Temporal and spatial toxicogenetic damage in estuarine catfish Cathorops spixii from a marine protected area with evidence of anthropogenic influences.

Taking action to contribute to the preservation of the natural coastal environment, which maintains a diversity of life, is important in today's world. In estuarine ecosystems that are included in marine protected areas (MPA), it is important to conduct biomonitoring over time to monitor the effects of xenobiotics on bioindicator species such as the catfish Cathorops spixii. This study aims to evaluate the toxicogenetic signals in C. spixii from a MPA (Cananéia-Iguape region, Brazilian coast). These genotoxic signals are demonstrated by the presence of micronuclei (MN) and abnormalities in erythrocytes (AE). This study also takes into account the environmental differences signaled by the water's properties (temperature, salinity, pH, DO, dissolved nutrients, and particulate material) over time (2009, 2014, 2017, and 2018). The results show that in the northern region of the estuary, and mainly in 2009, the water properties reveal the influence of the river in the form of the lowest levels of salinity and pH and the highest levels of nutrients. The most significant levels of suspended particulate material and chlorophyll-a are found in the southern region of the estuary, mainly in 2017 and 2018. In general, the C. spixii from the northern region had better well-being than catfish from the more preserved (southern) region. The data on MN and AE show evidence of toxicogenetic damage occurring over time and clastogenic/aneugenic effects mainly in fish sampled in 2018. The presence of a new AE, first recorded in C. spixii sampled in 2018, and named here as double bubbled, is another indicator of toxicogenetic effects. Finally, although the southern region of the estuary has better abiotic properties and conditions for primary production, the obtained toxicogenetic data for C. spixii reinforces the need for - and encourages the implementation of - monitoring actions in this marine protected area using bioindicator species of contamination.

Polymorphisms in the melatonin receptor gene promoter and their associations with fertility characteristics in buffalo herd in Eastern Amazon.

Buffalo production is spreading globally because of its economic advantage. Then, it has become necessary to improve the reproductive and productive efficiency of these animals, as well as to look for genetic factors that increase this efficiency. The objectives of this study were to characterize the promoter region of the melatonin 1A receptor gene (MTRN1A), to detect possible SNPs and associate them with fertility characteristics, and identify binding sites of transcription factors involved in the regulation of genetic expression in buffaloes in the Amazon. The conventional PCR method was carried out using the two primers designed from the reference sequence deposited in the GenBank AY52466.1. The products of the PCRs were purified, sequenced, and subsequently edited and aligned. Twenty-six SNPs were found, where 73% presented allele frequencies of wild nucleotides above 0.5, and 73% presented deviations from the Hardy-Weinberg equilibrium (P < 0.05) and FIS varying between 0.06 and 1.00, characterizing high degrees of inbreeding within the population. A block of ACAA deletion (position -1483) was observed in 25% of samples. The associations between these SNPs and reproductive characteristics were observed for calving interval and 5 SNPs: -1289, -1139, -911, -724, and -656 (P < 0.05), and three other SNPs: -1395, -724, and -94 (P < 0.05) were associated significantly with age at first calving, and were not associated with calving concentration. The promoter region was characterized by the different types of binding factors, where only 11 sites are significantly strong enough for transcription factor bindings. The ACAA deletion also exhibited a strong association with transcription factors. As a result, it would be necessary to test the SNPs above with other reproductive characteristics of economic relevance to approve the gene as a strong candidate for the selection of buffaloes in the Amazon.

Polymorphisms in the leptin gene promoter in Brazilian beef herds.

Brazil is the world's largest producer of beef cattle; however, the quality of its herds needs to be improved. The use of molecular markers as auxiliary tools in selecting animals for reproduction with high pattern for beef production would significantly improve the quality of the final beef product in Brazil. The leptin gene has been demonstrated to be an excellent candidate gene for bovine breeding. The objective of this study was to sequence and compare the leptin gene promoter of Brazil's important cattle breeds in order to identify polymorphisms in it. Blood samples of the Nellore, Guzerat, Tabapuã, and Senepol breeds were collected for genomic DNA extraction. The genomic DNA was used as a template for polymerase chain reaction (PCR) to amplify a 1575-bp fragment, which in turn was sequenced, aligned, and compared between animals of different breeds. Twenty-three single nucleotide polymorphic sites, including transitions and transversions, were detected at positions -1457, -1452, -1446, -1397, -1392, -1361, -1238, -963,-901, -578, -516, -483, -478, -470, -432, -430, -292, -282, -272, -211, -202, -170, and -147. Additionally, two insertion sites at positions -680 and -416 and two deletion sites at positions -1255 and -1059 were detected. As the promoter region of the leptin gene has been demonstrated to vary among breeds, these variations must be tested for their use as potential molecular markers for artificial selection of animals for enhanced beef production in different systems of bovine production in Brazil.

