Insights in the Size Heterogeneity of Lates calcarifer: Expression of Growth and Immune-related Genes

1Tropical Aquaculture Research and Development Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Sanya 572018, P.R. China 2Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou, 510300, P.R. China 3Sanya Tropical Fisheries Research Institute, Sanya 572018, P.R. China 4Ocean College, Hainan University, Haikou 570228, PR China Article Information Received 28 March 2020 Revised 18 May 2020 Accepted 30 May 2020 Available online 18 December 2020


INTRODUCTION
S ize heterogeneity exists in many species within a batch in the growing period. This phenomenon can be caused by multiple factors including internal and external factors (Geffen, 2002). Intrinsic factors include genotypic or phenotypic variation, including genetic factors and external causes include various biological factors and abiotic factors such as temperature, food, and stocking density (Baras et al., 2011;Conner and White, 1999;Sukumaran et al., 2011;Umanah and Nlewadim, 2019). The formation of general individual size heterogeneity comes from the interaction of internal and external factors. In a more intuitive way, size heterogeneity facilitate cannibalism, but It's also affected by cannibalism, since the smallest fish are consumed by the largest ones (Kestemont et al., 2003).
The existence of size heterogeneity contributed to the natural selection within the population (Conner and White, 1999). Cannibalism eliminated the individuals who were stunted by the above reasons of internal and external factors in the early stage of development, making the whole population develop away from recession and improving the survival ability of them (Baras et al., 2013;Ribeiro and Qin, 2016).
After animals are affected by some external factors, the internal environment will change accordingly, thus affecting the expression level of some genes (Gottlieb, 1998). Growth hormone/insulin-like growth factor (GH/ IGF) system is the key promoter to regulate growth in vertebrates. In fish, the GH induces muscle growth by modulating the expression several of genes belonging to the GH, IGF systems, myostatin (MSTN) and atrophy (Fuentes et al., 2013). These genes have been shown to be closely related to growth in many fish (Gahr et al., 2008;Peterson et al., 2004;Picha et al., 2008), but do they contribute to size heterogeneity.
The immune system of fish has synergistic effects of O n l i n e F i r s t A r t i c l e innate immunity and acquired immunity, but the innate immunity is relatively developed. The innate immune system includes lysozyme, complement system and some cytokines (Kiron, 2012). The mechanistic target of rapamycin (mTOR) signaling pathway can regulate growth and homeostasis in an organism by perceiving and integrating multiple environmental cues (Laplante and Sabatini, 2012). Heat shock proteins (HSPs) are a group of stress proteins which can be triggered by environmental stress, such as starvation, hypoxia, water deprivation and microbial infection (Roberts et al., 2010). In raising barramundi juveniles, we found that comparing to their small siblings, larger fish from the same batch were likely to die sudden death when external factors changed. The resons are still unknown. In order to explore the causes of size heterogeneity and sudden death in juvenile fish, we explored the expression of growth and immune-related genes in three sizes of fish and inorder to correlate size heterogeneity and gene expression levels. Results from the present study will provide the background information on the size heterogeneity in artificial cultured fish.

Incubation of fertilized eggs and larval rearing
Fertilized eggs from the same batch of barramundi broodstocks were obtained from the Tropical Fisheries Research and Development Center, Lingshui Town. Incubation of fertilized egg and for rearing of fish methods of Liu et al. (2018) were followed after entering the juvenile stage, all fish were transferred to 2000 L indoor tanks with the seawater recirculating aquaculture system. Stocking density was maintained at 0.2 fish L −1 . Fish were fed commercial diet twice a day at 0800 and 1600 h. Feces were cleaned up an h after each feeding. During the rearing period, the water parameters were measured daily and were maintained as ammonia nitrogen <0.1 mg L −1 , nitrite nitrogen <0.02 mg L −1 , pH 7.8, and dissolved oxygen >7.0 mg L −1 .

Grading and sampling
Ten weeks after rearing in hatchery, about 3000 juvenile fish were graded according to body length. In order to magnify the difference, the 0.15% fish were separated of the shortest and longest body lengths, respectively, and defined them as S grade and L grade, while the rest were M grade (Fig. 1). Nine fish were randomly sampled from each grade (body length and body weight in Table I). The fish were anaesthetized with 7 mg L −1 eugenol (Shangchi Dental Material Co., Ltd., Changshu, China) before handling and sampling. The tissues and organs sampled were brain, muscle, liver and kidney, which were snap-frozen in liquid nitrogen and then preserved in -80 o C until use.  The experimental procedure was complied with the standards of Institutional Animal Care and Use Committee guidelines (Suckow and Stewart, 2016). All experiments were conducted in line with the principles and guidelines for the care and use of live fish and the guidelines for animal experimentation approved by the Animal Experimental Council (AEC/NRIFS) of the National Research Institute of Fisheries Science, Fisheries Research Agency.

