Relative expression levels of growth hormone gene and growth rate in Indian major carp species

The phenomenon of growth is a leading factor for aquaculture success. The uneven growth of major Indian carps ( Labeo rohita, Catla catla, and Cirrhinus mrigala ) is a serious issue in fish culture from an economic point of view. The growth hormone (GH) gene is crucial for selection in commercially cultivated fish species for better growth and production. Indian major carp ( L. rohita, C. catla, and C. mrigala ) are commonly cultured in Pa-kistan. The GH expression was examined using qPCR to understand growth in fish species better. Muscle tissue samples ( n =480) from 160 individuals of the same age were collected from three species ( L. rohita, C. catla, and C. mrigala ). Individuals were divided into two groups (high-weight and low-weight groups), cultured under normal conditions. The housekeeping gene β-actin validated GH expression in fast and slow-growing fishes from the same species. Results showed that GH expression varies across species and fish specimens that over-weight their counterpart feature have higher GH expression. A selection for overweight fish in the aquaculture breeding systems is preferable as those fish could inherit their genomics to the future cohort, enhancing production, and commercial profit for farmers. Comprehensive research about different growth genes and the environmental aspects that influence fish growth is mandatory. No work has been reported regarding the growth gene analysis of fish from Pakistan. This report was Pakistan’s first and baseline study regarding growth analysis of main culturable fish species at the molecular level.


INTRODUCTION
Pakistan is blessed with fish fauna and water resources, but only nine species (7 warm and 2 cold water) are commercially cultured.Moreover, only carp culture is carried out through extensive farming depending on natural water resources.Among polyculture, carp poly-culture systems primarily consisting of major carps (Labeo rohita, Catla catla, and Cirrhinus mrigala) are more prevalent due to their high growth rate and ability to adapt in captive conditions (FAO, 2016;Wasim, 2007).Aquaculture success is mainly dependent on the maximum growth and production of fish.Vertebrate growth is regulated by several genes and various morphological characters (Dunham, 2011).The growth expression is correlated with a screening of candidate genes involved in the growth and selection of fish species.Higher fish growth is essential for boosting the aquaculture industry (Khan et al., 2023;Canestro et al., 2013).The role of the GH is extensively studied in many fishes like Mandarin fish (Siniperca chuatsi; Yi et al., 2015), Nile tilapia (Tilapia nilotica; Tanamati et al., 2015), Tench (Tinca tinca; Kocour et al., 2011), Ganges Jewfish (Nibea coibor; Zhang et al., 2009), and Mandarin fish (Siniperca chuatsi; Tian et al., 2014).In recent years, marker-assisted growth selection has been employed in the aquaculture and livestock sector for better performance (Ling et al., 2023;Biswas et al., 2003;Pal et al., 2004;De Faria et al., 2006).
Fish growth is a key feature for many fish biologists regarding fish production and the selective breeding program of many economically significant fish species (Gjedrem et al., 2012;Hulata, 2001).Growth is a quantitative trait controlled by many genes (Mullis, 2005) and environmental factors (Mackay, 2001;Moriyama et al., 2000).The GH, growth hormone receptor (GHR), insulin-like growth factors (IGF), and myostatin (MSTN) genes are observed in tamed animals and aquaculture species.Among them, GH is the main regulator of postnatal development.Moreover, it is also associated with strengthening anabolic functions such as protein interaction, cell growth, and muscle development (Reinecke et al., 2005).In addition to hormone-flagging procedures, it participates in other pathways that make its physiology challenging to understand (Vairamani et al., 2017).The primary genes that control fish growth have been studied using various experimental methods.Research on such fish ranges from those whose growth has been retarded through starvation during gametogenesis to those whose growth has been accelerated through refeeding after starvation, GH-transgenesis, or domestication.These studies also discovered a complicated association among factors that control growth and other biological processes.Moreover, it is also revealed that many somatotropic genes such as GH, GHR, and insulin-like growth factors IGFs are more expressed in fast-growing fishes, i.e., rainbow trout (Oncorynchus mykiss) (Devlin et al., 2013) and regulate muscle growth (Sheridan & Hagemeister, 2010).
GH is secreted by somatotropic cells and comprises a single-chain polypeptide of about 22kDa.It plays a vital role in metabolic processes, i.e., reproductive and immune systems and seawater adaptability.Moreover, it is also involved in problems related to appetite and food conversion (Rajesh & Majumdar, 2007;Canosa et al., 2007).Previously DNA and cDNA sequences of the GH have been well studied in various economically important fish species like Cyprinus carpio (Chiou et al., 1990), Protopterus annectens (May et al., 1999), Cynoglossus semilaevis (Ma et al., 2012), and Pelteobagrus fulvidraco (Li et al., 2017).The total length of the GH was almost 3-4kb and its structures differed in different fishes.The GH genome in some fishes consists of 5 exons and 4 introns; in other fish species, it was 6 exons and 5 introns (Chiou et al., 1990;Li et al., 2017).The current study was undertaken to determine the differential expression levels of the GH in fast and slow-growing fish species of Indian major carps (Labeo rohita, Catla catla, and Cirrhinus mrigala), which are commonly cultured in Pakistan.

