RESUMEN
Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.
Asunto(s)
Anopheles , Sistemas CRISPR-Cas , Razón de Masculinidad , Cromosoma Y , Animales , Anopheles/genética , Masculino , Femenino , Cromosoma Y/genética , Mosquitos Vectores/genética , Meiosis/genética , Espermatozoides/metabolismo , Edición Génica/métodos , Malaria/transmisión , Malaria/genéticaRESUMEN
The development and application of functional feed ingredients represents a great opportunity to advance fish growth and health, boost the immune system, and induce physiological benefits beyond those provided by traditional feeds. In the present study, we looked at the feasibility of in vitro methods for screening the qualities of functional feed ingredients using the fish cell line RTgill-W1, which has never been used in fish nutrition, and the culture of Paramoeba perurans. Five functional feed ingredients (arginine, ß-glucan, vitamin C, and two phytogenic feed additives) were selected to investigate their effects on cell viability and reactive oxygen species production. Three of the selected ingredients (arginine and two phytogenic feed additives) were additionally tested to assess their potential amoebicidal activity. As these functional ingredients are the core of a commercially available feed (Protec Gill, Skretting AS), their beneficial effects were further assessed in a field trial in fish affected by complex gill disease. Here, the analyzed parameters included the evaluation of macroscopic and histopathological gill conditions, pathogen detections, and analyses of plasma parameters. RTgill-W1 cell line assays were a good tool for screening functional ingredients and provided information about the optimal ingredient concentration ranges, which can be helpful for adjusting the concentrations in future feed diets. Through the culture of P. perurans, the tested ingredients showed a clear amoebicidal activity, suggesting that their inclusions in dietary supplements could be a viable way to prevent microbial infections. A three-week period of feeding Protec Gill slowed the disease progression, by reducing the pathogen load and significantly improving gill tissue conditions, as revealed by histological evaluation. The use of diets containing selected functional ingredients may be a feasible strategy for preventing or mitigating the increasingly common gill diseases, particularly in cases of complex gill disease, as documented in this study.