Genomic Selection of Large Yellow Croaker (Larimichthys crocea) with a High Plant Protein Diet Enhances the Growth Performance of Offspring.

Fishmeal is over-represented in the diets of large yellow croaker (Larimichthys crocea), and this farming mode, which relies heavily on fishmeal, is highly susceptible to the price of fishmeal and is unsustainable. Therefore, more and more studies on the large yellow croaker tend to replace fishmeal with land-based animal or plant proteins, but few studies have considered it from the genomic selection. In this study, we evaluated the survival rate (SR), final body weight (FBW), body weight gain (BWG), weight gain rate (WGR), and specific growth rate (SGR) of the large yellow croaker GS7 strain, which was obtained through genomic selection for tolerance to plant proteins and analyzed the differences in plant protein utilization between the GS7 strain and unselected commercial large yellow croaker (control group). The results of separate feeding for 60 days showed that although there was no significant difference in SR between the control and GS7 strains (P > 0.05), the BWG, WGR, and SGR of the control were significantly lower (P < 0.05) than those of the GS7 group. Results of mixed feeding after PIT marking showed that compared to the control fish, the GS7 strain had significantly higher BWG, WGR, and SGR (P < 0.0001). To make the experimental results more precise, we compared fishes with equivalent initial body weight (IBW) in the GS7 strain and the control group. The final fish body weight (FBW) of Ctrl-2 (IBW 300-399 g) and Ctrl-4 (IBW 500-599 g) was significantly lower than those of the corresponding GS7-2 and GS7-4 (P < 0.05), while the FBW of Ctrl-1 (IBW 200-299 g) and Ctrl-3 (IBW 400-499 g) was much significantly lower than the corresponding GS7-1 and GS7-3 (P < 0.01). The BWG, WGR, and SGR of Ctrl-1 and Ctrl-4 were more significantly lower than those of the corresponding GS7-1 and GS7-4 (P < 0.01), while the BWG, WGR, and SGR of Ctrl-2 and Ctrl-3 were more significantly different from the corresponding GS7-2 and GS7-3 (P < 0.0001). Our results seem to point toward the same conclusion that the GS7 strain is better adapted to high plant protein diets than the unselected commercial large yellow croaker. These results will provide a reference for the low-fishmeal culture industry of large yellow croakers and the selection and breeding of strains tolerant to a high percentage of plant proteins in other marine fishes.

Swimming Performance in Large Yellow Croaker: Effects of Group Size, Test Protocol, and Recovery Time On Critical Swimming Speed.

Swimming is critical for fish survival, and little attention has been paid to the swimming performance of large yellow croaker, the largest farmed marine fish in China. To address this gap, we conducted a study to measure the critical swimming speed (Ucrit) of 1050 croaker in a designed swim test flume. Our findings shed light on the effects of group size, Ucrit test protocol, and recovery time on swimming performance. The water flow in the swim flume increased steadily and linearly. The linear fit equation was y = 2.89x + 1.79 with an R2 of 0.99. With the help of the swim flume, we found that group size, and the Ucrit test protocol had a significant effect on the Ucrit values, except for the recovery time: The Ucrit values obtained in the ramp-Ucrit test averaged 28.32 ± 6.11 cm.s-1, which was significantly lower than that obtained in the traditional Ucrit test of 32.75 ± 7.60 cm.s-1; The Ucrit value of a group size of 50 fish was 33.51 ± 5.96 cm.s-1, which was significantly higher than that of a group of 200 fish (28.49 ± 6.37 cm.s-1). These results provide insights into the swimming performance of large yellow croaker and can be used to standardize the swimming test protocols.

Switch from fight-flight to freeze-hide: The impacts of severe stress and brain serotonin on behavioral adaptations in flatfish.

