Introduction

Malformation has adverse impact on the market value of marine fish (Cobcroft and Battaglene 2009, 2013; Sandel et al. 2010; Ma et al. 2014d). Malformed fish are usually sold at lower price or are manually removed before being sold on market (Koumoundouros et al. 1997). Furthermore, deformation can negatively impact fish growth, survival, swimming, food conversion, and susceptibility to stress and pathogens (Andrades et al. 1996; Koumoundourous et al. 1997; Boglione et al. 2001). Though the rearing environment (e.g., temperature, salinity, water current, dissolved oxygen, and tank color) (Koumoundouros et al. 1999, 2001; Hattori et al. 2004; Sfakianakis et al. 2004; Okamoto et al. 2009; Georgakopoulou et al. 2010; Owen et al. 2012), genetic factors (Ferguson and Danzmann 1998; Castro et al. 2007; Ma et al. 2014c), parasites and pesticides (Kusuda and Sugiyama 1981; Liang et al. 2012; Liu et al. 2012) can affect fish bone development, more and more evidence has demonstrated that nutritional factors during larval fish rearing can directly cause fish deformity (Andrades et al. 1996; Afonso et al. 2000; Cahu et al. 2003a; Sandel et al. 2010).

Morphogenesis of marine fish larvae can be altered by changing dietary micronutrients such as vitamin A (Villeneuve et al. 2006; Fernández et al. 2009; Mazurais et al. 2009), vitamins D and C (Darias et al. 2011), and lipids (Cahu et al. 2003b). Although previous studies have demonstrated that feeding with high levels of dietary nutrients (e.g., lipids, fatty acids, vitamins) can improve growth and survival and significantly reduce skeletal malformation (Koven et al. 2003; Izquierdo et al. 2013), excessive dietary lipids (e.g., DHA, DHA/EPA) and vitamins (e.g., vitamin A, C) can also lead to low survival (Fernández and Gisbert 2011; Hamre et al. 2013; Ma and Qin 2014a) and high malformation (Izquierdo et al. 2010; Haga et al. 2011; Izquierdo et al. 2013). Up to present, it is still unclear what the most suitable enrichment formula is for Artemia nauplii enrichment in rearing golden pompano larvae.

Lipids are the main source of energy supply for larval fish (Sargent et al. 1999a). Among different lipid components, fatty acids are an indispensable biological component having the ability to modulate the transcription of genes involved in metabolism (Kliewer et al. 1997). Peroxisome proliferator-activated receptors (PPAR) belong to nuclear hormone receptors, and these receptors are known to collaborate with other cellular receptors such as retinoid X receptors (RXR) through formation of dimers (Ross et al. 2000). These dimers modulate the expression of target genes through binding to the hormone response elements (HREs) in DNA, and perform a function in energy balance and morphogenesis (Mangelsdorf and Evans 1995; Kliewer et al. 1997; Ross et al. 2000).

RXR is a type nuclear receptor that is activated by retinoid acid, and there are three types of RXR encoded as RXRα, RXRβ, and RXRγ. This receptor is a critical component in heterodimer formation, which in turn controls hormone responses (Mangelsdorf and Evans 1995). RXRs have been studied in many aquatic animals such as zebrafish Danio rerio, European sea bass Dicentrarchus labrax, and crustacean Daphnia magna (Jones et al. 1995; Villeneuve et al. 2007; Wang et al. 2007), and they are associated with skeleton deformations, morphosis, and survival and co-work with other genes like IGF, BMP, Hox, or shh in morphogenesis (Villeneuve et al. 2007). Additionally, RARs are ligand-controlled transcription factors that function as heterodimers to regulate cell growth and survival (Altucci et al. 2007).

Golden pompano belongs to the family Carangidae and is a good candidate species for aquaculture due to fast growth and suitability for cage culture. Although the early ontogenetic development of digestive functionality (Ma et al. 2014a, b) and weaning protocols for rearing golden pompano have been studied (Ma et al. 2014e), high malformation during early development stage of this species has severely compromised its production efficiency in hatchery. Previous studies have identified the type, position, and frequency of jaw and skeleton malformations in hatchery reared golden pompano larvae (Ma et al. 2014d; Zheng et al. 2014), but factors causing malformations on this fish remain to be elucidated. The objective of the present study was first to explore the developmental pattern of RXR genes expression in fish larvae from 0 to 5 DPH, and then to evaluate the expression of RXRs during the development of golden pompano larvae in response to dietary treatments in the Artemia nauplii feeding phase. Such information may contribute to improvement in fish quality and production efficiency in golden pompano and other related species.

