Molecular cloning and expression characteristics of ghrl and ghsr genes in bighead carp (Hypophthalmichthys nobilis)

doi:10.21203/rs.3.rs-2445193/v1

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Molecular cloning and expression characteristics of ghrl and ghsr genes in bighead carp (Hypophthalmichthys nobilis)

https://doi.org/10.21203/rs.3.rs-2445193/v1

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published 01 Mar, 2024

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Backgroud

Ghrelin (ghrl) and its receptor gene, ghsr (growth hormone secretagogue receptor), regulate numerous physiological functions, including growth hormone secretion and food intake. In the present study, we investigated the sequences and expression characteristics of the two genes in bighead carp (Hypophthalmichthys nobilis).

Methods

Cloning and expression characteristics of the ghrl and ghsr gene were got and analyzed by using the RACE technique, qRT-PCR, and in situ hybridization. The sizes of the experimental fish were 1.21 ± 0.20 kg in winter (6–7 months old) and 2.34 ± 0.20 kg in summer (18–19 months old).

Results

The ghrl and ghsr genes, encoded 106 and 346 amino acids, respectively, and were 1208 bp and 2239 bp in length. Early blastocysts had the highest levels of ghrl and ghsr expression throughout early development (P < 0.01). The gut exhibited the highest levels of expression of ghrl, whereas significantly higher levels of ghsr (P < 0.01) were detected in the brain. Significantly higher levels of expression of ghrl and ghsr were detected in the intestine and muscle in summer, when compared with the levels in winter (P < 0.05). Based on in situ hybridization, intestine ghrl expression signals were more pronounced than intestinal ghsr expression signals, whereas ghsr expression signals were higher than those of ghrl in brain tissues. The co-expression patterns of related genes in the growth hormone synthesis, secretion and action pathway (ko04935), and cAMP signaling pathway (ko04024) also indicated that ghrl and ghsr are involved in the feeding regulation of bighead fish.

Conclusion

Our findings suggest that ghrl and ghsr potentially play roles in bighead carp feeding and associated energy metabolism activities, in addition to growth performance.

planktivorous fish

feeding-related gene

tissue specificity

spatiotemporal expression

Fish feed to obtain the nutrients and energy they require for growth and reproduction, and their feeding habits are controlled by both their central nervous system and external environmental cues [1], which are regulated by key functional genes related to feeding [2]. In recent years, numerous genes with key roles in feeding in teleost fishes have been identified, including npy8r (neuropeptide Y receptor y8), agrp (agouti-related protein), npy (neuropeptide y), in addition to other orexigenic agents that drive fish to eat after fasting [3–5]. And the feeding suppressants, such as mc4r (melanocortin-4 receptor) and leptin, reduce fish feeding [6, 7]. Additionally, the sequence polymorphism of penk (proenkephalin) is related to food habit domestication traits [8]. As one of the most extensively researched appetite-regulating peptides in vertebrates, Ghrelin is a gastric-derived acylated peptide that was first purified from rat stomachs and has been identified as an endogenous ligand for growth hormone secretagogue receptor (GHSR) [9]. Until then, GHSR was considered an endogenous receptor for artificial GH secretagogues (GHSs) [10]. Studies have recently shown that ghrl mediates ghsr to play crucial roles, including enhancing appetite, preservation of energy balance, and immunological and anti-inflammatory activities [11]. With advancements in research, fish feeding strategies, exogenous injection stimulation, and other topics, have been the focus of investigations [12–14]. Meanwhile, ghsr is a crucial component mediating the different physiological functions of ghrl. In particular, three out of four ghsr subtypes have been observed that can be activated by ghrl to induce feeding [15]; ghrl promotes nutrient degradation by regulating the gene and protein expression of digestive enzymes in the intestine and hepatopancreas of goldfish through ghsr 1a [16]. Although feeding-related genes (including ghrl and ghsr) have been investigated in fish feeding research, especially for filter-feeding fishes, more investigations are required to determine their roles and mechanisms in feed intake and other processes in different fishes, considering less information is available with regard to passive feeding fishes.

