Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized. We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD₅₀ (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues. Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process. In Brazil, accidents with scorpion stings have been a serious public health problem, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. Therefore, efforts have been made to understand the characteristics of the molecules present in scorpion venoms. These venoms are complex mixtures, in which neurotoxins are the main toxic components. Ts venom also contains enzymes, such as hyaluronidase, that have not been well characterized. In this study, we described for the first time two sequences of Ts hyaluronidase isoforms: TsHyal-1 and TsHyal-2. We purified native hyaluronidase from Ts venom and produced anti-hyaluronidase serum in rabbits. This serum neutralized hyaluronidase activity present in Ts venom. In vivo neutralization assays showed that anti-hyaluronidase serum inhibited and delayed mouse death after injection of a lethal dose (50% lethal dose, LD₅₀) of Ts venom. This work confirms the influence of hyaluronidase in Ts venom lethality and paves the way for the development of new strategies for scorpionism therapy.

Background: Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite.Methods: To test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay.Results: Data show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay.Conclusion: This is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid.

Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple loci from many samples. These processes can be laborious and expensive. The choice of microsatellites and PCR calibration for genotyping are particularly daunting. Here we optimized a low-cost genotyping method using multiple microsatellite loci for simultaneous genotyping of up to 384 samples using next-generation sequencing (NGS). We designed primers with adapters to the combinatorial barcoding amplicon library and sequenced samples by MiSeq. Next, we adapted a bioinformatics pipeline for genotyping microsatellites based on read-length and sequence content. Using primer pairs for eight microsatellite loci from the fish Prochilodus costatus, we amplified, sequenced, and analyzed the DNA of 96, 288, or 384 individuals for allele detection. The most cost-effective methodology was a pseudo-multiplex reaction using a low-throughput kit of 1 M reads (Nano) for 384 DNA samples. We observed an average of 325 reads per individual per locus when genotyping eight loci. Assuming a minimum requirement of 10 reads per loci, two to four times more loci could be tested in each run, depending on the quality of the PCR reaction of each locus. In conclusion, we present a novel method for microsatellite genotyping using Illumina combinatorial barcoding that dispenses exhaustive PCR calibrations, since non-specific amplicons can be eliminated by bioinformatics analyses. This methodology rapidly provides genotyping data and is therefore a promising development for large-scale conservation-genetics studies.