La mojarra tenguayaca (Petenia splendida) es una especie de cíclido carnívoro con excelente valor comercial en el sureste de México. A pesar de su potencial para la acuicultura, existe muy poca información sobre sus rutas metabólicas relacionadas con su nutrición, fisiología y reproducción. El presente estudio se enfoca en la expresión del transportador de glucosa (glut2) en embriones y larvas de 5, 10, 15, 20, 25 y 30 días post eclosión (dph) y en el hígado, intestino, riñón, músculo, corazón, testículo, branquias, estómago, páncreas y cerebro en peces adultos. Se diseñaron cebadores de qPCR específicos para glut2. La expresión en los embriones fue menor que en larvas a los 5, 15 y 20 dph. La expresión máxima en larvas se observó a los 20 dph y la mínima a los 25 y 30 dph. La expresión más alta en los adultos ocurrió en el hígado y el intestino. Nuestros resultados muestran que el gen glut2 se expresa de manera diferencial en los tejidos de adultos de la mojarra tenguayaca y su expresión fluctúa durante el desarrollo larvario.
Palabras clave: Expresión genómica, Ontogenia inicial, Peces adultos, Petenia splendida, Transportador de glucosa.
The Bay snook Petenia splendida Günther, 1862 is a carnivorous freshwater cichlid species distributed from Southeast Mexico to Central America (Álvarez-González et al., 2008). This species possesses an excellent economical value and is widely accepted in local markets (Pérez-Sánchez, Páramo-Delgadillo, 2008). Additionally, it presents suitable characteristics for aquaculture, including high growth rate, tolerance to overcrowding, relative low time of production (~1 year) and its meat has high nutrimental content (Uscanga-Martínez et al., 2011). Various studies have described different aspects of P. splendida,such as its biology and physiology (Álvarez-González et al., 2008; Jiménez-Martinez et al., 2019), taxonomy and ecology (Méndez et al., 2011), aquaculture technology (Pérez-Sánchez, Páramo-Delgadillo, 2008; Vidal-López et al., 2009; Treviño et al., 2011, Jiménez-Martínez et al., 2019), nutrition and digestive physiology (Álvarez-González et al., 2008; Uscanga-Martínez et al., 2011; Rodríguez-Estrada et al., 2020), and cytogenetics (Arias-Rodriguez et al., 2008). However, there is gap in the understanding of nutritional metabolic pathways, especially for glucose. Glucose is a primordial energy source for most physiological processes and the correct functioning of various tissues such as the brain, liver, gonads, and muscle (Hemre et al., 2002; Deng et al., 2020). This molecule is absorbed in the gut by enterocytes through specific glucose transporters (Blanco et al., 2017). Two types of transporters for glucose and other monosaccharides have been described: 1) sodium-glucose transporters (sglt) mainly related to renal glucose reabsorption, and 2) glucose transporters (glut) which facilitates the transport of glucose across the plasma membrane via facilitated diffusion (Thorens, 2015; Bertrand et al., 2020). In mammals, 14 glucose transporters (glut1-14) have been described (Wood, Trayhurn, 2003; Scheepers et al., 2004; Mueckler, Thorens, 2013; Thorens, 2015; Holman, 2020) and each GLUT isoform plays a specific role in glucose metabolism depending on tissue expression patterns, substrate specificity, and the regulation of the expression under different physiological conditions (Wright Jr. et al., 1998; Castillo et al., 2009; Gómez-Zorita, Urdampilleta, 2012). In teleost fish, glut1 has been characterized in common carp (Cyprinus carpio) (Teerijoki et al., 2001b), grass carp (Ctenopharyngodon idella) (Li et al., 2018), rainbow trout (Oncorhynchus mykiss) (Teerijoki et al., 2000, 2001a) and Atlantic cod (Gadus morhua) (Hall et al., 2004); glut3 in grass carp, (C. idella) (Zhang et al., 2003) and Atlantic cod (G. morhua) (Hall et al., 2005); and glut2 and glut4 in Atlantic salmon (Salmo salar) (Menoyo et al., 2006) and Atlantic cod (G. morhua) (Hall et al., 2006, 2014). In the case of glut1-6, glut8-13 and glut15 are reported in the spotted sea bass (Lateolabrax maculatus) (Fan et al., 2019).
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.