Materials and methods

Eggs hatching, larval rearing, and sample collection

Fertilized eggs of golden pompano were obtained from Guanghui Aquaculture Hatchery, Hainan Province, P.R. China, and were transported to Lingshui Town and hatched in 500-L fiberglass incubators at 26 °C with a hatch rate of 97.1 ± 1.9 % (mean ± SD). On day 2 post hatching (DPH), larvae were stocked into four 1000-L larval rearing tanks at a density of 60 fish L−1. Rearing tanks were supplied with filtered seawater (5-µm pore size) from the bottom of each tank through upwelling with a daily exchange rate of 200 % tank volume. Water was discharged through an outlet screen (300-µm mesh) on the upper side of each tank, and a screen was daily cleaned to reduce clogging. Two air stones were used in each tank to maintain dissolved oxygen close to saturation.

Light intensity was maintained at 2400 lux, and the light regime was controlled at 14 h light and 10 h dark. Salinity was maintained at 33 ± 0.8 ‰, and rearing temperature was 26.5 ± 1.0 °C throughout the experiment. Rotifers Brachionus rotundiformis at a density of 10–20 rotifers mL−1 were used to feed fish larvae from 2 DPH to 12 DPH. The rotifer-fed baker yeast were enriched with the DHA protein Selco (INVE Aquaculture, Salt Lake City, UT, USA) for 12 h before the rotifers were added into the larval rearing tanks. Instant microalgal paste (Nanocholoropsis sp., Qingdao Hong Bang Biological Technology Co., Ltd, Qingdao, China) was also added into larval fish tanks to create a green-water background. On the morning of 11 DPH, fish larvae were restocked into twelve 500-L larval rearing tanks at a density of 20 fish L−1.

Feeding protocol

This experiment included three dietary treatments with three replicates each. Artemia nauplii were treated in three methods (1) enriched with instant microalgal paste (Nannochloropsis sp, Qingdao Hong Bang Biological Technology Co., Ltd, Qingdao, China), (2) enriched with Algamac 3080® (Aquafauna, USA), and (3) without any enrichment as control. Artemia cysts (Great Salt Lake, UT, USA) were used in the present study (INVE Aquaculture). Artemia nauplii instar II were enriched for 12 h at 25 °C following the manufacturer’s instruction of enrichment products before feeding to fish. After harvest, pre-washed Artemia nauplii were fed directly to fish larvae. Artemia nauplii were fed to fish from 11 DPH to 27 DPH. On 11 DPH, Artemia nauplii were first introduced at 0.2 nauplii mL−1, and then added with a daily increment of 90 % by number. In each treatment, Artemia nauplii were enriched with each product following the manufacturer’s instruction. Each tank bottom was siphoned daily to remove dead fish and feces. For the analysis of gene expression, three replicates were used in this study.

Fish growth was determined by specific growth rate (SGR) as % day−1 (Hopkins 1992): SGR = 100 × (Ln(SL f) − Ln(SL i))/Δt, where SL f and SL i are the final and initial fish total length (mm), respectively, and Δt is the time interval (days) between samples. At the end of this experiment, 50 fish larvae from each tank were sampled for assessments of growth and skeletal malformation. The remaining fish in each rearing tank were harvested and counted for survival determination.

Total RNA extraction and reverse transcription

Fish larvae were sampled on 0, 1, 2, 3, 4, 5, and 28 DPH. In the first 6 days, a total of 200 larval fish were sampled on each of the sampling day, and approximately 50 individuals were collected on 28 DPH. Total RNA was extracted using TRIzol (Invitrogen, USA). RNA integrity was verified by electrophoresis on a formaldehyde–agarose gel (1.2 %). The RNA concentration was measured by absorbance at 260 nm, and the purity was determined at the OD 260/280 ratio and agarose gel electrophoresis. RNA was reverse-transcribed to cDNA with oligo (dT) primers using a PrimeScript 1st strand cDNA synthesis kit (TaKaRa Biotechnology, Dalian Co., Ltd). The cDNA was used as a template in subsequent PCR.

