Characterization and expression of the gene glucose transporter 2 (GLUT2) in embryonic, larval and adult Bay snook Petenia splendida (Cichliformes: Cichlidae)

Bay snook ( Petenia splendida ) is a carnivorous cichlid species with excellent economic value in Southeast Mexico. Although this species presents an excellent potential for commercial aquaculture, the information about its nutritional, physiological, and reproductive metabolic pathways is meager. The current study focuses on the expression of glucose transporter 2 ( glut2 ) in embryos and larvae at 5, 10, 15-, 20-, 25-, and 30-days post-hatch (dph) and in the liver, intestine, kidney, muscle, heart, testicle, gill, stomach, pancreas, and brain of adult fish. The partial sequence of glut2 was obtained, and specific qPCR primers were designed. In embryos, the expression was lower compared to larvae at 5, 15, and 20 dph. The highest expression in larvae occurred at 20 dph and the lowest at 25 and 30 dph. Maximum expression levels in adults occurred in the liver and intestine. Our results show that glut2 is expressed differentially across tissues of adult bay snook, and it fluctuates during larval development.

GLUT2 is considered the main isoform of glucose transporters in the liver, plays a role in regulating insulin, and removes excess glucose from the blood (Mueckler, Thorens, 2013). This molecule is involved in different processes, including intestinal and renal glucose absorption, stimulation of insulin secretion in pancreatic cells, and the glucose detection capacity in specific brain regions involved in the regulation of glucose and food metabolism (Castillo et al., 2009;Yan, 2017;Zhao et al., 2020). In fish, glut2 has been detected in different tissues (pancreatic cells, hypothalamus, pancreas, kidney, and liver), and its expression is related to feeding habits and nutrition in species such as zebrafish (Danio rerio), common carp (C. carpio), rainbow trout (Oncorhynchus mykiss), Nile tilapia (O. niloticus), blunt snout bream (M. amblycephala), grass carp (C. idella), and cobia (Rachycentron canadum) (Krasnov et al., 2001;Panserat et al., 2001;Castillo et al., 2009;Polakof et al., 2010;Liu et al., 2014;Liang et al., 2018;Deng et al., 2020;Zhao et al., 2020;Ye et al., 2020). In Atlantic cod (G. morhua), the expression of glut2 during larval development decreased with starvation because of changes in blood glucose (Hall et al., 2006). However, there is no available information regarding glut2 regulation during the larval development of the P. splendida. For this reason, this study examined the expression of glut2 in various organs of P. splendida adults and contributed to understanding the gene's regulation and dynamics during the early ontogeny of this species.
Sampling. After males were euthanized by cold thermal shock (at -4°C) after 24 h of fasting. Fish were dissected in ice to obtain the liver, intestine, kidney, muscle, heart, testicles, gills, stomach, pancreas, and brain. Larvae were sampled on different days after hatching (10 larvae per tank): before first feeding, starting from the embryo (considered as 0 dph), and 5, 10, 15, 20, 25, and 30 dph. Larvae were removed from each tank, rinsed in distilled water, and transferred to Eppendorf tubes with 1.5 mL of RNA Later and stored at -80°C.
RNA extraction and cDNA synthesis. The RNA extraction was performed from tissues and pooled larvae (10) using the Trizol Reagent (Invitrogen, Carlsbad, CA) method under the manufacturer's indications. One microgram of RNA was used for reverse transcription with iScript TM Select cDNA Synthesis Kit 170 -8,896 (BioRad, Hercules, CA). Subsequently, 1 µL of cDNA was used for the end-point Polymerase Chain Reaction (PCR). To obtain the partial sequence of glut2, samples were run in a 96-well thermocycler using the Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA). Amplification was conducted under the following conditions: 10 min at 95°C, followed by 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 50 s with a 5 min extension at 72°C using specific oligonucleotides previously obtained from alignment (using Clustal-W software, Infobiogen) of corresponding sequences available in the library from different species of cichlids including Nile tilapia (O. niloticus ACZ73587.1), Burton's mouthbrooder (Astatotilapia burtoni XP_005926097) and zebra mbuna (M. zebra XP_004540234.1) (Tab. 1). The amplification products were separated in 1.5% agarose gel stained with ethidium bromide. Observed bands under UV light (Biorad® Model Universal Hood II, Hercules, CA) were cut from the gel and purified using the PureLink® PCR Purification Kit (Invitrogen). The purified bands were sent to the Synthesis and Sequencing Unit of the Institute of Biotechnology of the Universidad Nacional Autónoma de México (UNAM) to be sequenced.

Sequence analysis.
Obtained partial sequences were edited and analyzed using ExPASy translation software to search for the open reading frame (ORF). Once the ORF was identified, it was translated to amino acid (AA) sequences using standard genetic codes. The nucleotide sequence was compared with DNA sequences from other fish available in the GenBank database network service at NCBI (https://blast.ncbi. nlm.nih.gov/Blast.cgi). Protein sequence alignments were performed by the multiple sequence alignment software BioEdit 7.2 (www.mbio.ncsu.edu/bioedit/bioedit.html). A phylogenetic tree was generated using neighbor-joining (NJ) methods based on the AA sequence using MEGA 7.0 software.