Sample Collection for Growth Expression Analysis
A total of 480 fish individuals (Labeo rohita/n=160, Cirrhinus mrigala/n=160, and Catla catla/n=160) of similar age were collected from the Umer Fish Farm, located at District Rajanpur, Pakistan.The capturing and experiments on fish species were conducted following relevant institutional, national, and international guidelines and legislation.Out of 160 muscle tissue samples (Skeletal Muscle, 5 g) from each fish species, 80 samples were captured from extremely high-weight fishes (≥1.3; ≥2 and ≥0.9 Kg) and 80 from extremely low-weight fishes (≤0.5; ≤1 and ≤0.5 kg) from L. rohita, C. catla, and C. mrigala, respectively.Fish samples were collected in a centrifuge tube (1.5 ml), containing RNA later solution.Samples were stored immediately in liquid nitrogen and then at -80°C for later analysis.During sampling, feeding and water quality data were continuously collected (Table 1).

Total RNA isolation and cDNA synthesis
Total RNA isolation from the muscle tissue sample was carried out using TRIzol reagent (Life Technologies cat no.15596026).Samples were ground in liquid nitrogen using a pre-chilled mortar and pestle.The homogenate of muscle tissue samples was made in a 2.0 ml microcentrifuge tube with 1.5 mL of TRIzol ® reagent and incubated for 5 minutes at room temperature.After shaking strongly for 30-40 seconds, 200 μl of chloroform was added per 1 ml of TRIzol ® reagent and the content was mixed vigorously for 15 seconds and incubated at room temperature for 10 minutes followed by centrifugation for 5 minutes at 13 000 rpm (at 4°C).As a result of centrifugation, three distinctive layers were formed consisting of the upper colorless, middle whitish layer and bottom dark layer.The upper clear phase containing nucleic acid was transferred to another nuclease-free 2.0 ml Eppendorf tube and 750 μl of pre-chilled isopropanol was added to precipitate the RNA and incubated for 10 minutes at room temperature followed by centrifugation.To clean the RNA pellet, 750 μl of 75% ethanol was used and centrifuged again at 13 000 rpm (at 4°C) for 15 minutes (2×) by discarding the upper content.The RNA pellet was airdried by incubating at room temperature for 10 minutes.The air-dried RNA samples were resuspended in 30 μl DEPC water and immediately stored at -70°C.The quality and quantity of the isolated RNA were determined by using Spectrophotometer ND 1000 Nan-oDrop ®. and the Size of RNA was determined by 1.5 % gel electrophoresis (Fig. 1).

Synthesis of complementary DNA (cDNA)
The cDNA was synthesized using the Revert Aid First-Strand cDNA synthesis kit (Thermo Fisher Sci-

Primer Designing and Optimization
Primer Express 3.0 software was used to design primers for the GH and housekeeping genes (β-actin).The β-actin was selected as an internal control for precise validation of qPCR (Table 2).Both primers (GH and β-actin) were optimized using certain qPCR conditions.The results of the optimized protocol were assessed by the gel documentation system (Fig. 3).

Amplification of targeted genes by reverse transcriptase Polymerase Chain Reaction (RT-PCR)
Real-time PCR was used to quantify differential GH expression of fishes with the highest and lowest weight under uniform pond conditions.The RT-PCR process was accomplished using PikoReal™ Q-PCR to quantify selected samples.Amplification of the targeted genes was carried out using gene-specific primers (Table 2) and DreamTaq Green PCR Master mix 2x (Thermo Fisher Scientific, cat no.K1081).The PCR reaction contained 10 μ-L of DreamTaq Green PCR master mix, 0.8 μ-L template cDNA and 0.4 μ-L of both forward and reverse primers (10 μ-mol) added to PCR tube (0.2 mL) and nuclease-free water to a final volume of 20 μ L. The PCR thermal conditions were; preliminary incubation for 4 minutes at 94°C, 30 cycles of amplifications with denaturation at 94°C for 45 seconds, primer binding at 56-58°C for 30 seconds and amplification at 72°C for 40 seconds and then ending extension for 5 minutes at 72°C (Fig. 4).The 2-ΔΔCT mode was selected to assess GH expression (Livak & Schmittgen, 2001).

