been claimed as a tool to biodiversity identification from distinct groups. Accordingly, the goal of this study was to identify juvenile fish species along an estuary of Caeté River in the Brazilian Blue Amazon based on. For this purpose, we applied the DNA barcoding and discuss this approach as a tool for discrimination of species in early ontogenetic stages. A 500-bp fragment was obtained from 74 individuals, belonging to 23 species, 20 genera, 13 families and seven orders. About 70% of the 46 haplotypes revealed congruence between morphological and molecular species identification, while 8% of them failed in identification of taxa and 22% demonstrated morphological misidentification. These results proved that COI fragments were effective to diagnose fish species at early life stages, allowing identifying all samples to a species-specific status, except for some taxa whose COI sequences remain unavailable in public databases. Therefore, we recommend the incorporation of DNA barcoding to provide additional support to traditional identification, especially in morphologically controversial groups. In addition, periodic updates and comparative analyses in public COI datasets are encouraged.

Molecular Evidence Supports Five Lineages within Chiropotes (Pitheciidae, Platyrrhini).

Pitheciines have unique dental specializations among New World monkeys that allow them to feed on fruits with hard pericarps, thus playing a major role as seed predators. The three extant pitheciine genera, Pithecia, Cacajao and Chiropotes, are all endemic to the Amazon region. Because of the uncertainties about interspecific relationships, we reviewed the systematics and taxonomy of the genus Chiropotes. The phylogenetic analyses were performed based on Maximum Likelihood and Bayesian Inference, while species delimitation analyses were carried out using multispecies coalescent methods. In addition, we estimated genetic distances, divergence time and the probable ancestral distribution of this genus. Our results support five species of Chiropotes that emerged during the Plio-Pleistocene. Biogeographic estimates suggest that the ancestor of the current Chiropotes species occupied the endemism areas from Rondônia and Tapajós. Later, subsequent radiation and founder effects associated with the formation of the Amazonian basins probably determined the speciation events within Chiropotes.

Genetic Differentiation of Aedes aegypti (Diptera: Culicidae) in Areas with High Rates of Infestation in Mid-North Region of Brazil.

Aedes aegypti is the principal vector of the arboviruses-yellow fever, dengue virus, chikungunya, and zika virus. Given the epidemiological importance of this mosquito, its capacity to adapt to different habitats, and its resistance to many types of control measures, systematic research into the genetic variability of the populations of this mosquito is one of the most important steps toward a better understanding of its population structure and vector competence. In this context, the present study verified the presence of distinct genetic lineages of Ae. aegypti in areas with high infestation rates, based on the analysis of microsatellite markers. The samples were collected in nine municipalities with high building infestation rates in the Mid-North region of Brazil. Six microsatellite loci were genotyped in the 138 samples, producing a total of 32 alleles, varying from one to nine alleles per locus in each of the different populations. The AMOVA revealed greater within-population genetic differentiation with high fixation rates. The general analysis of population structure, based on a Bayesian approach, revealed K = 2, with two Ae. aegypti lineages that were highly differentiated genetically. These data on the connectivity of the populations and the genetic isolation of the lineages provide important insights for the development of innovative strategies for the control of the populations of this important disease vector.

Development of rapid and cost-effective multiplex PCR assays to differentiate catfish of the genus Brachyplatystoma (Pimelodidae-Siluriformes) sold in Brazil.

The catfishes Brachyplatystoma filamentosum (Kumakuma), Brachyplatystoma vaillantii (Laulao catfish), and Brachyplatystoma rousseauxii (gilded catfish) are important fishery resources in Brazil, where they are sold both fresh and in the form of fillets or steaks. These species have morphological similarities, thus, they can be easily misidentified or substituted, especially after processed. Therefore, accurate, sensitive, and reliable methods are needed for the identification of these species to avoid commercial fraud. In the present study, we develop two multiplex PCR assays for the identification of the three catfish species. Each multiplex protocol combined three species-specific forward primers and a universal reverse primer to produce banding patterns able to discriminate the target species unequivocally. The length of the cytochrome C oxidase subunit I (COI) fragments was approximately 254 bp for B. rousseauxii, 405 bp for B. vaillantii, and 466 bp for B. filamentosum, while the control region (CR) assay produced fragments of approximately 290 bp for B. filamentosum, 451 bp for B. vaillantii, and 580 bp for B. rousseauxii. The protocols were sensitive enough to detect the target species at a DNA concentration of 1 ng/µL, with the exception of the CR of B. vaillantii, in which the fragment was only detectable at 10 ng/µL. Therefore, the multiplex assays developed in the present study were sensitive, accurate, efficient, rapid, and cost-effective for the unequivocal identification of the target species of Brachyplatystoma. They can be utilized by fish processing industries to certify their products, or by government agencies to authenticate products and prevent fraudulent commercial substitutions.