Characterization of the genomic responses in early Senegalese sole larvae fed diets with different dietary triacylglycerol and total lipids levels I. Hachero-Cruzado, A. Rodr´ıguez-Rua, J. Roman-Padilla, M. Ponce, C. Fern´andez-D´ıaz, M. Manchado PII: DOI: Reference:
S1744-117X(14)00051-3 doi: 10.1016/j.cbd.2014.09.005 CBD 345
To appear in:
Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics
Received date: Revised date: Accepted date:
5 July 2014 29 September 2014 30 September 2014
Please cite this article as: Hachero-Cruzado, I., Rodr´ıguez-Rua, A., Roman-Padilla, J., Ponce, M., Fern´andez-D´ıaz, C., Manchado, M., Characterization of the genomic responses in early Senegalese sole larvae fed diets with different dietary triacylglycerol and total lipids levels, Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics (2014), doi: 10.1016/j.cbd.2014.09.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Characterization of the genomic responses in early Senegalese sole larvae fed diets
PT
with different dietary triacylglycerol and total lipids levels
I. Hachero-Cruzado*#; A. Rodríguez-Rua; J. Roman-Padilla; M. Ponce; C. Fernández-
NU
SC
RI
Díaz and M. Manchado#
IFAPA Centro El Toruño, Junta de Andalucía, Camino Tiro Pichón s/n, 11500 El
D
MA
Puerto de Santa María, Cádiz
AC CE P
dietary triacylglycerol.
TE
Running title: Genomic responses in Senegalese sole larvae fed diets with different
# Theses authors contributed equally to the study ms 107 pages, 6 figures, 5 tables, 4 suppl. files
*Address for Correspondence: Ismael Hachero Cruzado. IFAPA Centro El Toruño. Camino Tiro de Pichón s/n. 11500 El Puerto de Santa María (Cádiz), Spain. Tel: +34 956011334. Fax: +34 956011324. Email: [email protected]
Abbreviations: CM, chylomicrons; DPH, days post hatch; DEPC, diethyl pyrocarbonate; DET, differentially expressed transcripts; DMA, dimethylacetal; FA, fatty acids; GO, gene ontology; HDL, high-density lipoproteins; HTAG, high triacylglycerol diet; KEGG, Kyoto Encyclopedia of Genes and Genomes; LDL, low-density lipoproteins; LTAG, low triacylglycerol diet; LV, lipid vacuoles; NGS, Next Generation Sequencing; NL, neutral lipids; PI: posterior intestine; PL, phospholipids; rRNA, ribosomal RNA; SD, standard deviation; SNI: supranuclear inclusions TAG, triacylglycerol; VLDL, very lowdensity lipoproteins, ZG, zymogen granules
1
1
ACCEPTED MANUSCRIPT Abstract The aim of this work was to evaluate the genomic responses of premetamorphic sole larvae (9 days post-hatching, dph) fed diets with different lipid and triacylglycerol
PT
(TAG) content. For this purpose, two diets with high (rotifers enriched with a fish oilbased emulsion; referred to as HTAG) and low (rotifers enriched with a krill oil-based
RI
emulsion; LTAG) levels of total lipids and TAG were evaluated. Lipid class and fatty
SC
acid (FA) profiles, histological characterization of intestine, liver and pancreas and expression patterns using RNA-seq were determined. Discriminant analysis results showed that larvae could be clearly differentiated on the basis of their FA profile as a
NU
function of the diet supplied until 9 dph although no difference in growth was observed. RNA-seq analysis showed that larvae fed HTAG activated coordinately the transcription
MA
of apolipoproteins (apob, apoa4, apoc2, apoe, and apobec2) and other related transcripts involved in chylomicron formation, likely to facilitate proper lipid absorption and delivery. In contrast, larvae fed LTAG showed higher mRNA levels of several
D
pancreatic enzymes (try1a, try2, cela1, cela3, cela4, chym1, chym2, amy2a and pnlip)
TE
and appetite modulators (agrp1) and some intra- and extracellular lipases. Moreover, KEGG analysis also showed that several transcripts related to lipid metabolism and
AC CE P
glycolysis were differentially expressed with a higher abundance in larvae fed LTAG diet. All these data suggest that early larvae were able to establish compensatory mechanisms for energy homeostasis regulating key molecules for FA and TAG biosynthesis, FA uptake and intracellular management of TAG and FA to warrant optimal growth rates.