Bighead carp (Hypophthalmichthys nobilis) is one of the major aquaculture fish species in China, and is very valuable economically and nutritionally [17]. It is primarily a filter-feeder; and is generally polycultured with other fish species (e.g., common carp Cyprinus carpio, grass carp Ctenopharyngodon idella, and tilapia Oreochromis mossambicus, etc.) in earthen ponds, facilitating full utilization of the available food and water space resources, and water quality maintenance in ponds. Furthermore, bighead carp is frequently used in water eutrophication control and water purification fisheries, particularly in open water bodies (e.g., lakes and reservoirs) [18]. The "Great Protection of the Yangtze River" proposal and the "ten-year ban on fishing" initiatives undertaken in the Yangtze River have led to increase in the demand for bighead carp production in semi-intensive ponds [19, 20]. Increasing feed conversion ratio (FCR) and lowering feed costs are key goals in traditional aquaculture. However, a few studies have been conducted on filter-feeding fishes, which are typically considered passive feeders, or how their regulatory mechanisms may respond to food abundance. The molecular functions of genes related to feeding and metabolism, including the roles of ghrl and ghsr, have been reported in many fishes [12–16, 21]; however, it remains unclear whether they serve similar roles in filter-feeding fish. Consequently, basic research on ghrl and ghsr in bighead carp could provide insights that could facilitate future aquaculture methods and fish management activities.

Feeding-related genes are the genetic basis for studying and exploring animal feeding-related life activities. Analysis of the underlying genetic mechanisms involved in food intake and energy metabolism regulation in different fish is critical for the understanding and improvement of feeding performance. In the present study, the full-length cDNAs of feeding-related genes, ghrl and its receptor gene, ghsr, in bighead carp were obtained using Rapid Amplification of cDNA Ends (RACE) technology. The spatiotemporal expression patterns of the two genes were studied using quantitative real-time polymerase chain reaction (qRT-PCR) and verified by in situ hybridization (ISH). Furthermore, co-expression analysis of related genes was used to explore their relationships across functional pathways. The findings of the present study shed light on the sequence characteristics, tissue distribution, and potential regulatory roles of the two genes in bighead carp, and serve as a basic foundation for further research on the functions of the feeding-related genes in bighead carp, and could facilitate the selection of improved varieties in future.

Experimental fish collection

The experimental fish were obtained from the Qiting Pilot Research Station (Yixing, Jiangsu, China), affiliated to the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China. The average body weight of the experimental fish was 1.21 ± 0.20 kg in winter (6–7 months old, November 5, 2021) and 2.34 ± 0.20 kg in summer (18–19 months old, June 5, 2022). The water temperature was measured at 9:00 A.M., with 12.3°C and 32.6°C in winter and summer, respectively. The fish were of good vitality and had no signs of disease. Nine tissues including kidney, spleen, liver, brain, heart, blood, muscle, foregut and gills were collected from five fish during winter and summer, respectively, and stored at -80°C after quick freezing in liquid nitrogen for use in subsequent RNA extractions. The four individuals of winter samples (w01-04) and four of summer samples (s01-04) were selected for spatiotemporal expression experiments, respectively.

To explore the gene expression profiles of bighead carp during embryonic development, samples of early embryos (blastula, gastrula, eye-primordia, muscle segment, and hatching stages) and larvae (1 dph [day post-hatching], 5 dph and 10 dph stages) were consecutively collected and numbered 1–8. A total of 15 embryos (or eggs) or 8–10 larvae were pooled in each stage. The 1 dph larvae were maintained in a concrete tank with no special feeding. The 5 dph and 10 dph larvae were reared in an earth pond, and were fed on natural plankton developed in advance, in addition to being sprayed soybean milk (3–4 kg/666.67 m²) every day in the first week. In addition, the foregut and whole brain of the summer fish were collected and fixed in 4% paraformaldehyde for 48 h, and then stored at -20°C for use in situ hybridization experiments.

Total Rna Extraction And Cdna Synthesis

The total RNA of winter and summer tissues were extracted using TRIzol reagent (Cowin Bio, China), and their concentrations and integrity were quantified using a NanoDrop UV spectrophotometer (Thermo Scientific, USA) and 1% agarose gel electrophoresis, respectively. After total RNA (500 ng) was treated with RNase-free DNase I (New England Biolabs), the first-strand cDNA was reversed using PrimeScript™ RT Master Mix Kit (Perfect Real Time) (Takara, Japan). The final concentrations were diluted for use as templates in real-time PCR analyses.

