Differentially expressed genes in the pituitary of the Amazonian fish Arapaima gigas

Arapaima gigas, known as pirarucu, is one of the largest tropical freshwater fish and an icon fish species of the Amazon. Many studies on reproduction of A. gigas have been developed but a significant gap persists. To analyze the gene expression profile of A. gigas from wild stocks, four cDNA libraries from pituitary of young and adults of males and females were developed. Among the differentially expressed genes, 97 genes related to several physiological functions were identified, especially related to growth and reproduction. The main ones were activin receptor IIB, estrogen receptor, follistatin, growth hormone receptor isoform 1, luteinizing hormone receptor, pituitary adenylate cyclaseactivating polypeptide, prolactin receptor, somatostatin receptor type 1 subtypes A and B. Gene expression in A. gigas differs strongly between adult and young, but presents high similarity between male and female. EST collections obtained in this study represent an important resource to better understand the mechanisms involved in the growth, cell development, reproduction and sexual differentiation of this species. In addition, the present set of results contributes towards improvement of breeding management of this important fish species for world aquaculture.


INTRODUCTION
Arapaima gigas is known as pirarucu in Brazil and paiche in other Amazonian countries.It is one of the largest freshwater fish in the world, reaching up to 3 m in length and over 250 kg.It is a bony-tongue fish belonging to Osteoglossomorpha, a group of fish that form a link between the ancient bony fish (ray-finned line) and the current specialized teleosts (Nelson, 1944).A. gigas has been overfished as a source of food, impacting their natural stocks (Imbiriba, 2001).Overfishing is the major reason that this fish species is among the freshwater fishes requiring extreme conservation procedures (Castello and Stewart, 2010).A. gigas has a great potential for the aquaculture industry in Amazonian countries.It is highly priced, growths up to 15 kg during its first year of life, and as an air-breather it can survive poorly oxygenated water (Val and Almeida-Val, 1995).However, sexual differentiation of pirarucu is unknown.Only scarce information is available on strategies for reproduction in captivity (Núñez et al., 2011), gonadal morphology (Godinho et al., 2005), general structure of the pituitary gland (Borella et al., 2009) and the molecular forms of neurohormones, as gonadotropinreleasing hormone (Okubo and Aida, 2001).Only empirical evidence or fragmented information on morphological differences between males and females of A. gigas are available what hamper its management and culture.Imbiriba (2001) reported that sexual dimorphism becomes apparent only when the animals are 4-5 years old and are sexually mature.
Teleost growth and reproduction are claimed to be controlled by environmental factors such as temperature, rain, river water level, among other factors (Weltzien et al., 2004).Endogenous factors control the synthesis and release of gonadotropins, gonadal steroid and modulators affecting growth and reproduction through the hypothalamic-pituitary axis (HPA) in several species (Levy et al., 2010).Growth factors, like growth hormone (GH) and prolactin (PRL), have been claimed to be involved in the reproduction process (Very et al., 2005).How the HPA govern all functions related to the growth and reproduction is a key point to manage breeding of A. gigas in captivity.It would also aid to reduce the pressure on natural stocks.
The molecular mechanisms underlying the development and functioning of different tissues depends on gene expression profiles, gene hierarchies and interactions, as well as on the proteins encoded by these genes.There are many methodologies available for gene identification and comparison of gene expression profile.Analysis of expressed sequence tags (ESTs) has proven to be an efficient methodology for different organisms (Baxendale et al., 2009;Qiu et al., 2008;Toro et al., 2009).
Therefore, considering that A. gigas is an endangered species, an important commercial fish species of the Amazon and a singular biological model, efforts to understand its reproductive biology are needed.In the present study, cDNA libraries were built to identify the gene expression profile in male, female, adult and young fishes, and to explore the molecular mechanisms potentially involved with growth and reproduction of this species.

