Introduction

Growth rate is an important trait with ecological, evolutionary, and conservation implications (Lorenzen, 2016). At the molecular level, body growth is promoted by growth hormones, growth factors, and other growth-related proteins and peptides. Growth factors regulate cell growth and proliferation by binding to receptors and activating signal transduction pathways (Homma et al., 2001). Among these, insulin-like growth factors (IGFs) are the most extensively studied and well-established regulators of animal growth, widely present in both vertebrates and invertebrates (Nakai et al., 2022). Additionally, adenosine deaminase-related growth (ADGF) is also well known for cell growth and proliferation. The ADGF superfamily possesses insect-derived growth factor (IDGF), tsetse salivary growth factor (TSGF), mollusk derived growth factor (MDGF), and mollusk-like growth factor (MLGF). Among the ADGF superfamily MLGF has been recently identified in Pacific abalone (Haliotis discus hannai) and this mollusk specific growth factor has been known to regulate embryonic and larval development of molluscan species (Hanif et al., 2022).

The growth of animals, both aquatic and terrestrial, is influenced by a wide range of biotic, abiotic, and nutritional factors that regulate the expression of growth-related genes and signaling pathways (Meyer et al., 2017). Among abiotic factors, temperature, salinity, and toxicants are the most significant. These factors can disrupt hormonal regulation and impacting the growth of aquatic organisms. Water temperature is a key environmental factor influencing the growth of aquatic animals (Reid et al., 2019). Aquatic animals during starvation depend on body energy reserves. Rates of protein synthesis and ATP production capacity fall during starvation which reduces the activity of most growth-related pathways (Salem et al., 2007).

The Pacific abalone is a extensively cultured shellfish species in Korea (Hanif et al., 2022). Abalone in natural environment faces frequent challenges (Sun et al., 2022). Abalone in aquaculture conditions frequently face challenges such as thermal stress and food scarcity (starvation), which ultimately affect their growth (Morash and Alter, 2016). Although the effects of different abiotic factors on the growth and development of aquatic organisms have been widely studied, the effects of these factors on growth and development related gene expression have been poorly studied. Thus, the present study aimed to investigate the growth-specific and stress-responsive expression patterns of growth regulatory gene Hailotis discus hannai-Mollusk Like Growth Factor in Pacific abalone.

Materials and methods

1. Abalone culture and growth-specific sample collection

Abalones were cultured in tanks for one year in Dolsan, Yeosu with adequate food and oxygen supply. After one year, abalones were collected and categorized based on their growth patterns: rapid growth (RG), average (normal) growth (AG), minimum growth (MG), and stunted growth (SG) following the previously published manuscript by Hanif et al. (2023). After anesthetization with 5% MgCl₂, muscle tissues were collected from three individuals (n=3) of each group. The tissue samples were flash-frozen in liquid nitrogen, and stored at –80°C until total RNA extraction.

2. Tissue samples from starved Pacific abalones

Three-year-old Pacific abalones (n = 60) were randomly collected from sea cages in Jindo-gun, South Korea, and transported to an abalone hatchery in Dolsan, Yeosu. The abalones were acclimated in rearing tanks with a continuous flow of seawater, proper aeration, and adequate feed. The abalones were then divided into two groups: Group A (control) and Group B (starvation treatment). Group A abalones were maintained with food while Group B abalones were subjected to a three-week starvation period. Muscle tissue were collected from five individuals of each group at each week. After three weeks, the starved abalones were reintroduced to feeding for one day, followed by another round of sampling. All samples were stored at –80°C until total RNA extraction after rinsed with PBS.

3. Tissue samples from Pacific abalones subjected to heat stress

Three-year-old Pacific abalones (n = 60) were randomly collected from sea cages in Jindo-gun, South Korea, and transported to an abalone hatchery in Dolsan, Yeosu and acclimatized for a week. After acclimation, abalones were divided into three groups: a control group (20 °C), a 25 °C group and a 30 °C Group. A total of 25 abalones were assigned to each treatment group and placed in separate tanks for 24 hours. Following this, the water temperatures in the 25 °C and 30 °C treatment tanks was gradually increase at a rate of 2 °C per hour. After reaching desired temperature, muscle tissue samples (n=3) were collected from abalones from each treatment group and the control group at 1, 6, 12, 24, and 48 hours. All samples were flash-frozen in liquid nitrogen, and stored at –80 °C for subsequent total RNA extraction.

4. Extraction of total RNA and cDNA synthesis

Total RNA was extracted from all sampled tissues using the ISOSPIN Cell & Tissue RNA Kit (Nippon Gene, Tokyo, Japan), following the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μL of total RNA using the SuperScript III First-Strand cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA), according to the manufacturer’s protocol.

5. Primer design and qRT-PCR analysis

Primers were designed based on the gene sequences of Hdh-MLGF and Hdh-β-actin available in the NCBI database (Table 1). qRT-PCR was performed by using high performance LightCycler^®96 machine in a 10 μL reaction mixture containing 1μL cDNA, 4μL SyGreen Mix (PCR Biosystem Inc., Wayne, USA), 2μL forward and reverse primer (Macrogen, Korea) mix, and 3 μL DDW (Nippon Gene, Japan). Each sample was analyzed in triplicate. The qRT-PCR amplification conditions were as follows: initial denaturation at 95 °C for 2 min., followed by 40 cycles of three-step amplification at 95 °C for 30 sec., 60 °C for 20 sec., and 72 °C for 30 sec. Relative mRNA expression levels were determined using the Hdh-β-actin gene as a reference gene, as described previously (Hanif et al., 2022).