Rapid amplification of ghrl and ghsr cDNAs

The partial cDNA sequences of ghrl and ghsr genes of bighead carp were obtained from transcriptomic data of bighead carp [22]. Specific primers (ghrl-F and ghrl-R; ghsr-F and ghsr-R) were designed to verify the sequences (Online Resource 1: Table S1). The cDNA of nine tissues were used as the templates to amplify partial fragments under the following PCR conditions: 94°C for 3 min, followed by 35 cycles including 94°C for 15 s, 60°C for 20 s, 72°C for 1 min, and a final extension at 72°C for 5 min. Eventually, we obtained and confirmed the partial cDNA sequences of the two genes after sending the purified PCR products to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

The specific primers were designed according to the intermediate sequences obtained after sequencing, and intestinal or muscle RNA was used as templates to synthesize the first strand cDNA using a HiScript®-TS 5'/3' RACE Kit (Vazyme, China) to amplify the 5' and 3 'ends. The first-round amplification PCR of 5′- and 3′-RACE was in a 50-µL reaction mixture consisting of 2.5 µL cDNA template, 1 µL 5'/3′ GSP (10 µM), 5 µL 10× Universal Primer Mix (UPM), 25 µL 2× PCR Mix, and 16.5 µL ddH2O, and was performed under the following reaction conditions: pre-denaturation at 98°C for 1 min, 5 cycles of 98°C for 10 s, 72°C for 3 min; 5 cycles of 98°C for 10 s, 70°C for 15 s, 72°C for 3 min; 25 cycles of 98°C for 10 s, 68°C for 15 s, 72°C for 3 min; and final extension at 72°C for 5 min. In the second-round PCR program, the PCR product obtained from the first round of reaction was diluted 50 times as a template. The reaction mixture consisted of 5 µL diluted PCR product, 1 µL Nested GSP (10 µM), 1 µL Nested Primer, 25 µL 2× PCR Mix, and 18 µL ddH2O. The PCR program was run with an initial denaturation at 98°C for 1 min; 25 cycles including 98°C for 10 s, 68°C for 15 s, 72°C for 3 min; and final extension at 72°C for 5 min. The target PCR products were purified using an agarose purification kit (Axygen, Union City, CA, USA) and cloned into the pMD19-T vector (Takara, Japan). Following transfection into Escherichia coli DH5-α competent cells, putative clones were further screened by PCR amplification. The products were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

Quantitative Real-time Pcr (Qrt-pcr)

Three primer pairs for qRT-PCR were designed and synthesized according to the cDNA sequences of ghrl, ghsr, and β-actin. Considering the fact that ghrl and ghsr are members of two Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways (www.kegg.jp), i.e., ko04935 (growth hormone synthesis, secretion and ac-tion pathway), and ko04024 (cAMP signaling pathway), 12 additional genes associated with ko04935 (ip₃r, gh, ghr, jak2, stat, igf 1, ghrh, pka, and vscc-l) and ko04024 (gpcr, pka, pparα, and aco) were selected for integration in co-expression analysis, and their qRT-PCR primers were also designed (Online Resource 1: Table S1) based on the transcriptomic sequences [22]. The amplification efficiencies of all primer pairs for qRT-PCR were checked using standard curve analysis.

TB Green™ Premix Ex Taq™ II (Takara, Japan) was selected as the reagent kit, and qRT-PCR experiments were completed using a CFX96TM Real-time System (Bio-Rad, Hercules, CA, USA) in 20-µL reaction systems comprising 10 µL TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X), 1 µL each of the forward and reverse primer, 2 µL cDNA template (80 ~ 100 ng/µL), and 6 µL ddH2O. The reaction conditions were as follows: 95°C for 30 s; followed by 40 cycles of 95°C for 5 s, and 60°C for 30 s; and then 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. Three experimental replicates were set up for each sample in the amplification reaction. Relative expression analyses were based on the levels of ghrl and ghsr mRNA expression relative to β-actin mRNA expression levels in the same samples, based on the 2^−∆∆Ct method [23].

In Situ Hybridization

T7 reverse transcriptase and digoxigenin-labeled UTP were used to synthesize antisense RNA probes in vitro. The intestine and brain were selected for ISH according to the results of relative expression of ghrl and ghsr. After washing, the tissue slides were fixed in the fixing solution for more than 12 h. After gradient dehydration with alcohol, soaking in wax, embedding, and slicing, the slides were baked at 62°C for 2 h.

