The aim of this study was to evaluate the antimicrobial activity of Lippia sidoides, Ocimum gratissimum and Zingiber officinale‘s essential oils against Aeromonas spp. isolates from Colossoma macropomum, and to determine their chemical composition. Essential oils were obtained through hydrodistillation and their chemical composition analyzed using gas chromatography coupled to mass spectrometry. The antimicrobial activity of the oils was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The major compounds were thymol (76.6%), p-cymene (6.3%) and (E)- β-caryophyllene (5.0%) in L. sidoides; eugenol (43.3%), 1,8-cineole (28.2%) and β-selinene (5.5%) in O. gratissimum; and geranial (23.2%), neral (16.7%) and 1,8-cineole (15.8%) in Z. officinale. All the essential oils evaluated showed bactericidal action against isolates of Aeromonas spp., with MIC and MBC ranging from 625 to 5,000 μg mL⁻¹. We highlight the L. sidoides oil with the best antimicrobial activity (MIC and MBC ranging from 625 to 1,250 μg mL⁻¹).

Early puberty poses a significant challenge for male Atlantic salmon in aquaculture due to its negative impact on growth and welfare. The regulation of puberty in vertebrates involves 2 key reproductive hormones: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their gonadal receptors. In male mice lacking FSH receptor, testes size is reduced, but fertility is maintained, while medaka and zebrafish with a disrupted fshr gene exhibit near normal testis size and fertility. In these fishes both Fsh and Lh are present during puberty and Lh may rescue fertility, while in salmonid fish only Fsh is present in the circulation during puberty. Using CRISPR-Cas9, we produced crispants with a high prevalence of fshr mutations at the target site, which remained fertile, although more than half showed a testis development deviating from wild-type (wt) males. Crossing out these F0 crispants to each other produced a viable F1 generation showing frameshift (fshr^−/−) or in-frame mutations (fshr^if/if). Nearly all wt males matured while all fshr^−/− males remained immature with small testes containing A spermatogonia as the furthest developed germ cell type and prepubertal plasma androgen levels. Also, the pituitary transcript levels of gnrhr2bba and lhb, but not for fshb, were reduced in the fshr^−/− males compared with maturing males. More than half of the fshr^if/if mutant males showed no or a delayed maturation. In conclusion, Atlantic salmon show the unique characteristic that loss of Fshr function alone results in male infertility, offering new opportunities to control precocious puberty or fertility in salmon.

Genetic introgression of farmed salmon into wild populations can damage the genetic integrity of wild stocks and is therefore considered as an environmental threat. One possible solution is to induce sterility in farmed salmon. We have searched for proteins potentially essential for germline survival in Atlantic salmon. One of these is the argonaute protein Piwil1, known to be required for germ cell survival. To examine Piwil1 function in salmon, we induced indels in the N domain by CRISPR-Cas9. The encoded domain is present in all vertebrate Piwi proteins and has been linked to Tdrd1 protein interaction and PAZ lobe structure. The F0 founder generation of piwil1 crispant males and females displayed a mosaic pattern of piwil1 mutations, exhibiting highly mutated alleles (53%–97%) in their fin gDNA samples. In general, piwil1 crispants carried germ cells, went through puberty and became fertile, although a transient and partial germ cell loss and delays during the spermatogenic process were observed in many male crispants, suggesting that Piwil1 functions during salmon spermatogenesis. By crossing highly mutated F0 founders, we produced F1 fish with a mixture of: loss-of-function alleles (⁻); functional in frame mutated alleles (⁺) and wt alleles (⁺). In F1, all piwil1^−/− fish lacked germ cells, while piwil1^+/+ siblings showed normal ovaries and testes. Yet, most juvenile F1 piwil1^+/−males and females displayed an intermediate phenotype with a higher somatic/germ cell ratio without an increase in germ cell apoptosis, suggestive of a gene dose effect on the number of germ cells and/or insufficient replacement of lost germ cells in heterozygous fish. Interestingly, the two longest in-frame indels in the N domain also ensured germ cell loss. Hence, the loss of 4–6 aa in this region Phe130-Ser136 may result in crucial changes of the protein structure, potentially affecting piRNA binding of the PAZ lobe, and/or affecting the binding of Piwil1 interacting proteins such as Tdrd protein, with critical consequences for the survival of primordial germ cells. In conclusion, we show that loss of piwil1 leads to loss of germ cells in salmon and that part of the N domain of Piwil1 is crucial for its function.