Cloning of RXRs genes and real-time PCR

Open reading frame was amplified with primers (Table 1) designed with Primer 5.0 (Premier Biosoft International, Palo Alto, CA, USA) based on the data of golden pompano sequence measured previously in our laboratory. PCR were conducted in the following g conditions: denaturation at 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 2 min, and a final extension at 72 °C for 10 min. The PCR products were recycled using a DNA pure kit (Sangon biotech Shanghai Co., Ltd) after electrophoresis on a 1.5 % agarose gel. The purified PCR conducted was cloned into a pMD18-t vector (TaKaRa Biotechnology (Dalian) Co., Ltd, China), and sequenced by Majorbio (Majorbio Bio-pharm Technology Co., Ltd, Guangzhou office, China). In quantitative real-time PCR, EF-1α was used as the internal reference and amplified. The cycling conditions for RXRs genes were as follows: 1 min at 95 °C, followed by 40-cycles 95 °C for 15 s, and 60 °C for 1 min. Dissociation curves were employed to ensure that only one single PCR product was amplified in each gene reaction. For each test, three replicates were performed in this study. The relative gene expression was calculated using the ΔCT (comparative threshold cycles) (ΔCT = CT of target gene − CT of EF-1α, ΔΔCT = sample CT − ΔCT of calibrator sample).

Table 1 Oligomeric primers used in PCR and real-time PCR
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Fatty acid analysis

The nutritional content of Artemia nauplii was assessed when fish larvae was at 18 DPH. After the enrichment procedure, Artemia (4-g wet weight) from each treatment in three replicates were collected by a using 100-µm screen. All Artemia samples were pre-washed using deionized water to remove salt, and paper tower was used to remove extra water before preservation in liquid nitrogen. Fatty acids were analyzed at South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China following the method described by Ma and Qin (2014b).

Jaw malformation

At the end of this experiment, a total of 100 fish larvae were randomly collected from each rearing tank to examine the incidence of malformation. Fish were anaesthetized with overdosed Aqui-S (AQUI-S New Zealand Ltd., Lower Hutt, New Zealand) and fixed in 10 % neutrally buffered formalin. Jaw deformity was assessed on a stereo microscope (Olympus SZ40, Tokyo, Japan) using the criteria described by Ma et al. (2014d). Jaw malformation (%) was calculated by the following equation: Jaw malformation = (malformed larvae/total larvae) × 100 %.

Statistical analysis

All percentage data were arcsine-transformed prior to analysis in this study. However, the data were presented as untransformed values in the figures. The data in this paper were expressed as mean ± SD and were tested by one-way ANOVA (PASW Statistics 18.0, Chicago, SPSS Inc.). When a significant treatment effect was found, Tukey’s test was performed for multiple range comparisons with the level of significant difference set at P < 0.05. All the data were tested for normality, homogeneity, and independence to satisfy the assumptions of ANOVA.

Results

The growth of golden pompano larvae was significantly affected by the enrichment products (P < 0.05; Fig. 1). The highest SGR was obtained in fish fed the Artemia nauplii enriched with Algamac 3080, and the lowest SGR was observed in fish fed non-enriched Artemia nauplii. The highest survival was achieved in the treatment of non-enriched Artemia and Nanochloropsis-enriched Artemia (P < 0.05; Fig. 1), and the lowest survival was observed when fish were fed with Algamac 3080-enriched Artemia nauplii (P < 0.05).

Fig. 1
figure 1

Growth and survival of golden pompano larvae in four enrichment treatments on 28 DPH. Different letters represent significant differences at P < 0.05

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Partial sequences of RXRα and RXRβ genes were obtained after sequencing analysis (Fig. 2). Homology alignment analysis was conducted by using nucleotide BLAST (http://www.ncbi.nlm.nih.gov/). Results showed that RXRα in golden pompano exhibited high sequence homology with fish species such as Stegastes partitus (95 %) and Dicentrarchus labrax (94 %, Table 2), while RXRβ showed high sequence homology with fish species such as Lateolabrax japonicas (95 %) and Stegastes partitus (91 %, Table 3). The cloned fragments included a partial zinc finger motive, corresponding to the ligand-binding domain.