The successfully processed slides were treated with sample fixation, gradient rehydration, and protease K digestion. Subsequently, they were put into the pre-hybridization solution without the probe and incubated at 37°C for 1 h. Afterward, the slides were put into the hybridization solution containing ghrl/ghsr probes. After incubation overnight at 42°C, the slides were washed with distilled water, blocked with the antibody, and dried. The slides were then put into the freshly prepared DAB chromogenic solution until the positive signal (bluish-violet color) appeared. The slides were washed with pure water to terminate the coloration. Finally, the color signal was observed and photographed under an optical microscope.

Bioinformatics And Statistical Analysis

The sequencing results were spliced using DNAMAN to obtain the full-length cDNA sequences of the two genes (http://en.bio-soft.net/format/DNAMAN.html). The ORF finder tool (http://ncbi.nlm.nih.gov/gorf/gorf.html) was used to search and predict the open reading frame (ORF) and corresponding amino acid sequences. Signalp 4.0 server (http://www.cbs.dtu.dk/services/SignalP/) was used to predict the sequences and positions of signal peptides; and Protscale (http://web.expasy.org/protscale/) was used to predict and analyze the hydrophobicity of amino acid residues. Pfam (https://pfam.xfam.org/) was used to predict protein functional domain, and swiss model (http://www.swissmodel.expasy.org/) was used to predict the tertiary structure of proteins. TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) was used to analyze transmembrane regions of the two genes. Multiple comparisons and homology analyses for amino acid sequences of the two genes with other species were carried out using DNAMAN. The phylogenetic trees were built using MEGA-X (https://www.megasoftware.net/index.html) with the Neighbor-Joining (NJ) method, and bootstrapped with 500 replicates. Based on gene expression (qRT-PCR data), the Pearson’s correlation analysis, heatmap construction, and linear correlation analyses were carried out and visualized using the states, corrplot, and pheatmap packages in R version 4.1.2 (http://www.R-project.org).

IBM SPSS Statistics 25 (IBM Corp., Armonk, NY, USA) was used for one-way Analysis of Variance and Duncan’s multiple comparative analyses. The relative expression data (mean ± SE) were processed and visualized using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). All primers used in the present study were synthesized by Sangon Biotech (Shanghai) Co. Ltd., and are listed in Supplemental Table S1. about GenBank accession numbers of other species involved was downloaded from the NCBI GenBank and is provided in the Online Resource 1 (Table S2).

Characterizations of ghrl and ghsr

The ghrl cDNA sequence of bighead carp had a length of 1208 bp (GenBank Accession No. OP039099), which included a 321 bp ORF (encoding 106 amino acids), 39 bp and 848 bp of the 5'-Untranslated Region (5'UTR) and 3'UTR, respectively, and ended with a putative polyadenylation consensus signal (poly-A: AATAAA) (Online Resource 2: Fig. S1a). The predicted protein of the GHRL sequence had an N-terminal signal peptide with 26 amino acid residues, followed by a mature peptide with 19 amino acid residues. The mature peptide’s C-terminal contained two typical cleavage-processing sites, denoted by the letters "GRR", following the 12th and 19th amino acids. Additionally, the third serine residue (Ser3) of the mature peptide exhibited potential acylation modification sites and contributed to the development of the shared structure of Cyprinidae fish active-centers (GTSF) (Fig. 1a). At the C-terminus of GHRL protein, Pfam projected that there was a conserved Motilin associated (Motilin_assoc) sequence between the 66th and 98th amino acid. There was no obvious transmembrane region and the hydrophobicity was − 0.139 on average.

The 2239 bp full-length ghsr cDNA (GenBank Accession No. OP068274) contained a 590 bp 5'UTR, a 1041 bp ORF encoding 346 amino acids, and a 608 bp 3'UTR, and also ended with poly-A (Online Resource 2: Fig. S1b). Its seven transmembrane (TM) structures, including TM1 to TM7, were identified using amino acid sequence analysis and were similar to the relevant TM region sequences of goldfish (Fig. 1b). The overall hydrophobicity was 0.501 on average.

