Molecular Characterization of Embryos with Different Buoyancy Levels in the Yellowtail Kingfish (Seriola lalandi).

The buoyancy of eggs and embryos is associated with successful development in pelagic fish. Buoyancy is the result of oocyte hydration, which depends on the osmotic force exerted by free amino acids (FAA) generated by yolk proteolysis, and cathepsins are the main enzymes involved in this process. Seriola lalandi is a pelagic fish whose farming has been hampered by development failure that have been partially attributed to decreased buoyancy of embryos. Therefore, the aim of this study was to compare the mRNA expression and activity of cathepsins B, D, and L, as well as the FAA content in floating and low-floating embryos at different developmental stages. The chosen stages were eggs, morula, blastula, gastrula and 24 h embryos. Complementary assessments showed that there were no differences attributed to buoyancy status in embryo and oil droplet diameters, as well as the transcriptional status at any developmental stage. Cathepsin B did not show differences in mRNA expression or activity related to buoyancy at any stage. Cathepsin D displayed higher transcript and activity levels only in low-floating eggs compared with those floating. Cathepsin L showed higher expression in floating eggs and 24 h embryos compared with that of low-floating, but the activity of this enzyme was higher in floating eggs and morula. Total FAA content constantly decreased throughout development in floating embryos, but it was always higher than low-floating embryos until gastrula stage. In 24 h embryos floating and low-floating embryos share similar quantities of FAA. In summary, differences in the expression and activity of cathepsins between floating and low-floating embryos could be revealed at specific embryonic stages, suggesting different functions of these enzymes throughout development. Besides 24 h embryos, FAA content seems to be a decisive factor for buoyancy of embryos during early development of S. lalandi. Overall, considering the main role of cathepsins and FAA in buoyancy acquisition process and therefore in both embryo quality and viability, our study identifies good marker candidates to evaluate embryo quality in the farming of this species.

Can Responsible Ownership Practices Influence Hunting Behavior of Owned Cats?: Results from a Survey of Cat Owners in Chile.

The domestic cat (Felis catus) has become a worldwide threat to wildlife. The potential impact of owned cats on wildlife in Chile has not been documented at a large scale. The purpose of this study was to investigate the number and type of prey that owned cats bring back in Chile and its relation with responsible ownership practices. An online survey was distributed to 5216 households that included questions about the type of pet, responsible ownership practices, and in the case of cats, the type of prey they brought home. Descriptive statistics as well as univariate and multivariate logistic regression analysis were applied. The results showed that 94.3% of respondents had a pet, and from these, 49.9% had at least one cat. A total of 84.1% of owners reported that their cats had brought back prey. Birds were the most common type of prey, followed by mammals and insects. Not being registered with a microchip, not having a litter box, living in a house with access to a garden, not having a hiding place for the cats, and having free access to the outdoors significantly increased the odds of cats bringing back prey. Body condition score or providing ad libitum food to cats did not have an effect on bringing prey.

Transcriptomic response of rainbow trout (Oncorhynchus mykiss) skeletal muscle to Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiologic agent of rainbow trout fry syndrome (RTFS). This pathogen infects a wide variety of salmonid species during freshwater stages, causing significant losses in the aquaculture industry. Rainbow trout (Oncorhynchus mykiss) infected with F. psychrophilum, presents as the main external clinical sign ulcerative lesions and necrotic myositis in skeletal muscle. We previously reported the in vitro cytotoxic activity of F. psychrophilum on rainbow trout myoblast, however little is known about the molecular mechanisms underlying the in vivo pathogenesis in skeletal muscle. In this study, we examined the transcriptomic profiles of skeletal muscle tissue of rainbow trout intraperitoneally challenged with low infection dose of F. psychrophilum. Using high-throughput RNA-seq, we found that 233 transcripts were up-regulated, mostly associated to ubiquitin mediated proteolysis and apoptosis. Conversely, 189 transcripts were down-regulated, associated to skeletal muscle contraction. This molecular signature was consistent with creatine kinase activity in plasma and oxidative damage in skeletal muscle. Moreover, the increased caspase activity suggests as a whole skeletal muscle atrophy induced by F. psychrophilum. This study offers an integrative analysis of the skeletal muscle response to F. psychrophilum infection and reveals unknown aspects of its pathogenesis in rainbow trout.

Stocking density induces differential expression of immune-related genes in skeletal muscle and head kidney of fine flounder (Paralichthys adspersus).

Immunity can be modulated by different internal and external factors, being stress one of the most important. However, the stress effects on the immunocompetence of the skeletal muscle has not been studied in detail in earlier vertebrates. Here, we examine the effect of chronic (4 and 7 weeks) crowding stress on the immunocompetence of skeletal muscle and head kidney in the fine flounder (Paralichthys adspersus). Corticosteroid receptor transcript levels and their target genes; pro-inflammatory cytokines, and Toll-, NOD-, and RIG-like receptors were quantified by qPCR. The results indicate that chronic stress down-regulates the expression of these genes in muscle, compromising skeletal muscle immunocompetence, while the expression of these genes is upregulated in head kidney after seven weeks of crowding stress. The data suggests that chronic stress modulates the expression of these immune-related genes in a tissue-specific manner.

Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function.