Data analysis
Data were coded and entered using the statistical package SPSS (IBM SPSS Statistics V.22).The data were checked for satisfying parametric test assumptions, and continuous variables were tested for normality using Shapiro-Wilk and Kolmogorov-Smirnov tests.Data were presented as mean and standard deviation.A student t-test was conducted for experimental groups, while a Tukey pairwise comparison was conducted in MiniTab V14 for post-hoc analysis.The P-value was considered significant at<0.05.Data was visualized when possible, using R studio V 2022.02.4.

RESULTS
In this study, we observed significant differences in body weights between high-weight and low-weight groups of similar ages in all three fish species (Fig. 5).

Labeo rohita
Average values of GH expression and body weight of selected fish samples were utilized to determine the relationship between GH expression and body weight.Larger body weight fishes (1.88±0.50kg) have significantly higher GH expression relative to lower body weight fishes (0.83±0.38 kg) (Fig. 6).
We found a very surprising correlation between GH expression and the body weight of selected fish species (Fig. 7).The analysis revealed a statistically significant positive correlation between body weight and gene expression for both groups of low and high body weight fish.The strength of association was greater in the highweight groups compared to the low-weight groups for all three species.For L. rohita, the correlation coefficient was 0.965 and 0.729 for the high and low-weight groups, respectively.Similarly, for C. catla the correlation coefficients were 0.744 and 0.696, and for C. mrigala they were 0.871 and 0.677 for the high and low-weight groups, respectively.In summary, the degree of correlation between body size and gene expression intensified as body weight increased for all species.
The analysis revealed a statistically significant positive correlation between body weight and gene expression for both groups of fish with low and high body weight.The strength of association was greater in the highweight groups compared to the low-weight groups for all three species.For L. rohita, the correlation coefficient was 0.965 and 0.729 for the high and low weight groups, respectively.Similarly, for C. catla the correlation coefficients were 0.744 and 0.696, and for C. mrigala they were 0.871 and 0.677 for the high and low weight groups, respectively.In summary, the degree of correlation between body size and gene expression intensified as body weight increased for all species.