Novel polymorphism in exon 1 of the melatonin receptor gene unassociated with reproductive characteristics of buffaloes in the Amazon Region.

The objective of this study was to sequence part of the exon 1 in the melatonin receptor 1A gene (MTRN1A) in buffaloes to detect a novel polymorphism with which to associate reproductive characteristics, such as age at first birth and the interval between births, in buffaloes from the northeastern region of the State of Pará (Brazil). Buffalo hair samples (77) were collected from the Terra Firme region of Pará. DNA was extracted and polymerase chain reactions (PCRs) were carried out with a primer that was designed using the GenBank accession No. AY524665 reference sequence. PCR products were purified and sequenced. After editing and analysis of the sequences, a mutation was observed at the 62nd position in exon 1 of MTRN1A (T↔C), which corresponded with a change in the 21st amino acid from leucine to proline. All possible genotypes were observed, with the most common being genotype CC (0.481). The allele frequencies were T = 0.377 and C = 0.623. Statistical analysis of FIS showed inbreeding within the sample group (FIS = 0.397) and deviations from the Hardy- Weinberg equilibrium were observed (P < 0.05). Associations between genotypes and reproductive characteristics were not significant (P > 0.05). Although the related SNP was not synonymous, there were no observable effects on the reproductive characteristics under investigation. As such, it would be ideal to detect other SNPs in exon 1 of the MTRN1A gene that can be associated with reproductive characteristics in Amazonian buffaloes.

Corrigendum Polymorphism in the melatonin receptor gene in buffalo populations of the Brazilian Amazon - Genet. Mol. Res. 15 (2): gmr.15027960.

The correction is only in the name of the first author and should be: E.M. Barbosa(1), B.B. Souza(2), R.C. Guimarães(2), J.S.N. Azevedo(3), E.C. Gonçalves(4), H.F.L. Ribeiro(2), S.T. Rolim Filho(2), E. Silva Filho(2).

Polymorphism in the melatonin receptor gene in buffalo populations of the Brazilian Amazon.

Buffalo farming in Brazil is increasing, as is the challenge of identifying molecular markers that will improve productivity. Therefore, the aim of this study was to analyze single nucleotide polymorphisms of the receptor gene for the hormone melatonin in buffaloes from northern Brazil by polymerase chain reactions (PCRs) and restriction fragment length polymorphism assays. The PCR products exhibited a cutting point for HpaI at the 318th position of the gene, indicating a transition substitution (T↔C). This substitution was synonymic, and did not alter the stability of the mRNA structure. Allelic and genotypic frequencies differed between the populations studied, and all of the populations demonstrated endogamy and were in Hardy-Weinberg equilibrium. Therefore, the HpaI restriction marker in the melatonin receptor gene cannot be used for genetic improvement, but is an excellent marker for population genetic studies.

Homology modeling and molecular dynamics of ß-defensin II variants in Amazonian sheep.

ß-defensins are capable of creating pores in the bacterial membrane. In this study, we aim to determine the structure of 3 different sheep ß-defensin 2 (SBD-2) sequences by molecular modeling. A herd of 47 sheep from the Centre for Ovine and Caprine Research of Pará was selected for this investigation. The AA, AG, and GG alleles were found on ß-defensin sequences. We used homology modeling and molecular dynamic simulations to generate 3D models of peptides and they were successfully validated. The proteins are structurally very similar to classic defensins composed of 3 ß-sheets and 3 disulfide bonds. Variations in the organization of the tertiary structure and distribution of charged residues were found between AA, AG, and GG alleles. In this study, we were able to characterize and show the structure of 3 SBD-2 gene variants for the first time in Amazonian sheep. Results demonstrated that these variants are similar in structures to classic ß-defensins, but contain more positives charges, which may indicate an increase in efficacy.

Genetic polymorphisms in ß-defensin II gene in Amazon sheep from Brazil.

The northern region of Brazil produces a large number of sheep, with Pará being the largest sheep breeding state in the region. In the Amazon region, livestock production is a challenge due to the high diversity of pathogens affecting humans and animals. Defensins are antimicrobial peptides acting as a first barrier against micro-organisms and present high variation in different organisms. The objective of this study was to detect polymorphisms in exon II in ß-defensin II in Amazon sheep. The gene was amplified by PCR from DNA extracted from 47 sheep blood samples from the Santa Inês breed. Products were sequenced, aligned and analyzed. Three single nucleotide polymorphism (SNP) positions were observed with transition substitutions (A↔G) at positions 1643, 1659, and 1750. The 1643 and 1750 SNPs showed a low variability and significant deviations from Hardy-Weinberg equilibrium (HWE) (P < 0.05) meanwhile the SNP 1659 showed moderate absence of genetic variability and deviation from HWE (P > 0.05). Polymorphisms at 1643 and 1659 were predicted to modify amino acids in the peptide chain (isoleucine to valine and arginine to lysine, respectively) with no effects on protein function. Results from this study suggest that SNPs are important markers for ß-defensin II efficiency studies on the immune system of sheep in the Brazilian Amazon.