RNA isolation
The harvested frozen tissues (the brain, muscle, liver and kidney) were homogenized in 1 mL Trizol (Invitrogen and Greenprima Instrumenta Co., Ltd., UK), and RNA was separated in the chloroform layer and precipitated by isopropanol. The RNA pellet was washed in 1-mL 75% ethanol, and air-dried before resuspension in RNase-free water (Fu et al., 2019). The quantity of isolated RNA was later determined by measuring their absorbance at 260 and 280 nm using a ND 5000 spectrophotometer (BioTeke Corporation, China). Finally, the integrity of RNA was assessed using agarose gel (1%) electrophoresis.

Genomic DNA removal and cDNA synthesis
The RNA was immediately used for cDNA synthesis. Subsequently, genomic DNA removal and reverse transcription was performed on 1 μg of total RNA using TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix, that were determined according to the manufacturer's instructions using commercial kits (Transgen Biotech Co., Ltd., China). The synthesized cDNA samples were stored at -20 o C until further use.

Calculations and statistical analysis
The ΔΔ Ct method was used to calculate the relative expression with β-actin as a reference gene, and normalized to control (Green and Sambrook, 2012). The data were expressed as the mean ± standard deviation (SD). Statistical analyses were carried out by PASW Statistics (version 18). Comparisons between different groups were conducted by one-way ANOVA and LSD test, and significant difference was set at P < 0.05.

The expression of growth-related genes
The GHRH expression in the S grade and the GH gene expression level in the M grade was significantly upregulated ( Fig. 2A, 2B, P < 0.05). There were significantly different in expression levels in three grades of GHR, IGF-I, IGF-II and MSTN-1 (P < 0.05), while the expression levels were upregulated with the increasing of body length (Fig. 2C, D, E, F). The MSTN-2 expression was significantly affected by the size grade (P < 0.05), and the expression upregulated in L grade and downregulated in M grade (Fig. 2G).

The expression of immune-related genes
In the mechanistic target of rapamycin (mTOR) signaling pathway genes of liver, the mTOR expression in the S grade and the L grade were significantly upregulated compared to the M grade (P < 0.05), and the expression level in L grade was significantly higher than those in S grade (Fig. 3A). The elf4e expression in the L grade was significantly up-regulated compared to the M grade (P < 0.05), while the S grade and the M grade showed no significant difference (Fig. 3B, P > 0.05). The mLST8 expression in the S grade and the L grade were significantly down-regulated compared to the M grade (P < 0.05, Fig. 3C).
In heat shock protein genes, the HSP70 expression in the S and L grade HSP70 were significantly downregulated compared to the M grade (P < 0.05), and the lowest expression level was observed in L grade (Fig.  3D). The HSP90 expression in the S and L grade was significantly up-regulated compared to the M grade (P < 0.05), and the highest expression level was observed in the Le (Fig. 3E).
In cytokine genes, the expression of TNF was significantly up-regulated in the L grade compared with the other two grades (P < 0.05), and there was no significant difference between the S and M grade (Fig. 3F, P > 0.05). In the expression of IFN-γ, the S grade was significantly down-regulated compared to the other two grades (P < 0.05), while there was no significant difference between the M and L grade (Fig. 3G, P > 0.05). The S grade in IL8 and IL10 was significantly up-regulated compared to the other two groups, and the highest expression of IL8 was observed in L grade (Fig. 3H, I, P < 0.05). In the expression of TGF, both the S and L grade were significantly downregulated compared to the M grade (P < 0.05), and the lowest expression was observed in L grade (Fig. 3J).
In inflammatory marker genes of liver, CRP expression level was significantly up-regulated by body length (Fig. 3K, P < 0.05). In complement system genes, the C3 and C4 expression in the L grade were significantly up-regulated compared to the other two groups, and the S grade was significantly up-regulated compared to the M grade in C3 (Fig. 3L, M, P < 0.05).

DISCUSSION
In teleost fishes, as in other vertebrates, growth is under the control of the growth hormone/insulin-like growth factor (GH/IGF) system, as well as insulin and other endocrine and local factors (Pierce et al., 2005;Rolland et al., 2015). In this study, the downstream genes of the growth axis, such as GHR, IGF-I and IGF-II, showed a significant increasing trend with the increase of body length. The expression of IGF-I and IGF-II genes is positively correlated with growth performance, that was observed in previous studies of clownfish (Amphiprion ocellaris) (Avella et al., 2009) and golden pompano (Trachinotus ovatus) (Tan et al., 2017). GH plays its role of biological actions attributed to the production of IGF-I when bound with GHR (de Azevedo Figueiredo et al., 2007). Although GH is a universal growth regulator, its regulation of growth rate is delayed and has negative feedback regulation with IGF-I (Gabillard et al., 2006). These reasons may be explained why GH did not show regular changes with body O n l i n e