Real-time polymerase chain reaction (q-PCR).
The resulting cDNA from adult tissues, embryos, and larvae were diluted in 200 μL of distilled water. The quantitative polymerase chain reactions (qPCRs) were performed in a 96-well thermocycler CFX96 Real-Time System Thermal Cycle (Model C1000, CA). The reaction mixture included 10 μL of Eva Green, 2 μL cDNA, and 0.2 μL of each primer (shown in Tab. 1). The thermal program included 2 min at 95°C, followed by 38 cycles at 95°C for 10 s, 60°C for 30 s, and extension at 70°C for 5 s. All reactions were performed by duplicates. The normalization of cDNA 18S rRNA was used as a constitutive gene and carried out in parallel with all samples, according to Wang et al. (2015) and Yang et al. (2013). A standard curve for each pair of primers was generated to estimate amplification efficiencies based on known amounts of cDNA (four serial dilutions corresponding to cDNA transcribed from 100 to 0.1 ng of total RNA). Relative gene expression of tissues and larval growth stages was calculated by the delta-delta copy threshold (CT) method (Pfaffl, 2001).

Statistical analysis.
The relative expression of glut2 between the different tissues of adult P. splendida and the comparison between embryos and the different dph of larvae were analyzed using the Kruskal-Wallis test. A posteriori Nemenyi test was performed to determine significant differences between tissues (adults) and developmental time (embryos and larvae) (P ≤ 0.05). All statistical analyses were performed using the software STATISTICA TM v. 7.0 (StatSoft, Inc., Tulsa, OK.).

PCR amplification and sequencing analysis.
A partial sequence for glut2 of 889 bp encodes 296 AA was obtained and registered in the GenBank (accession number QKG31965.1, MN792759.1; Fig. 1 (Fig. 2). According to the AA sequence of glut2, the phylogenetic three clusters P. splendida (bootstrap value of 74%) with the Nile tilapia (O. niloticus), blue tilapia, zebra mbuna (M. zebra), Burton's mouthbrooder (A. burtoni), flameback (P. nyererei) and princess cichlid (N. pulcher) (Fig. 3). Relative expression of glut2 in P. splendida adults and larvae. The highest expression of glut2 occurred in the liver, followed by the intestine, kidney, and muscle, while the lowest was in the pancreas, gill, heart, brain, testicle, and stomach, respectively (P ≤ 0.05) (Fig. 4). On the other hand, glut2 expression in embryos and larvae as a function of developmental time showed high variation (Fig. 5). Embryos had higher expression than larvae at 10, 25, and 30 dph and lower expression when compared to larvae at 5, 15, and 20 dph (P ≤ 0.05). The highest expression of glut2 occurred in 20 dph larvae and the lowest in 25 and 30 dph larvae (Fig. 5).

