The fish escape response is a kind of stress responses induced by various environmental factors such as rapid water flow and low oxygen. In Europe, frequent escape events of sea bream, Atlantic salmon, cod, and other fish not only cause huge economic losses, but also put strong competitive pressure on local wild fish, leading to a continued decline in the fitness of wild fish populations (Jackson et al. 2015; Svenning et al. 2017). This study focused on the common strain black carp, which owned the low-escape response property. Through multi-omics analysis, it was found that the strength of escape response was related to the expression of genes participating in brain signal transduction, and brain tissue and neuronal development, especially genes related to the GABA signaling pathway. In addition, the liver tryptophan metabolism pathway was also thought to affect the fish escape response.
The fish brain is the control center of fish behavior and the starting tissue of the HPI axis, which mediates stress responses (Lin et al. 2000; Wendelaar Bonga 1997). We analyzed and compared the transcriptome data of brain tissues from the BL and BH groups, and the transcriptome data of brain tissues from the JL and JH groups were also compared to reduce false positives. The DEGs from the common strain black carp and Jian carp were intersected, and 18 conserved DEGs were obtained (15 DEGs were annotated as 13 known genes), 17 of which performed low-expression levels in the low-escape groups, and only 1 of which showed high-expression levels in the low-escape groups. Most of the above genes are related to signal transduction, and brain tissue and neuronal development. mical2b is related to nervous system development and plays a role in axon guidance, myofilament organization, and synaptogenesis (Xue et al. 2010). Fibcd1 encodes a receptor for endocytosed glycosaminoglycans in the brain, and plays a role in nervous system structure, function and plasticity. Knocking down Fibcd1 leads to idiopathic complex neurodevelopmental disorder (NDD) (Fell et al. 2022). Eng2b is involved in the localization of diencephalon-mesencephalon boundary during brain development (Rampon et al. 2015). NHS-like participates in regulating actin dynamics and maintaining the correct shape of cells during development (Brooks et al. 2010). Pkd1l1 participates in regulating ciliary drive during development, and plays a key role in establishing left-right asymmetry in vertebrates (Field et al. 2011). Phyhipl affects the morphology and signal transduction of Purkinje cells (PC) in cerebellum, which is related to the motor learning ability of organisms (Sugimoto et al. 2021). sp8b takes part in the Wnt signaling pathway and affects neural posteriorization during the neural development stage (Mohanty 2017). CDHR1 encodes a calcium-dependent cell adhesion protein, which is located at the base of the junction between the outer and inner segments, and plays a crucial role in maintaining the structure and integrity of photoreceptors (Bessette et al. 2018). Pax2a is the key gene in the development of visual fissure in organisms, supporting the survival of outer cells in visual fissure and the abundance of periocular mesenchyme (POM) (Lusk and Kwan 2022). Eng2a is involved in the bone morphogenetic protein (BMP) signaling pathway in the myotome (Maurya et al. 2011). The protein encoded by MAP6 stabilizes microtubules and plays a role in neuronal differentiation and synapse formation (Sun et al. 2009). Fgf8 is involved in the morphogenetic process that establishes a defined relationship between axes of the embryo and the uterus at the onset of gastrulation (Guo and Li 2007). Trpc6 encodes a calcium-ion channel protein and plays a key role in maintaining the survival of neurons under cerebral ischemia (Du et al. 2010). The widespread low-expression of the above genes in the brain of fish from low-escape groups suggests that the brain activity and development level of low-escape fish may be lower than that of high-escape fish. Similar results have been found in Bahamas mosquitofish (Gambusia hubbsi), where mosquitofish with strong escape response had larger telencephalons and strong exploratory abilities than mosquitofish with weak escape response (Jenkins et al. 2021).