To ascertain the structural characteristics of the two proteins in bighead carp, we also examined the tertiary structures using Swiss model software. The tertiary structures of the GHRL (Fig. 2a) and GHSR (Fig. 2b) proteins in bighead carp were simply Helix and Folding.

We compared the homology of amino acid sequences of the two genes to those of other species using DNAMAN. Based on the amino acid sequences of GHRL, high similarities were detected between bighead carp and grass carp, goldfish (Carassius auratus), and zebrafish (Danio rerio), at 97.03%, 87.13% and 77.45%, respectively. Bighead carp had the closest genetic link to Cyprinidae fish, while Siluriformes and Perchformes species were distantly connected, according to the phylogenetic tree based on the GHRL data (Fig. 3a). With regard to the GHSR sequences, bighead carp and white minnow (Anabarilius grahami) had the highest homology (97.69%), followed by goldfish (91.49%) and common carp (90.88%), but had poor identity with the other fish (40–60%). Similar trends were observed in the GHSR evolutionary tree (Fig. 3b).

Relative Expression Of Two Genes In Different Early Developmental Stages And Different Tissues

Relative levels of expression of the two genes in different early developmental stages, and various tissues, were analyzed using qRT-PCR. During early development, ghrl expression exhibited a declining trend overall, and the highest expression was observed in the early embryonic stage (blastocyst stage), which was extremely higher than those in the other stages (P < 0.01) (Fig. 4a). The level of expression of ghsr was the highest in the blastocyst stage and dropped over the following three periods, similar to the ghrl expression trends in the early stages. After hatching, the expression levels grew and tended to remain constant (Fig. 4b).

The expression levels of two genes (ghrl and ghsr) were detected in all the sampled tissues in the present study. The highest ghrl expression level was detected in the intestine (P < 0.01), followed by in the muscle, heart, and blood, with hardly detectable expression in the kidney and gill (Fig. 4c). The levels of ghsr expression were moderate in the liver and muscle, relatively low in the kidney and heart, and significantly higher in the brain (P < 0.01) (Fig. 4d).

In Situ Hybridization

In the present study, we used ISH to confirm the expression of ghrl and ghsr in the intestine and brain of summer fish (Fig. 5). The ghrl signal could be detected in almost the entire section of the intestine, whereas ghsr expression was higher in the brain, which are consistent with the qRT-PCR results for the two genes.

Relative Expression Of Two Genes In Winter And In Summer

Due to the passive feeding characteristics of bighead carp, conducting reliable fasting and re-feeding experiments is challenging. Considering plankton abundance and food intake intensity are potentially influenced by water temperature, we collected individuals in winter and in summer, separately, which were used for further analyses.

Considering the relatively high levels of expression of two genes (ghrl and ghsr) in the brain, intestine, muscle, and liver observed in previous experiments, we further compared the expression trends of the two genes in four tissues between winter and summer. There were no significant differences in ghrl expression among the four tissues in winter; however, significantly higher expression levels were detected in the intestine (P < 0.01) and muscle (P < 0.05) in summer (Fig. 6a). ghsr levels of expression were higher in the brain and liver in summer (P < 0.01), and significantly higher levels of expressions were also observed in the intestine and muscle in summer (P < 0.05), when compared with the levels in winter (Fig. 6b).

Co-expression patterns of ghrl, ghsr, and related genes evaluated using qRT-PCR

The sampled tissues in winter and summer were also used to obtain insights on the co-expression patterns of ghrl and ghsr with related genes under different physiological statuses. Significant positive and linear correlations were observed in the relative levels of expression of ghrl (Fig. 7a) and ghsr (Fig. 7b) between the intestine and muscle in winter and summer (P < 0.05).

Considering the two genes participate in the ko04935 and ko04024 pathways, we selected a total of 14 genes from the two pathways for co-expression analysis, to explore the potential roles of the two genes in growth and metabolism. The heatmap also demonstrated that the relative levels of expression of ghrl, ghsr, and related genes (from ko04935 and ko04024) were significantly higher in summer than in winter in muscle (P < 0.05) (Fig. 7c). In addition, the genes generally exhibited positive pairwise linear relationships (Fig. 7d).

