The Hyacinth Macaw (Anodorhynchus hyacinthinus) is a threatened species of the family Psittacidae. Its classification as vulnerable by the International Union for Conservation of Nature is especially a result of habitat loss and intense illegal trade of this species. The total estimated number of wild individuals in Brazil is around 6500 birds, distributed in three regions: Pantanal, Pará State, and Northeast Brazil. Data on the biology, ecology, and genetic variability of this species are fundamental to the development of conservation strategies for its natural populations. Therefore, the present study aimed to analyze the genetic structure of different populations of A. hyacinthinus and examine the population dynamics throughout its distribution in four areas of Brazil. We analyzed 100 individuals by amplifying and sequencing the mitochondrial DNA control region (D-loop) and identified 17 polymorphic sites and 16 distinct haplotypes. Total nucleotide diversity was 0.0034 and haplotype diversity was 0.7832. The fixation index indicated significant levels of genetic differentiation among all sampled populations. Our results indicate that different populations of A. hyacinthinus should be considered as evolutionarily significant units and must be independently protected and managed to preserve the adaptive diversity and evolutionary processes of this species throughout its distribution.

Natural history studies can provide information that can be used in species conservation and management. The present study provides information about the nests and nest sites of Hyacinth Macaws (Anodorhynchus hyacinthinus) in a mosaic of protected areas in Carajás and surrounding areas in the Amazon region of the state of Pará, Brazil. Data were collected during four expeditions conducted in 2007, 2008, 2013 and 2014. A total of 66 tree cavities were monitored, and 28 of these were used by Hyacinth Macaws. In addition, 38 breeding events were recorded (some nests were active in multiple seasons), and 17 eggs and 33 nestlings were observed. Environmental variables for the 24 cavities that were actively used by Hyacinth Macaws were evaluated. Most of the nest cavities were located in Sterculia sp. trees (86.6%) and in open areas (e.g., near pastures). Nesting tree and cavity measurements were variable, but the mean diameter at breast height of the trees containing Hyacinth Macaw nest cavities was larger than that reported from other regions, such as the Pantanal. The present study describes the unique ecology and life history of Hyacinth Macaws in Carajás, for which information is scarce. This information will facilitate the conservation and management of Hyacinth Macaws and can be used locally for environmental education.

Atualmente, o mundo tem os “olhos” voltados à urgência de transformações em direção ao respeito pela vida e sustentabilidade. Assim, a utilização de estratégias de educação ambiental, visando reduzir risco de extinção de espécies animais pode contribuir enormemente para sua conservação. Entre as diversas aves nativas do Brasil destaca-se a arara-azul-grande (Anodorhynchus hyacinthinus).O presente trabalho descreve a elaboração e aplicação de um jogo de tabuleiro com informações lúdicas sobre os riscos de extinção e propostas de conservação da arara-azul-grande, sendo direcionado a alunos do ensino fundamental I e fundamental II. Este material de educação ambiental foi particularmente aplicado e avaliado na região do mosaico Carajás, no Pará.

Salminus brasiliensis, a characin regionally named dourado' (meaning goldfish), is the biggest characid fish, this species is cited in a list of fishes that are vulnerable. The goal of the present work was to try to identify, through AFLP technique to convert them into single locus markers (SCARs), in order to improve the sex identification in S.brasiliensis. Between possible sex-specific AFLP markers in this study, one male-specific were isolated (Les1) and converted into a SCAR marker. Les1 marker was confirmed sex-specific in all samples tested. A sex-specific DNA marker applicable to fish would be very useful for elucidating sex determination mechanism in fish.

In birds, there are two main models for the determination of sex: the Z Dosage model in which the number, or dose, of Z chromosomes determines sex, and the Dominant W model which argues that a specific gene in the W chromosome may influence Z gene expression and determine sex. The best evidence for W determination of sex comes from birds with 2 copies of the Z chromosome paired with a single W (e.g. ZZW) which are nonetheless females. Here, we expand the species where such a mechanism may operate by reporting a case of a triploid Neotropical passerine bird with sexually dimorphic plumage, the Sao Paulo marsh antwren Formicivora paludicola. Evidence from 17 autosomal unlinked microsatellite loci, and CHD1 sex-linked locus, indicate that this individual is a 3n ZZW triploid with intermediate plumage pattern. This example expands our knowledge of sex determination mechanisms in birds by demonstrating that both the W and the two Z chromosomes affect the expression of morphological secondary sexual traits in a non-galliform bird.