Material and methods
Fish acquisition. Twenty male individuals of P. splendida (450–490 g and 20–25 cm) were obtained from the Tropical Aquaculture Laboratory, División Académica de Ciencias Biológicas, Universidad Júarez Autónoma de Tabasco, Southeast Mexico. Fish were kept in circular 2000-L polyethylene tanks and were fed the rainbow trout diet (45% protein and 16% fat, El Pedregal® Silver Cup, Toluca, Mexico) with particle diameters ranging between 5.5- and 9.0-mm. Embryos and larvae were obtained from simultaneous spawning from the broodstock kept in the same facility. Six females and three males were transferred from holding tanks to a 2000-L breeding tank. Six acrylic sheets (one side smooth, one side rough) were placed in each tank to provide shelter and egg-laying surfaces (rough side). Right after hatching, larvae were separated from the adults. After 3 days 150 larvae per tank were placed in three 70-L oval tanks with constant aeration (~95% air saturation), pH ~8.0, connected to an open system, at 28ºC, with water changes (80%) every two or three days. Larvae were fed satiety with brine shrimp (Artemia sp.) nauplii five times per day (at 8:00, 11:00, 13:00, 15:00, and 18:00 h) for 7 days (until 10 days post-hatching, dph). From 11 to 13 dph, larvae were provided with a co-feeding of Artemia nauplii and trout feed (Silver Cup; Nelson and Sons, Inc; proximate composition: 45% proteins, 16% lipids, 21% carbohydrates, 9–12% ashes) and from day 14 dph, larvae were only provided with trout feed until 30 dph. Food was provided at apparent satiation, and particle size was adjusted according to larval growth (250–500, 500–750, and > 750 m). Temperature (28.0 ± 0.7°C), dissolved oxygen (5.9 ± 0.6 mg/L), and pH (7.1 ± 0.3) Water parameters were constantly assessed with a YSI 85® Meter, YSI Inc., Yellow Springs, OH (temperature (28.0 ± 0.7°C), dissolved oxygen (5.9 ± 0.6 mg/L), and pH (7.1 ± 0.3).
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.
TABLE 1 | Oligonucleotides used for glut2 gene sequencing and q-PCRs in Petenia splendida.
|Primer name||Forward primer (5’-3’)||Reverse primer (5’-3’)||Size, pb||Step|
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.).
FIGURE 1 | Partial sequence of nucleotides and amino acids (AA) encoding glut2 (glucose transporter 2) from Petenia splendida taken from Gen Bank to design specific oligonucleotides for qPCR.
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). The alignment of P. splendida AA concerning other fishes exhibited conserved regions of glut2. Identity values were shown as 98.5 % for Nile tilapia (Oreochromis niloticus), 97.97% for zebra mbuna (Maylandia zebra), 93.24% for Burton’s mouthbrooder (Astatotilapia burtoni), 88.85% for Turquoise killifish (Nothobranchius furzeri), 93.58% for flameback (Pundamilia nyererei), 92.57% for princess cichlid (Neolamprologus pulcher), 90.20% for flier cichlid (Archocentrus centrarchus), 88.51% for yellow perch (Perca flavescens), 43.7% for zebrafish (Danio rerio), 43.6% for common carp (Cyprinus carpio) 43.5 % for blunt snout bream (Megalobrama amblycephala), 43.2 % for grass carp (Ctenopharyngodon idella) and 29.5% for rainbow trout (Oncorhynchus mykiss) (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).
FIGURE 2 | Amino acid sequence of glut2 in Petenia splendida aligned with other species other species of teleost fish. Identical amino acids are presented in black, and the high and less conserved amino acids are presented in gray and period, respectively.
FIGURE 3 | Phylogenetic tree based on the sequence of glut2 (glucose transporter 2) from Petenia splendida and other teleosts using the neighbor-joining (NJ) method. Values at branch points represent percentage frequencies for tree topology after 1,000 interations.
FIGURE 4 | Relative expression of glut2 in kidney (K), liver (L), muscle (M), brain (B), pancreas (P), gill (G), heart (H), intestine (I), stomach (S) and testis (T) of adult Petenia splendida (mean ± SEM; n = 3). Lowercase letters indicate significant differences between the expression level in the tissues (p < 0.05).
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).
FIGURE 5 | Relative expression of glut2 during the early ontogeny of Petenia splendida. Lowercase letters indicate significant differences in the expression of glut2 as a function of developmental time (p < 0.05). AN: Artemia nauplii (3–10 dph), CF: cofeeding (Artemia nauplii and tilapia feed) (11–13 dph), TF: trout feed (14–30 dph) (mean ± SEM; n = 3).
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.
Gratitude to the technician Vicente Garcia Morales in charge of the DAMC teaching laboratory for his support in carrying out this research.
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Alejandra del Carmen Castillo-Collado1, Carlos Alfonso Frías-Quintana2, Vicente Morales-Garcia3, Carina Shianya Alvarez-Villagomez4, Gloria Asencio-Alcudia4, Emyr Saul Peña-Marín4,5, Gil Martínez-Bautista6, Luis Daniel Jiménez-Martinez1 and Carlos Alfonso Álvarez-González4
 Laboratorio de Fisiología en Recursos Acuáticos, DACBIOL-UJAT, Villahermosa, México. (CSAV) carina.alvarez@Ujat.mx, (GAA) firstname.lastname@example.org, (ESPM) email@example.com, (CAAG) firstname.lastname@example.org.
 Cátedra-CONACyT-DACBiol-UJAT. Av. Crédito Constructor, CDMX, Mexico.
 Developmental Physiology Laboratory, Developmental Integrative Biology Research Group, Department of Biological Sciences,University of North Texas, Denton, TX 76203-5017, USA. (GMB) email@example.com.