Biochemical changes in the liver and gill of Cathorops spixii collected seasonally in two Brazilian estuaries under varying influences of anthropogenic activities.

In order to understand environmental health by the use of a bioindicator species in estuaries, biochemical responses observed in the catfish Cathorops spixii such as catalase (CAT), glutathione-S-transferase (GST), lipid peroxidation (LPO) and acetylcholinesterase (AChE) were evaluated in liver and muscle. Furthermore, histological changes were also verified in liver and gills preparations. Fish were collected in three sites of the Santos-São Vicente estuary located at São Paulo (Brazil), subjected to varying levels of inputs of pollutants. For a reference site, specimens were sampled at Cananéia estuary at southern coast of São Paulo, a region with low anthropogenic influence. In general, no significant seasonal differences in antioxidant enzymes and lipid peroxidation responses were found in the organisms from the Cananéia estuary. However, in the polluted estuary (Santos-São Vicente), biochemical responses were observed by increases in GST hydroperoxides and decreases in AChE activities in the summer. Inhibition of AChE expression in fish from different areas of the Santos-São Vicente estuary in the summer was also found and can indicate neurotoxic effects in these organisms. Histopathological observation of gill and liver showed severe lesions, such as lamellar fusion and necrosis.

New molecular variants of epsilon and beta IncP-1 plasmids are present in estuarine waters.

In this work the presence of broad-host-plasmids in an estuary in Portugal has been investigated. Pseudomonas putida KT2442 was used as model recipient bacteria in biparental matings with tetracycline and mercury to select for resistance phenotypes. As a result, 7 transconjugants were shown to carry broad-host-plasmids from the IncP-1 group, as seen by PCR amplification of the trfA gene. Sequence analysis confirmed the isolation of 4 plasmids from ß-1 subgroup and 3 assigned to the recently described ε subgroup. To our knowledge this is the first report concerning the detection and isolation of IncP-1ß and ε plasmids in estuarine waters. Moreover it is shown that, even though the retrieved plasmids are phylogenetically close to previously characterized plasmids, such as pB10 and pKJK5, respectively, they constitute new molecular variants.

Biomarkers of exposure to metal contamination and lipid peroxidation in the benthic fish Cathorops spixii from two estuaries in South America, Brazil.

Biomarkers as lipid peroxidation, metallothionein and delta-aminolevulinic acid dehydratase were determined in Cathorops spixii to compare the biological responses of this fish from estuaries with distinct anthropogenic influence. Three areas were selected in two estuaries in accordance with the levels of contamination for the polluted (Santos/São Vicente) and with the hydrodynamic characteristics for the non-polluted (Cananéia) estuary. Water characteristics and mercury levels in C. spixii confirmed a high human influence in the polluted system. In general, the biomarkers showed differences between the estuaries, suggesting disturbances in the specific cell mechanisms due to the presence of multiple xenobiotics in the contaminated system. Therefore, these biomarkers are recommended to promote more accurate information about the exposure to pollutants. Additionally, the study of the effect of the multiple xenobiotics on resident species such as the benthic fish C. spixii can favor a better assessment of the environmental quality of these systems.

Use of Cathorops spixii as bioindicator of pollution of trace metals in the Santos Bay, Brazil.

In the present study Cathorops spixii, was evaluated as a bioindicator fish for trace metal pollution. Concentrations of cobalt (Co), iron (Fe), selenium (Se) and zinc (Zn) were determined by Instrumental Neutron Activation Analysis in liver. Mercury (Hg) and methyl-mercury (MeHg) were analyzed by Cold Vapor Atomic Absorption Spectrometry in muscles and livers. High concentrations of Co, Fe, Se and Zn were observed in C. spixii from Santos Bay in comparison to fish collected in a non-polluted site in the same Brazilian coast. These trace metal concentrations were out of the permissible levels for human consumption. Although, Hg and MeHg levels were low, the C. spixii could still be used as an effective bioindicator to observe trace metal behaviors in the environment in function of the bioaccumulation process observed mainly by other analyzed trace metals. Thus, the use of this species is strongly recommended to monitor the effects and behavior of trace metal pollution in aquatic ecosystems in Brazil due to its bioaccumulation function.

Effect of parasitism on plasma sex-specific proteins in Cyphocarax gilbert (Teleost, Curimatidae).