Small populations of endangered species can be impacted by genetic processes such as drift and inbreeding that reduce population viability. As such, conservation genetic analyses that assess population levels of genetic variation and levels of gene flow can provide important information for managing threatened species. The São Paulo Marsh Antwren (Formicivora paludicola) is a recently-described and critically endangered bird from São Paulo State (Brazil) whose total estimated population is around 250-300 individuals, distributed in only 15 isolated marshes around São Paulo metropolitan region. We used microsatellite DNA markers to estimate the population genetic characteristics of the three largest remaining populations of this species all within 60 km of each other. We detected a high and significant genetic structure between all populations (overall FST = 0.103) which is comparable to the highest levels of differentiation ever documented for birds, (e.g., endangered birds found in isolated populations on the tops of African mountains), but also evidence for first-generation immigrants, likely from small local unsampled populations. Effective population sizes were small (between 28.8-99.9 individuals) yet there are high levels of genetic variability within populations and no evidence for inbreeding. Conservation implications of this work are that the high levels of genetic structure suggests that translocations between populations need to be carefully considered in light of possible local adaptation and that remaining populations of these birds should be managed as conservation units that contain both main populations studied here but also small outlying populations which may be a source of immigrants.

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

RESUMO Dois genes de α-actina do peixe Leporinus macrocephalus, referindo-se aos tecidos musculares branco e vermelho, foram isolados. Isoformas de actinas, que principalmente diferiram por uma substituição Ser/Ala155, podem ter uma significância funcional relacionada à interação entre actina e ATP. Um resíduo de Ala155, como observado na α-actina esquelética do músculo vermelho, resulta em uma diminuição da afinidade da actina pelo ATP, o que também pode estar associado à ação contrátil lenta desse tecido. Adicionalmente, uma substituição Phe/Ile262 na actina do músculo vermelho leva a uma variação na hidrofobicidade no "plug-D" da proteína, o que pode alterar sua estabilidade. Dados de qRT-PCR evidenciaram significante maior nível de actina RNAm em músculo branco, quando comparado ao músculo vermelho (T=105 Mann Whitney; p=<0,001). Este resultado pode estar relacionado às demandas energéticas do tecido muscular branco, com fibras de contração rápida e metabolismo glicolítico para fornecimento de energia. Os dados disponíveis sobre actinas musculares levam a propor que as α-actinas esqueléticas dos músculos branco e vermelho são geneticamente e funcionalmente distinguíveis em espécies de peixes, uma característica não encontrada em outros grupos de vertebrados.

B chromosomes are dispensable genomic elements found in different groups of animals and plants. In the present study, a whole chromosome probe was generated from a specific heterochromatic B chromosome occurring in cells of the characidae fish Moenkhausia sanctaefilomenae (Steindachner, 1907). The chromosome painting probes were used in fluorescence in situ hybridization (FISH) experiments for the assessment of metaphase chromosomes obtained from individuals from three populations of M. sanctaefilomenae. The results revealed that DNA sequences were shared between a specific B chromosome and many chromosomes of the A complement in all populations analyzed, suggesting a possible intra-specific origin of these B chromosomes. However, no hybridization signals were observed in other B chromosomes found in the same individuals, implying a possible independent origin of B chromosome variants in this species. FISH experiments using 18S rDNA probes revealed the presence of non-active ribosomal genes in some B chromosomes and in some chromosomes of the A complement, suggesting that at least two types of B chromosomes had an independent origin. The role of heterochromatic segments and ribosomal sequences in the origin of B chromosomes were discussed.

Hyacinth macaw Anodorhynchus hyacinthinus is the largest parrot of the world and is considered vulnerable to extinction due to its habitat loss and illegal trade associated to the international pet market demand. Genetic studies on this species are still incipient to generate a consistent characterization of the population dynamics and to develop appropriate conservation strategies. In this sense, microsatellite markers may support the detection of a population genetic structure for this bird species. However, at this time, none Hyacinth macaw species-specific primers for microsatellite loci have been so far established. This study aimed to develop and characterize polymorphic microsatellite markers for A. hyacinthinus and to check for their cross-amplification in other parrot species.

The São Paulo marsh antwren (Formicivora paludicola) is a critically endangered bird endemic to marshes in the metropolitan region of São Paulo city, Brazil. The total population is estimated to be around 300 individuals, distributed among 15 small (<50 ha) fragments, suggesting that loss of genetic variability may affect the long-term viability of this species. To develop genetic tools for gaining information on effective population sizes, inbreeding and gene flow between populations, we describe nine polymorphic microsatellite loci isolated from a F. paludicola library using next-generation sequencing. We report on levels of variation in these novel microsatellites and eight additional heterologous loci in these birds. Expected (H E) and observed (H O) heterozygosities averaged 0.72 and 0.70, respectively, and the number of alleles per locus ranged from 3 to 10. These loci will permit evaluation of whether artificial translocations are necessary for long-term viability of this rare bird.