Tissue samples
Twenty-four specimens of A. gigas were collected from wild stocks at the Mamirauá Sustainable Development Reserve (Solimões River, State of Amazonas, Brazil, 02°22' S; 66°18' W), being six adult males and six adult females with 1.8 m of length and weighting between 50 and 75 kg, and six young males and six young females with one meter of length and weighting between 9 to 12 kg.The pituitaries were excised, immediately frozen in liquid nitrogen and transported to the Laboratory of Ecophysiology and Molecular Evolution at INPA, in Manaus, Amazonas, Brazil, where the samples were stored at -80°C until mRNA extraction.The sex and reproductive stage were externally and internally checked visually and photographed on the field.

RNA isolation and library construction
The poly (A+) mRNA was purified from each pituitary using FastTrack ® MAG mRNA Isolation kit (Invitrogen Corp, Carlsbad, CA, USA), according to manufacturer's instructions.The purity and integrity for all poly (A+) mRNA samples were checked, respectively, by spectrophotometry in a ND-2000 spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA)) and by electrophoresis in agarose denaturing gel under UV light.
Four pools were produced using the same quantity of Poly (A+) mRNA from each one of six samples of purified mRNA: adult male, adult female, young male and young female.These pools were used to construct four cDNA libraries through of Creator SMART cDNA Library Construction Kit (Clontech Lab, Inc., Palo Alto, CA, USA), according to manufacturer's instructions.All four libraries were built using 1 µg Poly (A+) mRNA from each pool.After transformation of mRNA into double strand cDNA, fragments between 0.1 to 0.5 kb were selected and purified from an agarose gel (Figure 1) using Wizard ® SV Gel and PCR Clean-Up (Promega, Madison, WI, USA).The products were then digested with Sfi I restriction enzyme to generate shorter, asymmetrical blunt-ended, double-stranded cDNA fragments, necessary for cloning vector ligation.

Sequencing of clones and bioinformatics analysis
The products from all four libraries were ligated to pDNR-LIB cloning vector and transformed into Escherichia coli XL-1 Blue bacteria.Plasmid DNA was extracted from clones and unidirectionally sequenced from the 3' ends using the Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) in conjunction with primer M13 reverse in a 3130 sequencing platform (Applied Biosystems, Foster City, CA, USA).
The sequences generated from all four libraries were then analyzed for nucleotide quality with PHRED/PHRAP software (Ewing and Green, 1998) and screened for vector contamination using Cross Match software.Sequence clustering and assembly was performed using CAP3 (Huang and Madan, 1999).Nucleotide sequences of assembled and non-assembled ESTs were compared with GenBank data using BlastN and BlastX.Significantly matches to nucleotides were considered when the e-value was <1x10 -4 .Blast2GO was used for additional annotation of biological activities of BlastX matched sequences, enabling Gene Ontology (GO) prediction and categorization of the result with respect to functional terms by biological process, cellular component, and molecular function (Conesa et al., 2005).ESTs clustering were performed with repeat masked sequences using the TIGR Gene Indices Clustering Tools (TGICL).The resulting TGICL consensus sequences were parsed and loaded into a MySQL EST database for further analysis.

Characteristics of the cDNA libraries
The pools of mRNA from pituitaries of A. gigas were used to generate four cDNA libraries: Adult male (AM), adult female (AF), young male (YM) and young female (YF).generated which were then compared with public databases of the GenBank from NCBI.Of this total, 1773 sequences, 54.5%, showed no similarities and, therefore, will be used for further studies, and 736 ESTs were associated to 97 genes (DNA Data Bank of Japan AB922020 -AB922116) related to several physiological functions of A. gigas, especially growth and reproduction.

