Introduction
Senegalese sole (Solea senegalensis) is an emerging aquaculture species in Europe. However, despite large investments, bottlenecks in reproduction still curtail the expansion of this industry. This flatfish can reproduce in captivity when captured from the wild, but males reared in captivity (F1) fail to reproduce despite they produce viable sperm for fecundation . So reproduction is the main constraint in Senegalese sole aquaculture and consequently, t he pursuit for potential solutions has been approached through different fields. It has been hypothesized that chemic al communication might underlie sole courtship failure and that environmental factors associated with different life stories of F1 vs wild males may underlie the low performance of F1 males.
The fish olfactory organ, an essential player for chemical communication, is a multilamellar and rosette-shaped structure, composed of an epith elium embedded with olfactory sensory neurons expressing individual olfactory receptors . Despite its critical role in social relationships and individuals’ interaction with their environment, the olfactory system remains understudied. In particular, while its role in mammalian reproduction is well-established , its involvement in fish reproduction is poorly characterized. The aim of this work is to improve our knowledge of the fish olfactory system , and in particular, in the context of reproduction. To do so, we conducted a single- nuclei RNAseq (snRNAseq) analysis of the olfactory organ, enabling the characterization of transcriptomic profiles at the individual cell level featuring the different cell populations and their heterogeneity.
Material and Methods
The olfactory organ was collected from 3 different Senegalese sole fish (1 wild male, 1 wild female & 1 F1 male - born in captivity). A total of 6 samples, two samples per individual considering dorsal and ventral olfactory rosettes , were dissected and flash frozen separately. Nuclei were extracted using a modified protocol (REF) and processed using the Single-cell/nuclei RNA-seq Chromium 10X pipeline. Single-nuclei RNA-seq libraries were sequenced in a Novaseq 6000 as 150PE reads. Raw sequencing was aligned against the most recent Senegalese sole genome (GCA_919967415.2 , de la Herrán et al. 2022 ) using STARsolo (Kaminow et al. 2021 ) and analysed using the R package Seurat (Stuart et al. 2019).
Results
The six Senegalese sole single-nuclei libraries (wild female, wild male and F1 male x dorsal and ventral olfactory rosettes) were successfully sequenced, generating a total of 59,616 high-quality cells after filtering and quality controls. C ells were clustered according to their transcriptomic profile, resulting in the identification of 19 different cell populations present in the olfactory organ of Senegalese sole. A first inspection revealed no differences between the dorsal and ventral olfactory rosettes. The characterization of the different cell clusters showed a diverse set of cell types, including several groups of neuronal cells, immune cells, blood cells, secretory cells, ciliated cells, and fibroblasts, among others. This cell diversity along with the complex organization of the organ suggests that multiple biological processes are supported by the olfactory organ . In particular, we identified four ORA genes (ora1, ora2, ora3, ora4), which belong to an olfactory receptor (OR) family directly associated to chemical communication and pheromone perception . These genes exhibited low expression levels, which directly correlate to the rule ‘one receptor-one neuron’ applied to all ORs, and were concentrated in two clusters, cluster 9 (ora1, ora2), and cluster 10 (ora3, ora4) . We characterized these clusters as neural cell types since ORs are always expressed in neurons (Fig. 1). Further analyses are in progress to functionally characterize the different cell populations, especially those neural subtypes potentially involved in reproduction.
Conclusions
The olfactory rosette of Senega lese sole is a complex and heterogeneous organ consisting of 19 cellular clusters with a diverse range of functions. The presence of different types of olfactory sensory neurons points to the importance of olfaction in fish, potentially underlying social behaviors such as reproduction.
References
Hao et al. (2021). Integrated analysis of multimodal single-cell data. Cell , 184(13), 3573- 3587.
Kaminow et al. (2021) STARsolo: accurate, fast and versatile mapping/quantification of single-cell and single-nucleus RNA-seq data. Biorxiv , 2021- 05. doi : https://doi.org/10.1101/2021.05.05.442755.
Kowatschew et al. (2022) Spatial organization of olfactory receptor gene choice in the complete V1R- related ORA family of zebrafish. Scientific Reports , 12(1), 14816.
Ruiz Daniels et al. (2022) A versatile nuclei extraction protocol for single nucleus sequencing in fish species–optimization in various Atlantic salmon tissues. Protocols.io. dx.doi.org/10.17504/protocols.io.261genwm7g47/v2.
Stuart et al. (2019) Comprehensive integration of single-cell data. Cell, 177(7), 1888-1902.
Funding : Royal Society of Edinburgh Saltire International Collaboration (Reference Number 1856) to the University of Edinburgh and the University of Santiago de Compostela.