DISCUSSION
Growth in vertebrates is mainly controlled by GH, a vital gene influencing growth physiology (Quik et al., 2010;Clayton et al., 2010).Indian major carp species (Labeo rohita, Catla catla, Cirrhinus mrigala) are widely cultured in Asia.GH might directly or indirectly affect the growth mechanism of these economically important fish species.We therefore investigated the connection of GH expression with the growth characteristics of carp species.In recent years, GH association with growth traits  has been studied well in domestic animals (An et al., 2011;Sugita et al., 2014) and poultry (Xu et al., 2007;Wu et al., 2012).The GH is the chief controller of IGF-I production in fry, fingerling, and adult fishes.Studies reported that injecting GH boosts the expression of IGF-I in salmon, (Duguay et al., 1994), rainbow trout (Shamblott et al., 1995), and gilthead sea bream (Duguay et al., 1996), zebrafish (Maures et al., 2002).The IGF-I mRNA levels in plasma or serum and its expression in tissues also confirmed its dependency on GH as observed in goldfish (Marchant & Moroz, 1993), salmonids (Moriyama et al., 1995) and tilapia (Kajimura et al., 2001).
We reported that GH expression varies in high and low-body-weight fish species of Indian major carp.The variable expression of growth-related genes has also been observed in fish and other animals in previous scientific studies (Carnevali et al., 2006;Filby & Tyler, 2007;Opazo et al., 2017).In fish, somatic growth is accelerated at the juvenile stage while it slows down during the adult stage as fish utilizes its energy for gonadal development and sexual maturation (Le Gac et al., 1993;Chen et al., 2018).In the present study, fish with the same age class and different body weights exhibit high GH expression as fish weight increases.Differences in recorded body weight of fish within the same age group could rely on stress factors, that can minimise fish growth and production.This stress may be caused due to internal factors (stocking density, fish species' competition for space and food) or external factors (low water quality) (Karakatsouli et al., 2008).If the stress factor is negated, then the role of the gene can be observed and explained well in experimental fishes, which enlightens the present study findings.Considering this scenario, present cultured fishes undergo uniform culturing conditions by following good feeding practices and healthy water quality management so that possible genetic effects on fish growth can be assessed effectively.The differential expression level of GH in three fish species defined that fish growth rates are different concerning GH content in muscle tissue.Similar observations were recorded in red spotted grouper (Epinephelus akaara) regarding expression analysis of growth genes like GH, GHR, and IGF1 from fish tissue (Mun et al., 2019).
A direct relationship was noticed between body weight and GH expression in L. rohita, which depicts that expression of the GH increases or decreases with the gradual increase or decrease in body weight of fish samples.GH-mRNA expression was found higher in a high-weight group while lower in low-weight populations, similar to those mentioned for crucian carp C. auratus (Zhong et al., 2012); European sea bass (Dicentrarchus labrax), which had a significantly higher GH-mRNA expression in fast-growing as compared to slow-growing fish samples (Carnevali et al., 2006).However, such differential GH-mRNA expression level was not obvious in zebrafish, Daniorerio (Opazo et al., 2017).Similar to L. rohita, we found the same findings for C. catla.Results of GH expression concerning the body weight of C. catla fish samples were very interesting as we found the same high per unit expression of GH with respect to a gradual increase in weight in higher body weight fishes.Such kind of growth pattern may reflect some ongoing interconnected morphological as well as physiological changes inside the body of C. catla.In contrast, a low level of GH expression was reported in high-body weight fish of Nile Tilapia (Oreochromis niloticus), which may be compensated by expression in some other tissues of the fish body (Aboukila et al., 2021).The low level of GH expression may depict its low synthesis from the pituitary due to the influence of endocrine and environmental aspects correlated with the body physiology, suggesting that GH expression in fish is a very complicated phenomenon (Tymchuk et al., 2009).
A direct correlation was also identified between body weight and GH expression in collected C. mrigala.Such results indicated a higher chance of reciprocal effect of GH expression in low-body-weight fish than in the highbody-weight group.A previous study on GH expression analysis of red-spotted grouper revealed that GH-mRNA expression was slightly higher in higher body weight samples than in moderate and slow-growing samples (Mun et al., 2019).Likewise, in C. mrigala GH expression results, a marine water fish named gilthead sea bream (Sparus aurata) possessed similar influential growth patterns of GH on high and low-weight fish tissues (Perez-Sanchez et al., 1994).Comparing GH expression among high and low body weight Indian major carp species revealed that GH expression varies from species to species in these fishes, however, it follows a similar trend with increasing or decreasing body weight.Certain other vital growth genes and various ambient environmental factors important for fish's body development and growth may cause current variation in expression results.The intricate phenomenon of the endocrine system in fishes reveals that the growth pattern with respect to hormonal state is very complex.This pattern gradually fluctuates among fresh and marine water fish species.More research in this area is necessary to properly understand growth governing endocrinology in fish.

CONCLUSION
Our research focused on Labeo rohita, Catla catla, and Cirrhinus mrigala, which are major Indian carp species commonly cultured in Pakistan.The uneven growth of these species is a significant challenge in aquaculture, impacting economic outcomes.The growth hormone (GH) gene is pivotal in selecting fish with superior growth and production potential in aquaculture.By validating GH expression using the housekeeping gene β-actin, we discovered that GH expression varies not only among species but also within the same species based on weight, with better-weight fish exhibiting higher GH expression levels.The significance of our findings lies in their potential application within the aquaculture sector in Pakistan.Selecting and breeding better-weight fish, which demonstrate higher GH expression can lead to improved production outcomes and increased profitability for farmers.Furthermore, this study represents the first molecular-level analysis of growth genes in fish species from Pakistan, making it a baseline study for future research in this region.

Declarations
Ethical Approval.It has been confirmed that the experimental sample, including the collection of fish blood samples, complied with relevant institutional, national, and international guidelines and legislation with appropriate approval from the Ethical Review Committee of the Department of Zoology, Government College University Lahore, Pakistan (Approval ID: Fish2570).It has been confirmed that the study was reported following the Guidelines for the Use of Fishes in Research (AFS 2014).
Competing interests.There are no competing interests regarding the publication of this manuscript.
Funding.Currently, no funding is available.

Figure 2 .
Figure 2. Thermal conditions for cDNA synthesis

Figure
Figure 4. RT-PCR Amplification thermal conditions to amplify targeted genes

Table 1 . Feeding and water quality monitoring data during tissue sampling.
*DO, dissolved oxygen; *TDS, total dissolved solid