F i r s t A r t i c l e
Insights in the Size Heterogeneity of Lates calcarifer 7 length grade in this study. GHRH is specially interacted with the GHRH receptor (GHRHR) to have contributed to stimulating GH synthesis and releasing from anterior pituitary cells (Nam et al., 2011). In this study, there was no consistency between the expressions of GHRH and GH. The neuroendocrine regulation of GH secretion in fish has been recognized as multifactorial, with a balance between stimulatory and inhibitory factors acting on the somatotrope (Canosa et al., 2007). Further work may need to be conducted to fully assess the control of GH expression in different body length grades of barramundi.
Myostatin is an identified member of the transforming growth factor-β (TGF-β) superfamily (Østbye et al., 2001). In teleost fishes, MSTN-1 appears to primarily inhibit muscle hyperplasia, but perhaps not hypertrophy, and the down-regulation of MSTN-2 is the main cause of grown through muscle hypertrophy (de Santis et al., 2012). However, there was no inhibition of growth at the expression level in this study, suggesting that the barramundi may not have entered a period of rapid muscle hyperplasia at this stage (Johnston, 1999).
The mechanistic target of rapamycin (mTOR) signaling pathway can sense and integrate a variety of environmental cues to affect energy metabolism protein synthesis and lipid synthesis, thus regulating homeostasis and growth in an organism (Laplante and Sabatini, 2012). The mTOR is known as an atypical serine/threonine protein kinase and has the ability of forming two distinct complexes named mTOR complex 1 (mTORC1) and 2 (mTORC2) by interacting with several proteins. The mTORC1 directly phosphorylates eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and S6 kinase 1, the translational regulators, which, in turn, promote protein synthesis (Ma and Blenis, 2009). The mLST8 is involved in both mTORC1 and mTORC2, and it is necessary for the mTORC2, but its activity shortage does not affect the activity of mTORC1 (Jacinto et al., 2004;Kim et al., 2003). In this study, mTOR pathway related genes were relatively active in L grade, and there was no abnormal up-regulated expression, indicating that L grade was relatively active in protein translation, metabolism and other aspects.
In aquatic animals, HSPs associated with the host response to environmental pollutants and food toxins, particular in the specific and non-specific immune responses to bacterial and viral infections and the development of inflammation, so they have been shown to play a significant role in health (Basu et al., 2002). HSPs was less expressed in the absence of environmental stimulation (Roberts et al., 2010). In this study, there was no consistent rule between the body length levels of HSP70 and HSP90, which may be the expression level fluctuated in a low range or that was not correlated with body length.
Pro-inflammatory cytokines and anti-inflammatory cytokine balance is maintained between the body's normal immune status, disease resistance, its stability and normal physiological activities of the key factors, inflammatory cytokines to activate the body is mainly responsible for the pathogens, congenital and acquired immune system, to destroy the intruder, and anti-inflammatory cytokine mainly was eliminated after the intruder to eliminate inflammation, make the body return to normal immune and physiological level (Bird et al., 2006;Secombes et al., 2001). In this study, pro-inflammatory cytokines (TNF and IL8) were up-regulated, but anti-inflammatory cytokine (TGFβ1) was significantly down-regulated in L grade. This is not conducive to balance between cytokines, the risk of inflammatory disease would be increased.
The CRP is an important inflammatory marker and it has a pleiotropic effect. Both "pro-inflammatory" and "anti-inflammatory" activities have been described (Black et al., 2004). The CRP is capable of activating complement, opsonizing bacteria, fungi and parasites and agglutinating particles (Ballou and Lozanski, 1992). Complement proteins C3 and C4 are also classified as acute phase reactants as their synthesis is upregulated during inflammation (Watts et al., 2001). In this study, the expression levels of CRP, complement gene C3 and C4 in the L grade were uniformly up-regulated, it further confirm the existence of inflammatory response in grade L, which may be the reason for its reduced tolerance to the environment CONCLUSIONS Collectively, our gene expression analyses suggest that the expression level of GHR, IGF-I and IGF-II is consistent with body length, but there is no similar rule between GHRH, GH, MSTN-1 and MSTN-2. TOR pathway related genes were more actively expressed in L grade. Heat shock protein has no obvious rule in body length grade. L grade pro-inflammatory cytokines were up-regulated, but anti-inflammatory cytokines were down-regulated and inflammatory markers were up-regulated, indicating an obvious inflammatory response. The expression levels of S and M grade anti-inflammatory and proinflammatory cytokines was more balanced. Our results would pave the way to further understand of the relationship between size heterogeneity and gene expression in barramundi juveniles.

Statement of conflict of interest
The authors have declared no conflict of interest.

O n l i n e F i r s t A r t i c l e
Insights in the Size Heterogeneity of Lates calcarifer O n l i n e