Fig. 2
figure 2

Partial sequences of RXRα and RXRβ genes of golden pompano Trachinotus ovatus

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Table 2 Homology alignment analysis of RXRα in golden pompano using nucleotide BLAST
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Table 3 Homology alignment analysis of RXRβ in golden pompano using nucleotide BLAST
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At hatching, the expression level of RXRα was 3.12 times higher than that on 1 DPH (Fig. 3). On 3 DPH, the expression level of RXRα reached the lowest level and increased significantly on 4 DPH (P < 0.05; Fig. 3). On 1 DPH, the relative expression level of RXRβ was 1.99 times higher than at hatching (0 DPH), and then shapely decreased by 2 DPH (P < 0.05; Fig. 3). Starting from 2 DPH, the relative expression level of RXRβ reduced with the increase in fish age, and reached the lowest level on 4 DPH (P < 0.05; Fig. 3).

Fig. 3
figure 3

Relative expressions of RXRα and RXRβ in the early developmental stage of golden pompano Trachinotus ovatus larvae

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Fatty acids composition in enriched and non-enriched Artemia nauplii

The specific fatty acid composition in Artemia nauplii significantly varied between treatments (Table 4). The amount of EPA (20:5n-3) in the Artemia nauplii enriched with Nannochloropsis (8.49 %) was significantly higher than in non-enriched Artemia nauplii (P < 0.05; Table 4). The EPA was not significantly different between the treatments of Nannochloropsis and Algamac 3080, or between the treatments of Algamac 3080 and non-enrichment (P > 0.05). After enrichment, the highest amount of DHA (22:6n-3) was obtained in Artemia nauplii enriched with Algamac 3080 (2.56 %, P < 0.05), and the lowest DHA level was observed in non-enriched Artemia nauplii. After enrichment, the DHA/EPA ratio was improved in all the enrichment treatments, and the highest DHA/EPA ratio was observed in the treatment of Algamac 3080 (0.36, P < 0.05). Similarly, the DHA/ARA ratio was improved after enrichment, but no significant differences were found between these enrichment treatments (P > 0.05). After enrichment, the EPA/ARA ratio in Artemia nauplii was only improved in the treatment of Nannochloropsis (Table 4; P < 0.05).

Table 4 Fatty acid composition (% of total fatty acids) of enriched and non-enriched Artemia nauplii
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Jaw malformation of golden pompano larvae fed with different diets

Artemia enrichments significantly affected the jaw deformity of golden pompano larvae (P < 0.05, Fig. 4). On 28 DPH, the jaw deformity of golden pompano larvae fed Algamac 3080-enriched Artemia nauplii was significantly lower than fish fed non-enriched Artemia nauplii or Nannochloropsis-enriched Artemia nauplii (P < 0.05). The jaw deformity was not significantly different between fish fed non-enriched Artemia nauplii and Nannochloropsis-enriched Artemia nauplii (P > 0.05).

Fig. 4
figure 4

Jaw malformation relative expressions of RXRα and RXRβ at 28 DPH in golden pompano fed with non-enriched Artemia nauplii, Nannochloropsis sp.-enriched Artemia nauplii, and Algamac 3080-enriched Artemia nauplii

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Gene expression of RXRα and RXRβ in fish fed enhanced Artemia nauplii

Nutrient enhancements significantly affected the gene expression of RXRα and RXRβ (P < 0.05). The expression level of RXRα in fish fed non-enriched Artemia nauplii was significantly higher than fish fed Nannochloropsis or Algamac 3080-enriched Artemia nauplii (P < 0.05; Fig. 4). The lowest expression of RXRα was observed when fish were fed with Nannochloropsis-enriched Artemia nauplii. The lowest expression of RXRβ was observed in fish fed non-enriched Artemia nauplii, and the highest expression of RXRβ was found in fish fed Nannochloropsis-enriched Artemia nauplii (Fig. 4).