Gene ontology
The gene ontology (GO) tool, used to classify gene products, showed that most of the genes detected in all for libraries were related to molecular function category, varying, respectively, from 37.8 to 42.6%, in AM and AF (Figures 2 to 5).The other two categories presented wider variation with the genes related to cellular components varying, respectively, from 18.7 to 31.3% in YF and AM, and those related to biological processes varying from 28.8 to 40.1% in AF and YF.Analysis of the distribution of GO categories (Figures 2 to 5) shows that the Biological processes cell communication (GO:0007154) and signal transduction (GO:0007165), the cellular component related to cell (GO:0005623) and the molecular function related to binding (GO:0005488) were the most common annotation categories for adult's libraries (AM and AF), while the Biological process related to metabolism (GO:0008152), the Cellular component related to cell (GO:0005623) and the Molecular function related to binding (GO:0005488) were the most common for young's libraries (YM and YF).

Genes related to growth and reproduction of A. gigas
The analysis of cDNA libraries from pituitaries of A. gigas  allowed the identification of 97 genes (Table 2).Among them the following genes related to growth and reproduction of A. gigas were found: activin receptor IIB, estrogen receptor, follistatin, growth hormone receptor isoform 1, luteinizing hormone receptor, pituitary adenylate cyclase-activating polypeptide, prolactin receptor, somatostatin receptor type 1 subtypes A and B.