DISCUSSION
Characterization and expression of glut2 in tissues from P. splendida adults. In the current study, the partial sequence of glut2 was isolated and identified from the liver of P. splendida. Amino acid alignment of glut2 showed a great identity and highly conserved regions among cichlids and other teleosts. These results are consistent with O. niloticus, M. amblycephala, and C. carpio, where glut2 contains 12 transmembrane domains (Liu et al., 2014;Liang et al., 2018;Deng et al., 2020). Moreover, the exact phylogenetic trend, including high values of convergence, is undeviating with results from Liu et al. (2014).
Furthermore, expression of glut2 was observed in all the analyzed tissues from P. splendida (liver, intestine, kidney, muscle, heart, testicle, gill, stomach, pancreas, and brain). The highest mRNA expression occurred in the liver, followed by the intestine and kidney. These results agree with studies in other teleosts, including O. niloticus (Liu et al., 2014), C. carpio (Deng et al., 2020), M. amblycephala (Liang et al., 2018), O. mykiss (Panserat et al., 2001;Krasnov et al., 2001), G. morhua (Hall et al., 2006 and European sea bass Dicentrarchus labrax (Terova et al., 2009). In addition, studies in mammals show that the highest expression of glut2 occurs in the liver, pancreas, intestine, and kidney (Karim et al., 2012;Thorens, 2015), which suggests that the patterns of glut2 expression are highly conserved within vertebrates. One possible explanation could be the similarity between the aminoacid sequences in fish and mammals, including humans (Krasnov et al., 2001;Castillo et al., 2009).
The increased expression of glut2 in the liver may occur since this organ is responsible for the synthesis, storage, and redistribution of glucose in the form of glycogen (Polakof et al., 2010;Karim et al., 2012;Liang et al., 2018). Furthermore, the intestine is a vital organ for glucose absorption, and its dynamics and mechanisms are critical endpoints in elaborating specific diets for cultured fish species (Thorens, Mueckler, 2010;Blanco et al., 2017;Zhao et al., 2020). In zebrafish, glut2 expresses in the brush border and basolateral membranes of the intestines. However, metabolic and endocrine factors regulate mRNA levels and the distribution of molecules of this gene to the apical membrane (Cheeseman, 2002;Castillo et al., 2009). In contrast, the expression of glut2 in the kidney has been detected in other species such as O. mykiss, D. rerio, and G. morhua (Krasnov et al., 2001;Panserat et al., 2001;Castillo et al., 2009;Hall et al., 2014), where they mention that the kidney is involved in the regulation of glucose homeostasis through 3 primary mechanisms: 1) the release of glucose into the bloodstream through gluconeogenesis, 2) the consumption of glucose to meet the renal energy needs, and 3) glucose reabsorption in the proximal tubule (Segura, Ruilope, 2013).
Expression of glut2 in embryos and larvae of P. splendida. In our study, glut2 expression was observed from the embryonic period. Expression in embryos could be attributed to zygotic gene activation during early development or maternal mRNA transference; however, details about gene activation in P. splendida zygotes are unknown. Fish energy reserves such as proteins, carbohydrates, and lipids are found in yolk and oil droplets in larvae, and their functions are namely structural and for the maintenance of metabolic pathways, which depend on both genetic and epigenetic factors (Burggren, Blank, 2009;Treviño et al., 2011;Lubzens et al., 2017). The low expression of glut2 in 5 dph larvae may be related to the poor differentiation of the digestive system, where the intestine is a straight tube (dorsally to the liver) connected directly to the esophagus (Treviño et al., 2011). Our results are consistent with previous reports in zebrafish, where glut2 expression is detected in 5 dph by foregut development anterior intestine (intestinal bulb), which plays an essential role in the absorption of glucose via facilitated diffusion occur, especially in the enterocytes of the luminal and basolateral membrane (Castillo et al., 2009;Polakof et al., 2010;Blanco et al., 2017). Therefore, Holmberg et al. (2004) mention that the efficiency of carbohydrate utilization in fish larvae depends on an adequate development of the digestive system and the concentration of this nutrient in live prey, which in the case of Artemia nauplii, ranges from 11 to 17% (Guevara, Lodeiros, 2003). For P. splendida larvae, the intestine is fully functional on day 5 dph when a regular movement pattern marks exogenous feeding is visible, being the moment where glut2 expression increases when the Artemia nauplii are provided. Similarly, to the results obtained with the cobia Rachycentron canadum larvae when they were fed with a diet rich in carbohydrates by the addition of rotifers and Artemia (Hall, 2006). In this sense, the regulation of several genes for carbohydrate metabolism in fish larvae is related to innate genomic expression and the external stimuli when the live prey is offered (Darias et al., 2006).
Expression of glut2 in P. splendida decreased at 10 dph and subsequently increased at 15 dph. The increment in glut2 expression could be related to the change in the diet (co-feeding). By 15 dph, larvae were fed with a balanced commercial diet, increasing the glucose intake using carbohydrates in the formulation. For example, wheat, soy, sorghum meals, and starch are used as binders (Kamalam et al., 2017). Uscanga-Martínez et al. (2011) mentioned that 15 dph P. splendida larvae present a digestive system formed with three well-differentiated segments in the intestine (anterior, middle, and posterior). The liver occupies the liver most of the anterior part of the abdominal cavity. In this regard, the maximum glut2 expression was registered at 20 dph, where P. splendida can be considered a juvenile with all its organs fully formed and functional, especially the liver, intestine, and endocrine pancreas, where pancreatic hormones including insulin, glucagon, and somatostatin are expressed (Treviño et al., 2011;Liu et al., 2014;Liang et al., 2018;Deng et al., 2020). The maximum glut2 expression was detected in P. splendida larvae at 20 dph. It can be attributed to the use of balanced feeds because their composition contains high concentrations of vegetable ingredients (up to 21% carbohydrate content), resulting in a considerable accumulation of glycogen in the liver. Although, high carbohydrate accumulation did not show histological damage (Treviño et al., 2011). Additionally, the high content of carbohydrates in diets for O. niloticus larvae is frequent since many cichlids are omnivorous species and can quickly assimilate these molecules (El-Sayed, 2006;Stickney, 2006). In contrast, P. splendida is a carnivorous fish and cannot tolerate a carbohydrate-rich diet such as other freshwater or marine carnivorous teleosts (Polakof et al., 2012;Thorens, 2015;Marandel et al., 2016).
In summary, the maximum glut2 expression in bay snook larvae occurred when organogenesis was completed (20 dph), especially in the liver and intestine. Similarly, glut2 expression in adults of P. splendida is mainly expressed in the liver and intestines to facilitate glucose absorption. Moreover, the decrease in glucose generated by gluconeogenesis and glut2 expression can be influenced by diet composition.

ACKNOWLEDGMENTS
Gratitude to the technician Vicente Garcia Morales in charge of the DAMC teaching laboratory for his support in carrying out this research.