In the present study, the full cDNA sequences of ghrl and ghsr and their encoded amino acid sequences were identified in bighead carp. Previous studies have confirmed that n-octanoic acid or n-decanoic acid modifies the Ser3 of the mature peptide, and that the modification is necessary for ghrl to bind to the receptor and trigger various physiological effects [9]. The first four amino acids of GHRL mature peptide are "GTSF", which are relatively conserved in Cypriniformes species [24]. There are two cleavage sites at the C-terminal of the GHRL mature peptide. This structural characteristic indicates that bighead carp may have various mature peptide versions of GHRL. In addition, Motilin_assoc is located at the C-terminal of the GHRL precursor peptide in bighead carp. Due to the similar structure to the motilin-related peptide (MTLRP), the particular site was considered to be potentially involved in the control of gastrointestinal physiological processes [25, 26]. The present study also yielded the first cDNA sequence of ghsr for bighead carp, which has already been reported for other fish species [27–29]. The TM1 to TM7 structures in GHSR were relatively conserved in Cypriniformes [30–32]. Additionally, GHSR may function in the cytoplasm because it is a hydrophobic protein without a signal peptide. GHRL and GHSR are typically conserved throughout the evolution of vertebrates according to the phylogenetic tree, which may be related to the evolution or evolutionary adaptation of fish feeding habits under complex living environments.

During the early stages of development, higher levels of mRNA expression of the two genes were detected as early as the blastocyst stage, and decreased rapidly over the course of the following three periods to the lowest level. After hatching, the levels of expression increased and tended to remain constant. In the early stages of development of fish, the blastocyst stage is a node for the initial formation of the embryo, and the hatching stage is one of the periods when higher energy consumption is observed. The high levels of expression of the genes in the two stages may be associated with the unique energy metabolism at the stage [33]. Previous studies have found that ghrl can regulate the expression of the GH gene, as well as its metabolism and release, in zebrafish embryos [34]. Therefore, we speculated that ghrl and ghsr may contribute to embryonic development by regulating the energy metabolism of early embryos. The mRNA expression levels of the two genes increased at 5 dph after hatching. During the early stages of fish development, the energy source switched from endogenous nutrition to external nutrition as the yolk was exhausted and food intake increased gradually. According to the findings of studies on fish species, the upregulated expression trends may be connected to a change in the larval diet [27, 33, 35]. The consistency and conservation of functional genes in the regulation of certain physiological process can be observed in the similarities of the expression patterns above.

In terms of tissue-specific expression, there are differences in the expression patterns of two genes in different fish species [30, 31, 36]. In the present study, bighead carp ghrl was highly expressed in the intestine, while higher levels of ghsr expression were detected in the brain, which are consistent with the findings in other bony fishes. The gut anatomy of fish is intimately tied to their feeding habits. Bighead carp is a typical filter-feeding fish and one of the stomach-less fish, so it mostly relies on the gut's absorption and digestion processes to obtain nutrients. According to recent studies, ghrl promotes intestinal cell proliferation and influences the expression of functional genes related to protein expression and metabolism [21, 37]. The intestinal expression of ghrl was much higher than those in other tissues based on the results of ghrl differential expression and ISH, which indicated that ghrl gene may be strongly associated with the feeding, digestion, and absorption functions of bighead carp. The high levels of expression of ghsr in the brain, liver, and muscle suggested it may have more biological activities. In addition, the high levels of ghsr expression detected in the brain are consistent with findings on other fish species [15, 26–29, 32]. Several hormones that the brain secretes play a role in energy homeostasis, including weight gain and food intake regulation, and they effectively control the expression of corresponding genes during negative energy balance (such as long-term fasting) to facilitate energy balance [39]. In addition, the study found that ghrl induced lipogenesis and glycogenesis in the liver via the receptor ghsr, increased triglyceride contents, and increased liver weight and total fat content in fish livers [40–41]. ghrl stimulates the generation of superoxide through its receptor ghsr, increasing the phagocytic activity of leukocytes in rainbow trout [42]. Furthermore, high ghsr expression in liver might be related to fish immune defense and energy storage. Overall, the tissue-specific expression characteristics of the two genes highlighted the variations and potential adaptations in the regulation of physiological activities in different fish.