Cyphocarax gilbert (Szidat, L., 1948) is a fish commonly found in coastal drainage of eastern Brazil. This fish is sometimes caught with signs of infection by the crustacean Riggia paranensis, a haematophagous parasite. A remarkable feature of infected fish is that they lack gonads. In this paper we have analysed the frequency of parasitism, the gonadal development of non-infected fish and the profile of plasma proteins in both infected and non-infected specimens. Two reproductive periods/year were observed, beginning in February and August. On average, 40% of fish were infected, in the Itabapoana River (Brazil). Sex-specific proteins were identified by electrophoresis. SDS-PAGE analysis demonstrated that a 143 kDa female-specific glycolipoprotein (FSP) is a calcium-binding phosphoprotein. FSP was isolated through ultracentrifugation and SDS-PAGE analysis showed that the native protein is composed of three polypeptides of 143, 100 and 70 kDa. Both FSP and a 33 kDa male-specific protein (MSP) are absent from infected fish plasma. FSP levels in female plasma changes with the developmental stage of gonads. Altogether these data suggest that the FSP corresponds to fish vitellogenin. Furthermore, the absence of the above-mentioned proteins in infected fish suggests that R. paranensis might interfere with the regular hormonal process of fish vitellogenesis.

Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification

Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems. The complete genome sequence of C. violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use. We examined the biotechnological potential of C. violaceum for environmental detoxification. Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail. Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins

UVA/UVB sunscreen determination by second-order derivative ultraviolet spectrophotometry.

UVA/UVB protective agents are important constituents in many cosmetics and sunscreen preparations. Analytical methodologies which enable the determination of combined substances with different spectrophotometric characteristics are therefore useful and cost-effective. To this end, the following substances in a sunscreen gel were analyzed: the UVB and UVA protective substances, 2-hydroxy-4-methoxy-benzophenone-5-sulphonic acid (BZP) and 2-phenylbenzymidazole-5-sulphonic acid (PBS), respectively, were evaluated by second-order derivative ultraviolet spectrophotometry in distinct solvents and wavelengths. The method used for determination of BZP in 95% ethanol was the zero-peak (ZP) at 356.0 nm. For PBS, the solvent used was 0.1 mol l-1 triethanolamine, and the methods were the ZP at 317.5 nm and the peak-peak (PP) at 317.5-323.0 nm. Statistical results for the methods showed linearity of 0.9999 and an average precision of 1.12% (ZP) for BZP, and 0.55% (ZP) and 0.63% (PP) for PBS. The average recovery for BZP and PBS were 97.28% (ZP), 103.13% (ZP) and 103.71% (PP), respectively.

Relationships and influences between Boophilus microplus characteristics in tick-naive or repeatedly infested cattle.

Six tick-naive male Hereford calves were infested once a month for 6 months with 18,000 Boophilus microplus larvae on the back and with 400 larvae in a cloth bag glued on the lumbar region. Working with the bag ticks, 12 tick characteristics were recorded for each infestation. Each tick attribute was analyzed for significant differences with those of the first infestation (analysis of variance), and for similarity (clustering), degree of relationship (correlation), and concomitant variation (regression) against all the other attributes during the first, third, and sixth infestations. Some attributes were affected maximally by host immunity about the third infestation but recovered later (length of feeding, detachment weight, egg weight, start of oviposition, fertility efficiency index), whereas others continued to be affected until the last infestation (length of oviposition, corpse weight, start of hatching, feeding efficiency index). All analyses showed that weight at detachment and egg weight were closely related, and corpse weight was partially related to these two. All other natural characteristics were largely independent. Length of feeding showed no significant relation with weight at detachment nor length of oviposition with egg weight. These findings suggest that different tick functions are independently affected by host immunity and recommends against estimating general anti-tick resistance by the evaluation of only a few tick characteristics.

Inhibition and recovery of tick functions in cattle repeatedly infested with Boophilus microplus.

Six tick-naive male Hereford calves were infested with about 18,000 Boophilus microplus larvae, 400 in a cloth bag, once a month for 6 mo. Working with the bag ticks, 12 tick characteristics were recorded for each infestation. Manifestations of resistance to some attributes appeared by the second infestation, peaked by the third (detachment weight, egg weight, fertility index) or fourth (feeding length, start of oviposition), and vanished by the fifth or sixth. Resistance to corpse weight, oviposition length, start of eclosion, feeding index, and survival developed more slowly but continued until the last infestation. Lymphocyte transformation after the fifth infestation was normal for concanavalin A and augmented in only 2 calves for phytohemagglutinin. In each infestation and at the end of all infestations some calves were significantly more resistant to some tick characteristics. There was no relationship between natural resistance and the ability to mount an acquired resistance. We concluded that B. microplus can depress or evade host's immunity, that resistance to different tick functions occurs independently, that natural and acquired resistance are unrelated, and that there exist genetic differences in natural or acquired resistance to the tick among seemingly homogeneous animals.