DISCUSSION
The HPA is considered the main link between the central nervous and endocrine systems regulating the secretion and release of several hormones acting on homeostasis, growth, reproduction, and several other physiological functions in fishes (Levy et al., 2010).The control occurs   through the production of hormones that stimulate or inhibit the synthesis and release of adenohypophysial hormones.The hypothalamus, through its neuronal projections into the neurohypophysis, produce hormones as isotocin and vasopressin and control the adenohypophysis (anterior pituitary lobe) that produces and secretes hormones responsible, among other functions, by growth and reproductive cycle (Unger and Glasgow, 2003).Therefore, in the present study four cDNA libraries from pituitary of A. gigas were built to analyze differentially expressed genes between males and females and young and adults.To our knowledge, this is the first study to analyze genes involved with growth and reproduction of A. gigas using a high throughput technology.
The construction of cDNA library is part of a method   AM / AF / YM / YF GO:0016020 Zonadhesin-like 7e -11 AF / YM / YF GO:0016020 (a) E-value obtained with BlastX of GenBank. (b) Indicates that gene is expressed in adult male library (AM), adult female library (AF), young male library (YM) or young female library (YF) of A. gigas.designed for comparing gene expression at various developmental stages or at altered physiological conditions (Diatchenko et al., 1996).In the present work, 3251 ESTs were generated from four cDNA libraries and 97 genes were identified, most of them involved with biological processes according GO tools (Figures 2 to 5).Note that genes related to cellular components showed larger relative expression in adult's libraries (AM and AF) compared to young fish libraries (YM and YF).In contrast, genes involved with metabolism (GO:0008152), transcription (GO:0006350), and nucleic acid metabolism (GO:0006139) presented an opposite situation, with larger relative expression in young fish libraries (YM and YF).As adult cells reduce their growth rate and present increased activities related to homeostasis, genes responsible for cytoskeleton (GO:0005856) and cytoplasm (GO:0005737) were found primarily in adult's libraries (AM and AF).Analyzing the ontology of genes identified in all four libraries from A. gigas, it is clear that adult (AM and AF) greatly differ from young (YM and YF) libraries.
The growth hormone (GH) binds to a specific GH receptor (GHR1 or GHR2), localized on the cell membrane of the target-tissues, and a cascade of physiological events takes place (Herrington and Carter-Su, 2001).The secretion of GH in fishes is up regulated by growth hormone-releasing hormone (GHRH) and down-regulated by somatostatin (SST).GH was isolated from several teleosts, being considered the major controller of growth of many species (Schalburg et al., 2008;Very et al., 2005).It acts on several physiological processes as gonadal development, osmoregulation, appetite, morphology, social behavior and immunology (Pérez-Sánchez et al., 2002).In many fishes, the GH has functions that lacks in others vertebrates as are its effects on sexual maturation (Levy et al., 2010;Schalburg et al., 2008).The SST is classically known to inhibit the secretion of a wide range of hormones including the GH and prolactin.There are five subtypes of SST receptors (SSTR1 to SSTR5) well characterized in vertebrates including fishes (Panetta et al., 1994).Two forms of SSTR1 (SSTR1A and SSTR1B) and one GHR1 were evidenced only in adult's libraries of A. gigas (AM and AF), indicating the presence of growth regulatory mechanisms in adults of this species, as described for other fish species (Toro et al., 2009).However, the absence of these receptors in libraries of young A. gigas (YM and YF) was not expected, but corroborate results  obtained by Borella et al. (2009) that found few cells producing GH in pituitary of young fish of A. gigas.
Further studies are needed to understand the distribution of different types of GHR and SSTR in A. gigas from larval and adult phase.
Contrasting to GHR1 and SSTR1, PRLR was found only in AF library, probably due to the presence of mature eggs in the ovaries of females at the moment of capture, indicating the importance of PRLR and PRL in reproductive physiology of A. gigas female.Its classical action, mediated by prolactin hormone binding, is directly related to reproduction, osmoregulation, and metabolism in several species of teleosts, as sea bream (Sparus auratus) (Power and Canario 1992), tilapia (Oreochromis mossambicus) (Weber and Grau, 1999), and zebrafish (Danio rerio) (Nguyen et al., 2008).
The LHR, found only in AM and AF libraries, has been classically described in the ovary and testis.It controls the reproductive process in several fishes by binding to the luteinizing hormone (LH) what causes gonadal maturation in young and gonadal steroidogenesis in adults (Pandolfi et al., 2006).The expression of LHR is induced by the follicle-stimulating hormone (FSH) and by LH (Dufau 1998).In the hermaphrodite fish Trimma okinawae, when in female phase, high levels of LHR occur in ovaries but only baseline levels are present in testes, contrasting with male phase when the levels of LHR is greater in the testes than in ovaries.These results indicate the importance of these receptors in determining the sexual characteristics of fishes (Kobayashi et al., 2009).Because of this, the absence of LHR in the libraries of young A. gigas (YM and YF) indicates gonad immaturity and can be used as a marker for sexual phases, since this species shows no sexual dimorphism before the reproductive period that starts when the fish is four or five years old.
The PACAP has been shown to be an important neuroendocrine regulator of reproduction and growth in vertebrates, including fish, by inducing secretion of various hormones such as GH and gonadotropin (Wong et al., 2000), LH (Miyata et al., 1989), thyrotropin (Okada et al., 2006), and prolactin (Rawlings and Hezareh, 1996).PACAP has been found and isolated in several fish species such as carp (Ctenopharyngodon idellus) (Wong et al., 2005), goldfish (Carassius auratus) (Wong et al., 2000), tilapia (hybrid of Oreochromis niloticus x O. aureus) (Gur et al., 2001), zebrafish (Toro et al., 2009), and marbled electric ray (Torpedo marmorata) (Valiante et al., 2006).In zebrafish the PACAP functions are extensive, starting in embryonic development and continuing throughout adult life, indicating its importance for growth and hormonal regulation of fishes (Toro et al., 2009).In tilapia, the PACAP controls several intracellular signaling pathways, causing increased expression of several gonadotropin genes (Gur et al., 2002).In goldfish PACAP stimulates the release of GH and LH from the pituitary (Sawisky and Chang, 2005).Interestingly, in A. gigas the PACAP was found in AF library but not in YF indicating its importance during reproductive stage of female.This is in agreement with the presence of mature eggs found only in adults female.When PACAP starts to be expressed in A. gigas, must be investigated.
The ERs act as ligand-dependent transcription factors that regulate the expression of their target-genes either by binding to specific sequences (estrogen response elements) or by interfering with other transcription factors (Bardet et al., 2002).Estrogens linked to their receptors influence growth, differentiation and functioning of many reproductive and non-reproductive tissues, breeding behavior, and secretion of pituitary gonadotropin (Ma et al., 2000).Vertebrate genomes contain three ER subtypes, termed ERα, ERβ, and ERγ, which have been found from teleosts to mammals (Hawkins et al., 2000).The ERs were found in several species of fishes such as Atlantic croaker (Micropogonias undulatus) (Hawkins et al., 2000), zebrafish (Bardet et al., 2002;Tingaud-Sequeira et al., 2004), and goldfish (Carassius auratus) (Ma et al., 2000).In A. gigas, however, we found only the most common subtype of ER, the ERα.More studies are needed to understand the distribution of ER types in ancient teleosts, as is the case of A. gigas.
The biological effects of Activin are mediated through specific activin receptors type I (ActR-IA and -IB) and type II (ActR-IIA and -IIB).Activin acts on the HPA, controlling the reproduction and development of different organisms, including teleosts as zebrafish and goldfish.ActRIIB was firstly found in ovaries and also in brain, heart, muscle, liver and testes.However its highest expression in ovaries, suggests its importance for fish breeding (Garg et al., 1999;Ge, 2000).The ActRIIB was identified in zebrafish embryos and was related to early development (Ge, 2000).We found ActRIIB only in youngs of A. gigas, suggesting its importance for the development of this species too.
The classical function of FST is the suppressive effect on FSH secretion by its binding to Activin with high affinity, rendering bound Activin unavailable for binding to its own receptor (Act-RIIA or B) (Phillips, 2005).FST, identified in fishes as zebrafish (Wu et al., 2000) and S. aurata (Funkenstein et al., 2009) from embryos to adult, confirms its importance along embryonic development and its inhibition effects on FSH.Others studies have indicated that FST act also in areas outside the reproductive system, such as neurons, muscle growth and development (Irwin and Kraak, 2012;Phillips, 2005).We identified FST only in the AF library, thought it has been found in teleost in different life stages.In summary, comparisons performed among the four libraries showed that gene expression in A. gigas differ strongly between adults and young, but is similar between males and females, observing, indeed, the sexual differences.EST collections obtained in this study allowed the identification of genes expressed in the pituitary of adult, young, male, and female of A. gigas, thus providing an important first step for studies to understand the mechanisms involved in the growth, cell development, reproduction and sexual differentiation of this species.Further studies will provide useful information on expression of other genes on specific cell types involved in the growth and reproduction of A. gigas.