Discussion

In marine fish larvae, both DHA and EPA in the diet are essential to fish growth (Rezek et al. 2010). Improvement in fish growth with increasing levels of dietary DHA has been observed in species such as yellowtail Seiola quiqueradiata (Furuita et al. 1996), striped jack Caranx vinctus (Takeuchi et al. 1996), and Japanese flounder Paralichthys olivaceus (Izquierdo et al. 1992). Previous studies have clearly demonstrated that the growth response of fish larvae to different enrichment products is varied among species. For instance, the growth of fish larvae such as striped bass Morone saxatilis and gilthead seabream Sparus aurata larvae was not affected by feeding the Artemia nauplii enriched with Algamac 2000 or PL-Cr (DHA-rich phospholipid extract of Crypthecodinium sp.), but the growth rate of halibut Hippoglossus hippoglossus larvae fed Artemia nauplii enriched with DHA Seleco was lower than the larvae fed PL-Cr (Harel et al. 2002). In the present study, fish growth was enhanced when fish larvae were fed with Artemia nauplii enriched with Algamac 3080 or Nannochloropsis. The best fish SGR was achieved in the treatment of Algamac 3080, which is consistent with the high dietary DHA levels in live feed. However, low survival in the Algamac 3080 treatment may also contribute to the high SGR due to the death of small larvae in this treatment.

Highly unsaturated fatty acids, especially EPA, DHA, and ARA, are essential to growth, development, and survival in marine fish (Sargent et al. 1999b; Cahu et al. 2003a; Rezek et al. 2010). To develop lipid-enriched food for fish larvae, the requirements of essential fatty acids in fish larvae have been extensively studied by using live prey enriched with different oils and micronutrients, aiming to increase the essential fatty acids content in live prey (Sargent et al. 1997; Takeuchi 1997; Izquierdo et al. 2000). However, excessive dietary lipid content or unbalanced of lipid class composition has led to poor growth and skeletal malformation in species such as yellowtail kingfish Seriola lalandi (Ma and Qin 2014a), gilthead seabream Sparus aurata (Salhi et al. 1999), Atlantic halibut Hippoglossus hippoglossus (Olsen et al. 2000), and Atlantic cod Gadus morhua (Kjørsvik et al. 2009). In the present study, enrichment did not change the DHA/ARA ratio, but a higher DHA/EPA ratio (0.36:1) was achieved by enriching Artemia nauplii with Algamac 3080. The high DHA/EPA ratio observed in the Algamac 3080 treatment led to fast growth but low survival. On the contrary, a better survival was obtained in the non-enriched and Nannochloropsis treatments where the DHA/EPA ratio was 0.07:1–0.22:1. Low fish survival in the Algamac 3080 treatment supports the claim in a previous study that a high DHA content and a high DHA/EPA ratio may reduce larval fish survival (Planas and Cunha 1999) as unbalanced lipid class composition in the diet affects digestion and absorption of fatty acids in fish larvae (Salhi et al. 1997, 1999).

Jaw malformations are a common type of skeletal malformation and exhibit in several forms (Cobcroft et al. 2001) in both artificial reared and wild caught marine fish (Boglione et al. 2001). Izquierdo et al. (2010) suggest that PUFA is important in bone formation, and dietary lipids can also affect the fatty acid composition in bone and cartilage (Kokkinos et al. 1993; Watkins et al. 1997; Liu et al. 2004). Hamre et al. (2002) suggests that the abnormal development of fish larvae may be triggered by insufficient dietary n-3 HUFA in Artemia nauplii. Recent research has demonstrated that fatty acids such as DHA, EPA, and ARA play an important role in bone development. For instance, a 50 % reduction in deformed fish was observed when fish larvae were fed with higher levels of dietary DHA (Izquierdo et al. 2010), and alterations in the dietary ARA/EPA ratio can indirectly affect osteoblasts and bone metabolism (Berge et al. 2009). In the present study, enrichment significantly affected jaw malformation. Fish fed Artemia nauplii enriched with Algamac 3080 showed two times lower jaw malformation than those fed non-enriched Artemia nauplii or Artemia nauplii enriched with Nannochloropsis. Skeletal malformation was reduced in fish fed Artemia enriched with Algamac 3080 which is coincident with high DHA in the feeds. This indicates that a dietary DHA level of 2.56 % may be suitable for skeletal development in golden pompano larvae.

In the present study, the gene expressions of retinoid X receptors (RXR) α and β in golden pompano were quantified at RNA level. The results indicate that the partially deduced amino sequences in RXRα and RXRβ genes present a high level of amino acid identity (>87 %) with the corresponding sequences in other fish species. Both RXRα and RXRβ contain a ligand-binding domain which involves in binding the retinoic acid (Villeneuve et al. 2004). Such ligand-binding domain has been found in many nuclear hormone receptors such as steroids and thyroid hormones receptors (Green and Chambon 1988; Tanenbaum et al. 1998).