6. Statistical analysis

The mRNA expression values were analyzed and reported as the mean ± standard error. Differences in mRNA expression levels were assessed using Analysis of Variance (ANOVA) in GraphPad Prism software (version 9.3.1). Statistical significance was set at p < 0.05, p < 0.01 and p < 0.001.

Results

1. Growth-specific expression pattern of Hdh-MLGF in Pacific abalone

The Hdh-MLGF mRNA expression increased in correlation with growth rate, from SG to RG. Specifically, abalone with RG exhibited significantly higher expression levels (p < 0.001), while those with SG showed the lowest expression levels (Fig. 1). Although the differences between MG and AG groups was not statistically significant (p > 0.05), expression level were higher in the AG group compared to MG group abalone.

2. Expression of Hdh-MLGF during starvation and refeeding in Pacific abalone

In the control group, no significant changes in Hdh-MLGF mRNA expression were observed throughout the experimental period. However, significant differences in Hdh-MLGF mRNA expression were detected in Pacific abalone subjected to starvation-induced nutritional stress, except during the first and second weeks. Expression levels gradually declined over the three-week starvation period, reaching a minimum in the third week (Fig. 2). Upon refeeding, however, Hdh-MLGF expression increased significantly (p < 0.001) compared to the starvation phase (Fig. 2). Throughout the study, Hdh-MLGF mRNA expression in the starvation group was consistently lower than in the control group, but it increased markedly after refeeding (Fig. 2).

3. Thermal stress influences Hdh-MLGF expression in Pacific abalone

Under thermal stress, Hdh-MLGF was varied throughout the experimental period. At 25 °C, expression significant increased (p < 0.001), peaking at 12 hours before gradually retuning to control levels at 24 and 48 hours (Fig. 3A). At 30 °C, expression peaked at 6 hours (p < 0.001) but declined sharply afterward, nearing zero by 48 hours (Fig. 3B).

Discussion

The present study examined the growth-specific expression of the Hdh-MLGF gene and its regulatory factors in Pacific abalone. Previously, Hdh-MLGF was identified as a secreted protein with both growth factor and adenosine deaminase (ADA) activity, playing a role in metamorphosis in Pacific abalone (Hanif et al., 2022). Secreted growth factors generally regulate growth functions via signal transduction pathways. Although the specific pathway for Hdh-MLGF remains unidentified, molecular docking suggests a potential interaction with a G-protein-coupled receptor (GPCR). As Hdh-MLGF contains an ADA domain and GPCRs are known adenosine receptors (Moreno et al., 2018), it is likely that Hdh-MLGF influences growth by promoting cell proliferation through a GPCR-mediated signaling pathway (Kumari et al., 2021).

Growth-specific expression analysis revealed that Hdh-MLGF is significantly expressed in rapid-growing abalone compared to other growth types, indicating its role in abalone growth performance. This growth process may occur through cell proliferation or by directly promoting the growth of specific cell types via the GPCR signaling pathway (New and Wong, 2007). However, the precise mechanism of its growth factor activity remains unknown.

Starvation or low food availability is a common environmental challenge for marine invertebrates, affecting their physiological state and tissue quality (Xie et al., 2022). In this study, we observed significant changes in gene expression in Pacific abalone subjected to long-term starvation (three weeks). Similarly, starved and refed Haliotis asinina exhibited slower growth rates after repeated starvation and refeeding cycles (Fermin, 2002). In disk abalone, starvation leads to glycogen and triglyceride depletion within 30 days, with increased mortality observed after 40 days (Takami et al., 1995). Previous studies have reported that hdhHGAP (haliotid growth-associated peptide) and hdhSMP5 (shell matrix protein 5) are downregulated under starvation in Pacific abalone. However, the exact mechanism underlying this reduction in expression remains unknown, warranting further investigation.

As poikilothermic animals, abalone growth and development is closely linked to temperature (Mzozo et al., 2021). This study found that Hdh-MLGF expression upregulated after thermal stress at 25 °C until 12 hours, and then stabilized. Since the optimal growth temperature for Pacific abalone is 18–22 °C, slight deviations had little effect on gene expressions. Pacific abalone can tolerate temperatures up to 28 °C, whereas 30 °C is considered a chronic stress temperature (Lee et al., 2023). However, expression of Hdh-MLGF declined at 30 °C indicating reduced activity under chronic heat stress. While moderate heat stress can upregulate some factors like nerve growth factor inducible (VGF) (Thompson et al., 2018). Heat stress has been reported to impact signaling pathways and the biological activity of epidermal growth factor (EGF) (Wang et al., 2022). In contrast, insulin-like growth factor-1 (IGF-1) has been shown to increase even under chronic heat stress (Xin et al., 2018), possibly due to elevated insulin levels resulting from glycogen degradation induced by heat stress..

In conclusion, the expression analysis in this study suggests that Hdh-MLGF regulates the growth of Pacific abalone, most likely through the GPCR signaling pathway. However, fluctuation in environmental condition such as temperature and nutritional deficiency (starvation) can alter the expression of this growth factor, potentially hindering the normal growth performance of Pacific abalone in aquaculture systems.