Animals often experience changes in their environment that can be perceived as stressful. Previous evidence indicates that different individuals may have distinct stress responses. The role of serotonin (5-HT) in stress adaptation is well established, but its relationship with different defense strategies and the persistence of physiological and behavioral responses in different individuals during repeated acute stress remain unclear. In this study, using olive flounder (Paralichthys olivaceus) as a model, we analyzed the relationship between boldness and neurotransmitter 5-HT activity. We found that 5-HT suppression with 5-HT synthesis inhibitor p-chlorophenylalanine (pCPA) and 5-HT receptor subtype 1A (5-HT1A) antagonist WAY-100635 increased their oxygen consumption rates and the boldness of shy individuals. We determined the metabolic and behavioral changes in bold and shy individuals to repeated acute stress. The results suggest that bold individuals switch on passive "energy-saving" personality by changing their defense behavior from "fight-flight" to "freeze-hide" during a threat encounter, which manifests high behavioral plasticity. Both behavioral types decreased their spontaneous activity levels, which were also strengthened by limiting metabolic rate. Interestingly, treatment with pCPA and WAY-100635 before stress procedure attenuated stress and increased the boldness across diverse behavioral types. This study provides the initial empirical evidence of how perception of stress impacts both individual defense behavior and personality in this species. These findings can enhance our comprehension of individual variability and behavioral plasticity in animals, thereby improving our ability to develop effective adaptive management strategies.

The excitatory effect of 5-HT1A and 5-HT2B receptors on the caudal neurosecretory system Dahlgren cells in olive flounder, Paralichthys olivaceus.

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an essential role in the regulation of neural activity via multiple receptors. Here, we investigated the functional role of serotoninergic input on the Dahlgren cell population in the caudal neurosecretory system (CNSS) of olive flounder. In this study, the effect of 5-HT on the firing activity of Dahlgren cells was explored in terms of changes in firing frequency and firing pattern using multicellular recording electrophysiology ex vivo, and the role of several 5-HT receptor subtypes in the regulation was determined. The results revealed that 5-HT increased the firing frequency in a concentration-dependent manner and altered the firing pattern of Dahlgren cells. The effect of 5-HT on the firing activity of Dahlgren cells was mediated through the 5-HT1A and 5-HT2B receptors, selective agonists of both receptors effectively increased the firing frequency of Dahlgren cells, and selective receptor antagonists could also effectively inhibit the increase in firing frequency caused by 5-HT. In addition, the mRNA levels of major signaling pathway-related genes, ion channels, and major secretion hormone genes were significantly upregulated in CNSS after treatment with 5-HT. These findings demonstrate that 5-HT acts as an excitatory neuromodulator on Dahlgren cells and enhances neuroendocrine activity in CNSS.

Integrated mRNA and miRNA expression analyses for Cryptocaryon irritans resistance in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. However, cryptocaryonosis caused by Cryptocryon irritans infection has brought huge economic losses and threatened the healthy and sustainable development of L. crocea industry. Recently, a new C. irritans resistance strain of L. crocea (RS) has been bred using genomic selection technology in our laboratory work. However, the molecular mechanisms for C. irritans resistance of RS have not been fully understood. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that are post-transcriptional regulators, and they play vital roles in immune process of bony fish. Identification of anti-C.irritans relevant miRNA signatures could, therefore, be of tremendous translational value. In the present study, integrated mRNA and miRNA expression analysis was used to explore C. irritans resistance mechanisms of the L. crocea. RS as well as a control strain (CS) of L. crocea, were artificially infected with C. irritans for 100 h, and their gill was collected at 0 h (pre-infection), 24 h (initial infection), and 72 h (peak infection) time points. The total RNA from gill tissues was extracted and used for transcriptome sequencing and small RNA sequencing. After sequencing, 23,172 known mRNAs and 289 known miRNAs were identified. The differential expression was analyzed in these mRNAs and mRNAs and the interactions of miRNA-mRNA pairs were constructed. KEGG pathway enrichment analyses showed that these putative target mRNAs of differentially expressed miRNAs (DEMs) were enriched in different immune-related pathways after C. irritans infection in RS and CS. Among them, necroptosis was the immune-related pathway that was only significantly enriched at two infection stages of RS group (RS-24 h/RS-0h and RS-72 h/RS-0h). Further investigation indicates that necroptosis may be activated by DEMs such as miR-133a-3p, miR-142a-3p and miR-135c, this promotes inflammation responses and pathogen elimination. These DEMs were selected as miRNAs that could potentially regulate the C. irritans resistance of L. crocea. Though these inferences need to be further verified, these findings will be helpful for the research of the molecular mechanism of C. irritans resistance of L. crocea and miRNA-assisted molecular breeding of aquatic animals.

Neuromodulatory effects of GnRH on the caudal neurosecretory Dahlgren cells in female olive flounder.