In pond aquaculture, the plankton community typically exhibits spatiotemporal variation, which is particularly evident in variations in dominant species and their numbers over time. Additionally, fish under different culture conditions would exhibit different feeding habits in response to the photoperiod, aquatic environment, and food abundance, and are mainly affected by temperature [38, 43]. In the present study, the expression of the two genes increased significantly from winter to summer in both the intestines and muscles of bighead carp. We hypothesize that because of seasonal temperature change, plankton communities tend to be abundant in the summer and scarce in the winter, which stimulates food intake and the growth of bighead carp in summer. According to the ghrl and ghsr expression trends observed in the present study, combined with the biological functions of these genes discussed above, the seasonal variations in energy metabolism, growth performance, and immunological defense activities of fish may be related to the up-regulated expression of ghrl and ghsr during winter.

Ghrelin regulates GH secretion in conjunction with somatostatin (SST) and growth hormone-releasing hormone (GHRH) to control cell growth, development, and systemic metabolism [10]. In addition, ghrl can activate the ghsr in the hypothalamic-pituitary axis, which in turn enhances GH release [44]. Once GH has been released into the bloodstream, it will react with GHR on the cell surfaces of target tissues (such as the liver and muscle) to activate JAK2 and set off signal cascades, including STAT, which encourages development by producing and secreting IGF-1 [45]. In addition, GHRH triggers G protein-coupled receptor (GHRHR) in growth hormone cells to activate the cAMP signaling pathway and control various physiological activities, such as metabolism, secretion, fatty acid breakdown, and muscle contraction.

ghrl and ghsr are participants in the ko04935 and ko04024 KEGG pathways. In the present study, the relative levels of expression of muscle ghrl, ghsr, and associated genes were increased significantly from winter to summer (P<0.05), and higher positive linear correlations were observed among the 14 genes. The co-expression patterns indicated that the genes related to certain metabolic activities generally work collaboratively. The findings of the present study demonstrate that the feeding and growth of bighead carp are regulated by the ghrelin–GHSR system and that the effect varies markedly across seasons.

The present study investigated the sequences and expression patterns of ghrl and ghsr, which measured 1208 bp and 2239 bp based on full length cDNAs, respectively, with high similarity to Cyprinidae fishes. The highest levels of ghrl and ghsr expression were observed at the blastocyst stage, indicating that the genes are critical for early development. Both are expressed in all tissues, with the intestine and the brain having the highest levels of expression of ghrl and ghsr, which were partially confirmed using ISH experiments. Significant positive and linear correlations were detected in the levels of expression of ghrl and ghsr between the intestine and muscle, in summer and in winter. The co-expression patterns of the two genes and related genes were associated with two functional pathways (ko04935, and ko04024) in both winter and summer. The findings imply that the two genes may be critically involved in the regulation of bighead carp growth and energy metabolism. Further comprehensive studies of the regulatory mechanisms of the two genes should be undertaken to explore the potential feeding-related genetic regulation activities of the genes.

Funding

This work was financially supported by the Earmarked Fund for China Agriculture Research System (No. CARS-45–05) and the Central Public-Interest Scientiﬁc Institution Basal Research Fund, Freshwater Fisheries Research Center, CAFS (No.2021JBFM01).

Competing Interests

The authors declare no conflicts of interest.

Author contributions

All authors contributed to the conception and design. Material preparation, data collection and analysis were performed by Pan Wang, Xiulan Shi, Shichang Shu, Wenbin Zhu and Lanmei Wang. The first draft of the manuscript was written by Pan Wang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics statement

All the trials were performed in accordance with Bioethical Committee of the Freshwater Fisheries Research Center (FFRC) of the Chinese Academy of Fishery Sciences (CAFS) (BC2013863, 9/2013). Sampling, handling, and experimental procedures were carried out according to the guidelines for the care and use of animals for scientific purposes issued by the Ministry of Science and Technology, Beijing China (No.398, 2006).

Supplementary Information

Supplementary files for this article can be found online.

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Molecular cloning and expression characteristics of ghrl and ghsr genes in bighead carp (Hypophthalmichthys nobilis)

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Abstract

Backgroud

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Experimental fish collection

Total Rna Extraction And Cdna Synthesis

Quantitative Real-time Pcr (Qrt-pcr)

In Situ Hybridization

Bioinformatics And Statistical Analysis

Results

Relative Expression Of Two Genes In Different Early Developmental Stages And Different Tissues

In Situ Hybridization

Relative Expression Of Two Genes In Winter And In Summer

Discussion

Conclusions

Declarations

References

Supplementary Files

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