O Projeto de Extensao Universitaria Difundindo e Popularizando a Ciencia na UNESP: Interacao entre Pos-Graduacao e Ensino Basico - Area de tem como objetivo difundir topicos de Genetica do conteudo curricular do ensino medio, recentes avancos da area de Biologia Molecular e Biotecnologia e o conceito e a aplicacao do Metodo Cientifico. Entre as diversas atividades deste programa de extensao, inserem-se a elaboracao, aplicacao e distribuicao de materiais didaticos que possam ser utilizados em atividades teoricopraticas com o objetivo de promover uma acao de ensino-aprendizado mais ativa. Desta forma, o objetivo do presente trabalho foi a elaboracao de materiais didaticos de baixo custo, facil compreensao e adequada aplicacao em atividades para o ensino medio para assimilacao de conceitos associados a eletroforese em gel para analise de DNA. Os materiais desenvolvidos encontram-se em conformidade com a missao institucional da CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior) de aplicar a experiencia adquirida na graduacao e pos-graduacao para melhorar a qualidade do ensino basico.

The ability of a population to adapt to a changing environment depends on its genetic variation. Thus, the study of genetic diversity within and among species or populations is especially important on conservation biology scopes. One way to assess the genetic diversity is through the use of microsatellite molecular markers. Microsatellites have been widely used to answer population genetics issues as gene flow, parentage, and population structure, mostly resulting in data on the distribution of genetic variability within and among natural populations, which are essential for ex situ and in situ conservation procedures. As the Psittacidae family comprehends one of the birds group with the largest number of endangered species, studies that aim to investigate the genetic diversity of these animals may support their conservation. This article is a review of genetic data on parrots, through the use of microsatellite markers, that have been published since 2004.

Universidade Estadual Paulista (UNESP), Instituto de Biociencias (IBB), Departamento de Botânica, Botucatu, SP, Brasil

Microsatellite markers were analyzed in Japanese quails, Coturnix japonica, using different methodologies (PAGE and automated genotyping), in order to evaluate their use in paternity testing. Ten animal triplets composed by a female and two males were used to mate and generate an offspring. Paternity was determined in five-day-old embryos, and the data generated by fluorescent labeled and tailored primers in PCR and further automated genotyping were robust. Three microsatellite markers were polymorphic (Na = 5-8, H-E = 0.75) and no loci were found to deviate significantly from Hardy-Weinberg equilibrium or showed any evidence of linkage disequilibrium (p > 0.05). A slight heterozygote deficiency and some incompatibilities between the female known parent and its offspring that involved homozygous genotypes were observed at GUJ0001 locus and may indicate the presence of null alleles. Although a reduced set of microsatellite primers were applied, it was possible to determine the paternity of 96.87% of the embryos, using combined data of three loci. The approach was useful for parentage inferring in a captive population of C. japonica and the results evidenced a potential polyandric mating system in the species, in which no advantage mechanism of last-male sperm precedence seems to occur.

Entre os animais silvestres envolvidos em tráfico/comércio ilegal no Brasil, as aves compreendem um dos grupos mais atingidos, especialmente devido a características como canto e colorido das penas. Atualmente, análises genéticas compreendem uma das formas mais eficazes de gerar dados para solucionar e minimizar os resultados de crimes ambientais e comércio ilegal de animais silvestres. Uma das análises genéticas que podem ser utilizadas com o intuito de subsidiar programas conservacionistas associados ao tráfico ilegal de aves refere-se à sexagem molecular, dado que neste grupo de vertebrados nem sempre é possível identificar o gênero por meio de caracteres morfológicos. A sexagem molecular pode ser feita com base em amostras de DNA obtidas de diferentes fontes, como penas e sangue, e posterior amplificação de regiões dos genes CHD-Z e CHD-W (chromo helicase-DNA binding). Os dados de perfis genéticos sexoespecíficos servem de subsídio a programas conservacionistas de manutenção e/ou reprodução em cativeiro e posterior soltura ou reintrodução dos animais em ambiente natural.

The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.

The matrinchã Brycon amazonicus, a commercially important freshwater fish resource, has no heteromorphic sex chromosomes so far described. In the present study, we performed a screening of sex-associated DNA markers in this species, through the use of a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis. DNA digestions evidenced no differences between sexes. Sixty-six random primers were used in pooled and individual DNA samples of males and females, and the analysis of the RAPD fingerprints revealed one female sex-associated band. Cloning and sequencing of this band led to the identification of two distinct DNA segments. While one of the isolated fragments showed a significant identity with a described protein gene (phosphatidylinositol glycan anchor biosynthesis, class W), the other fragment, composed of 535 bp, corresponds to a novel DNA marker. Further experiments were performed with this second DNA fragment in order to verify its sex-specificity. Data on dot blot hybridization, using total DNA of both sexes, confirmed its female-specificity in B. amazonicus. A primer set was designed based on its sequence data and used in PCR with DNA samples of this species, leading to diagnose the animals' sexes with a 100 % overall accuracy through a sequence characterized amplified region approach. No amplification results were found for two other species of the genus--B. orbignyanus and B. lundii. The obtained data can lead to the hypothesis that B. amazonicus may present heteromorphic sex chromosomes that should be in an early phase of differentiation.