Gonadotropin-releasing hormone (GnRH) is considered a key player in reproduction. The caudal neurosecretory system (CNSS) is a unique neurosecretory structure of fish that may be involved in osmoregulation, nutrition, reproduction, and stress-related responses. However, a direct effect of GnRH on Dahlgren cells remains underexplored. Here, we examined the electrophysiological response of Dahlgren cell population of the CNSS to GnRH analog LHRH-A2 and the transcription of related key genes of CNSS. We found that GnRH increased overall firing frequency and may be changed the firing pattern from silent to burst or phasic firing in a subpopulation of Dahlgren cells. The effect of GnRH on a subpopulation of Dahlgren cells firing activity was blocked by the GnRH receptor (GnRH-R) antagonist cetrorelix. A positive correlation was observed between the UII and GnRH-R mRNA levels in CNSS or gonadosomatic index (GSI) during the breeding season. These findings are the first demonstration of the ability of GnRH acts as a modulator within the CNSS and add to our understanding of the physiological role of the CNSS in reproduction and seasonal adaptation.

Inhibitory role of taurine in the caudal neurosecretory Dahlgren cells of the olive flounder, Paralichthys olivaceus.

Taurine plays role in neural development and physiological functions such as endocrine regulation in the central nervous system (CNS), and it is one of the most abundant free amino acid there. We investigated its potential effect as a neurotransmitter in the group of neuroendocrine Dahlgren cells at flounder Paralichthys olivaceus caudal neurosecretory system (CNSS). The application of taurine in vitro led to a reduction in electrical activity of Dahlgren cells, followed by a rise in the number of silent cells, at the same time the frequency of all three activity patterns (tonic, phasic, bursting) in Dahlgren cells was reduced. Both strychnine (a glycine receptor antagonist) and bicuculline (a GABAA receptor antagonist) can block the response to taurine separately. Transcriptome sequencing analysis showed the existence of glycine receptor (GlyR) and GABAA receptor (GABAAR) in the flounder CNSS, and the GlyR, GABAAR, and Cl- channel mRNA expression were significantly raised after taurine superfusion according to quantitative RT-PCR results. These data indicate that taurine may mediate Dahlgren cell population of CNSS activity in vivo through GlyR and GABAAR, thereby, regulating stress-response.

Electrotransfer of single-chain LH gene into skeletal muscle induces early ovarian development of orange-spotted grouper (Epinephelus coioides).

Luteinizing hormone (LH) plays important roles in regulating steroidogenesis and reproductive development of vertebrates. In the present paper, we study function of LH on early ovarian development of orange-spotted grouper by electrotransfer of single-chain LH gene into skeletal muscle for the first time. Short-term and long-term injection experiments were performed in this work, respectively. For short-term injection experiments, fish received one electrotransfer with the plasmid in skeletal muscle, then blood and muscle around the injected area were sampled 1, 3, 5 and 7 days after the injection, mRNA expression levels of LH gene relative to 18S were determined by quantitative real-time PCR (RT-PCR) assays and serum 17ß-estradiol (E2) levels were quantified by ELISA method. The results showed that levels of mRNA of LH gene in muscle and serum E2 level increased from 1 day to 7 days after the injection. For long-term injection experiments, fish received electrotransfer with the plasmid 4 times at weekly intervals in skeletal muscle. 48 h after the last injection, blood, gonad and hypothalamus samples were collected. Transcripts of cyp19a1a, cyp19a1b and gnrh1 genes and levels of serum E2 were separately analyzed by RT-PCR assays and ELISA method, and ovarian tissues were made of paraffin sections and stained by hematoxylin-eosin by method and observed by optical microscopy. The results suggested that long-term injection of LH gene into muscle upregulated transcripts of cyp19a1a and cyp19a1b and downregulated that of gnrh1, and stimulated E2 production and early-stage oogenesis. Moreover, statistical data showed that 9 of 10 ovaries of injected fish with LH gene began to develop after the long-term experiments. These data suggest that single-chain LH gene introduced into skeletal muscle via electrotransfer can be expressed and induce the early ovarian development of juvenile orange-spotted grouper. This work contributes to solve reproductive dysfunctions associated with low hormone levels of teleosts, further it may represent the demonstration at regulation of LH on early ovarian development of orange-spotted grouper to a certain extent.