Figure 1 .
Figure 1.Electrophoresis on a 1% agarose gel showing the cDNA double strand from pituitary of A. gigas used for construction of libraries.AM, adult male; AF, adult female; YM, young male; YF, young female; L, DNA ladder marker.The arrow indicates the position of 500 bp.

Figure 2 .
Figure 2. Frequency (%) of annotated sequences assigned to a Gene Ontology (GO) classification for the adult male library of A. gigas.

Figure 3 .
Figure 3. Frequency (%) of annotated sequences assigned to a Gene Ontology (GO) classification for the young male library of A. gigas.
e p t o r a c t iv it y s ig n a l t r a n s d u c e r a c t iv it y s t r u c t u r a l l e o t i d e b i n d i n g r e c e p t o r a c t i v i t y s i g n a l t r a n s d u c e r a c t i v i t y t r a n s c r i p t i o n f a c t o r a c t i v i t y t r a n s c r i p t i o n r e g u l a t o r a c t i v i t y t r a n s p o r t e r a c t i v i t y

Figure 4 .
Figure 4. Frequency (%) of annotated sequences assigned to a Gene Ontology (GO) classification for the adult female library of A. gigas.

Figure 5 .
Figure 5. Frequency (%) of annotated sequences assigned to a Gene Ontology (GO) classification for the young female library of A. gigas.
Raw number of libraries of pooled samples for sequenced clones, EST generated sequences, clustered EST and number of consensus are shown in Table 1.Out of 3857 sequenced clones, 3251 valid ESTs were

Table 1 .
Libraries statistics of A.

Table 2 .
Genes identified in pituitary of A. gigas.