Retinoid X receptors RXRα and RXRβ play key roles in several nuclear receptor signaling pathways (Wendling et al. 1999). Both RXRα and RXRβ can regulate the expression of target genes by binding to the DNA sequence in the regulatory region and alter biological processes such as blood vessel formation, lipid metabolism, energy balance, and morphogenesis (Kliewer et al. 1997; Ross et al. 2000; Balmer and Blomhoff 2002; Hayashida et al. 2004). Previous studies have demonstrated that RXRs play an essential role in the early development of vertebrates (Kastner et al. 1997; Wendling et al. 1999; Shi et al. 2012). In the present study, the highest expression of RXRα was observed at fish hatching and sharply reduced before first feeding. While the expression of RXRβ reached the highest level on 1 DPH and reduced to a low level after first feeding. Like most marine fish larvae, significant structural changes occurred in golden pompano larvae before first feeding (Ma et al. 2012, 2014b; Zheng et al. 2014). Higher expressions before first feeding observed in the present study may indicate that both RXRα and RXRβ are essential during the early development in golden pompano larvae when an intense process of body differentiation occurs (Villeneuve et al. 2004).

Retinoids are essential for normal embryonic development via directly or indirectly affecting gene families such as BMPs, IGFs, Hox, and shh that are involved in morphogenesis (Joore et al. 1994; Helms et al. 1997; Yates et al. 2002; Allan et al. 2003). Previous studies demonstrate that PUFA can activate RXRα expression, and direct activation of the transcription of RXRα by fatty acids has been shown under an experimental condition (Steineger et al. 1998; Mata de Urquiza et al. 2000). It is known that RXRα preferentially binds the α isoform of RAR (Ross et al. 2000; Egea et al. 2001). Therefore, the increase in RXRα could lead to the upregulation of RARα, and these two kinds of receptors might have formed active dimers leading to apoptosis (Glozak and Rogers 1998), which is a potential cause of skeleton malformation (Villeneuve et al. 2005). In the present study, the levels of DHA and DHA/EPA ratio in non-enriched Artemia nauplii were significantly lower than those enriched with Nannochloropsis or Algamac 3080. The highest expression level of RXRα was observed in fish fed non-enriched Artemia nauplii, and a high rate jaw malformation was also found in this feeding group. These results may suggest that nutrient deficiency may increase the expression of RXRα in golden pompano larvae and cause jaw malformation (Haga et al. 2003). In the present study, although the levels of DHA and DHA/EPA ratio in Artemia nauplii enriched with Nannochloropsis were significantly higher than in non-enriched Artemia nauplii, jaw malformation was not significantly different between fish fed non-enriched Artemia nauplii and Nannochloropsis-enriched Artemia nauplii. Furthermore, the expression level of RXRα in fish fed Nannochloropsis-enriched Artemia nauplii was significantly lower than the expression level in non-enriched feeding group. This may suggest that other nutrients may also regulate the expression level of RXRα and affect jaw malformation in golden pompano. However, the actual mechanism may need further investigation.

Although RXRβ plays important role in the early development of fish (He et al. 2009), it receives less attention in fish malformation studies compared with RXRα. In the present study, the expression levels of RXRβ in fish larvae were not consistent with jaw malformation rates observed in the feeding groups. The highest expression level of RXRβ was observed in fish fed Nannochloropsis-enriched Artemia nauplii, and the lowest expression level of RXRβ was recorded in the non-enriched feeding group. However, the jaw malformation rates were not statistically different between these two feeding groups. This may suggest that the impact between RXRβ expression and jaw malformation in golden pompano larvae is not clearly distinguished in fish larvae at the Artemia nauplii feeding phase when fish was subjected to nutrient challenge. However, as a possible transient regulation of RXRβ may exist in the early stage of larval development (Cahu et al. 2009), further study should be toward comparing the impact of nutrient on jaw malformation of golden pompano larvae at the rotifer feeding phase.

In summary, nutrient enhancement can affect the jaw malformation of golden pompano larvae during the Artemia nauplii feeding phase. The expressions of RXRα and RXRβ were varied between different dietary treatments, and there were no direct relationship between the expression of RXRs and jaw malformation of golden pompano larvae during the Artemia nauplii feeding phase.