We conducted signaling pathway and functional annotations on DEGs from the common strain black carp and Jian carp, respectively, and the results of KEGG signaling pathway annotation, KOG functional annotation, and topGO enrichment analysis were basically consistent. The KEGG signaling pathway annotation showed that DEGs from the common strain black carp and Jian carp had a significant enrichment in signal transduction, and signal molecule and interaction pathways. The KOG functional annotation was consistent with above results, and the most annotated function of DEGs from both sources was signal transduction. The topGO enrichment analysis exhibited that DEGs from the common strain black carp and Jian carp were significantly enriched at the signal transduction node (GO: 0007165) and its sub-node: G protein-coupled receptor signaling pathway node (GO: 0007186). Due to the greater differences in escape responses between the JL and JH groups, more DEGs were obtained through screening, and the results of topGO enrichment analysis were more detailed in Jian carp. DEGs enriched in the G protein-coupled receptor signaling pathway node were further enriched in its sub-node: GABA signaling pathway node (GO: 0007214). GABA is the main inhibitory neurotransmitter in the brain of vertebrates, and current research suggests that GABA has potential therapeutic effects in protecting animals from inflammatory damage and improving growth performance under stress (Dourado et al. 2023; Zhang et al. 2022). In Sus scrofa f. domestica, feeding GABA supplemented feed could improve the antioxidant status caused by transport stress (Bi et al. 2020). In Gallus gallus domesticus, adding GABA to the diet reduced the heat stress response, increased the number of goblet cells, and improved egg production and egg quality (Chen et al. 2013). In Litopenaeus vannamei, adding GABA to feed enabled to improve the growth performance, endocrine hormones and stress tolerance of juvenile shrimp (Xie et al. 2017). In Ctenopharyngodon idella, feeding feed containing GABA could also reduce the antioxidant status caused by stress, and improve their survival rate in juvenile fish (Wu et al. 2016). It was hypothesized that the activity of the GABA signaling pathway in the brains could affect the varying degrees of escape response under flood.
The liver is the organ where most nutrient utilization and metabolic regulation initially occurs, then influences cascades of events in other tissues and organs (Enes et al. 2009). We used LC-MS/MS detection to analyze the differences in metabolites in the livers of individuals from the BL and BH groups. The results exhibited that the tryptophan content in the BL group was significantly higher than that in the BH group. The KEGG signaling pathway annotation also showed that the differential metabolites were mainly enriched in the tryptophan metabolism pathway. Tryptophan is an essential amino acid that is a precursor to the synthesis of neurotransmitter/neuromodulator 5-hydroxytryptamine (5HT), the hormone melatonin and kynurenine and related compounds such as kynurenic acid, quinolinic acid or niacin (Hoseini et al. 2019). 5HT is the main regulatory molecule on the HPI axis that mediates stress response, and can bi-directional regulate the HPI axis (Winberg et al. 2001; Winberg and Lepage 1998). Melatonin is also involved in regulating the HPI axis. In teleost fish, melatonin has been shown to inhibit or delay the glucocorticoid response caused by stress (Azpeleta et al. 2010). In Solea senegalensis, adding tryptophan to the diet alleviated the stress response caused by high-density feeding, and reduce mortality (Martos-Sitcha et al. 2020). In Cirrhinus mrigala and Labeo rohita, under sustained stress conditions, adding tryptophan to the feed could significantly inhibit cortisol and glucose responses, and improve the growth performance of C. mrigala and L. rohita (Akhtar et al. 2013; Kumar et al. 2014). The above results showed that, compared with the BH group, the common strain black carps in the BL group contained more tryptophan in the liver, which subsequently convert into 5HT, melatonin and other substances to effectively alleviate the adverse reactions of stress responses.
The current view is that, the intestine microbiota plays a role in the early programming and later response of the stress system, and the stress responses can also lead to increased intestinal permeability, allowing bacteria and bacterial antigens to cross the epithelial barrier and activate mucosal immune responses, thereby altering the composition of intestinal microorganisms (Dinan and Cryan 2012). We used 16s sequencing to analyze the composition of intestinal microbiota in fish from the BL and BH groups, but no significant difference was found between the two groups.