Keywords: Senegalese sole; apolipoproteins; proteases; dietary triacylglycerol; expression
2
ACCEPTED MANUSCRIPT 1. Introduction Lipids represent a structurally and functionally diverse class of metabolites that, along with proteins, are the major constituents of cells. They play different important
PT
functional roles including the formation and maintenance of cell membranes, energy storage and cellular signalling (Sargent et al., 2002). Among all lipid classes, neutral
RI
glycerolipids and phospholipids (PL) play a pivotal role during larval development
SC
promoting cell proliferation and providing the energy required for an active anabolic metabolism during exponential growth (Tocher, 2003). Absorption of dietary lipids implies their active hydrolization by pancreatic lipases in the gut lumen. Released fatty
NU
acids (FA) are later taken up and re-esterified into triacylglycerols (TAG) and PL in the endoplasmatic reticulum of enterocytes. To be transported in the blood, lipids are
MA
assembled into lipoproteins, macromolecular complexes formed by specific carrier proteins, apolipoproteins, with varying amounts of PL, cholesterol esters, and TAG. As a result, four main types of lipoproteins with different density and size can be
D
synthetized including chylomicrons (CM), very low-density lipoproteins (VLDL), low-
TE
density lipoproteins (LDL) and high-density lipoproteins (HDL). Apolipoproteins are major components of these particles and they can be classified into five major classes, A
AC CE P
through E, according to their structure and function, similarly to mammals (Chapman, 1980). Apolipoproteins not only act as lipid transporters but can also target lipoproteins to specific tissues throughout specific binding to lipoprotein receptors and activation of lipolytic enzymes (Gursky, 2005). Mechanisms behind lipid digestion, absorption and transport have not been extensively studied in fish and they are generally assumed to be similar to those described in mammals (Tocher, 2003; Tocher et al., 2008). Nevertheless, a better knowledge of the mechanisms modulating lipid digestion and transport is required to produce high-quality larvae. Lipids and amino acids are the main energy sources during larval development although TAGs are considered as the most relevant lipid class for energy provision (Hamre et al., 2013; Mourente and Vázquez, 1996; Tocher et al., 2008). However, dietary TAG levels must be provided to larvae within an optimum range since several authors have reported detrimental effects on larval growth and development when they are provided in excess (Gawlicka et al., 2002; Izquierdo et al., 2000; Olsen et al., 2000; Pousão-Ferreira et al., 1999). Larvae fed high levels of TAG exhibit a high fat accumulation in the enterocytes that interfere with nutrients traffic throughout the gut 3
ACCEPTED MANUSCRIPT and, consequently, an inadequate absorption of protein and other essential nutrients can occur (Gisbert et al., 2008; Hamre et al., 2013; Izquierdo et al., 2000; Morais et al., 2007). Moreover, a reduced capacity of Senegalese sole (Solea senegalensis) larvae to
PT
digest and absorb FA esterified to TAG compared to PL has been suggested probably associated to a deficient emulsification of TAG and a lower activity of neutral lipases
RI
versus phospholipases (Morais et al., 2005a).
the pelagic stage
SC
Senegalese sole is an important species in aquaculture with high growth rates in and negligible mortalities until complete metamorphosis
(approximately three weeks after hatching) (Parra and Yúfera, 2001; Yufera et al.,
NU
1999). This high performance during early development has been associated to an early maturation of the digestive capacity as well as to an adequate management of energy
MA
reserves to accomplish successfully the metamorphic process. Studies on pancreatic enzymes showed that the activation of the expression and activity of zymogens occurs during development, including pancreatic proteases, amylases and lipases (Conceição et
D
al., 2007; Gamboa-Delgado et al., 2011; Manchado et al., 2008) making feasible the
TE
early assimilation of lipids and proteins at similar rates (Yufera et al., 1999). Moreover, some studies have evaluated lipid requirements and the role of essential FA, PL and
AC CE P
neutral lipids (NL) in sole larvae, providing new relevant information on the effects of these lipids on the digestive capacity, lipid absorption, FA composition and their effects on sole larvae development (Boglino et al., 2012; Morais et al., 2006; Morais et al., 2005a; Morais et al., 2005b; Mourente and Vázquez, 1996; Navarro-Guillen et al., 2014; Parra et al., 1999; Vazquez et al., 1994; Villalta et al., 2005a; Villalta et al., 2005b). Nevertheless, little is known about the overall physiological and genomic responses associated to dietary lipid components. To address this question, Next Generation Sequencing (NGS) technologies offer important advantages to evaluate wide transcriptomic responses in non-model species in a cost-effective manner (Cerda and Manchado, 2013). The aim of this work was to evaluate the physiological, metabolic and molecular responses induced by two diets differing in their TAG and total lipid contents in young sole larvae at 9 days post-hatching (dph). Lipid classes, FA profiles and wide-gene expression patterns using RNA-seq were determined. Histological characterization of the intestine, liver and pancreas and RT-qPCR were also carried out to evaluate the results. 4
ACCEPTED MANUSCRIPT 2. Material and methods 2.1 Larval rearing and experimental diets
PT
Fertilized eggs were obtained from naturally spawning Senegalese sole broodstock (IFAPA Centro El Toruño). Eggs were collected early in the morning (9:00 a.m.) and
RI
transferred to a 1,000 ml measuring cylinder to separate buoyant (viable) from nonbuoyant (non-viable) eggs. The number of eggs in each fraction was estimated using
SC
volumetric methods (1,100 eggs ml–1). Eggs were incubated at a density of 2,000 embryos L-1 in 300 L cylinder-conical tanks with gentle aeration and complete water
NU
exchange every two hours. Newly hatched larvae (one day post-hatch (dph)) were then transferred to six 300 L tanks (three replicates per treatment) at an initial density of 30
MA
larvae L-1. Lights were kept off until the onset of external feeding at 3 dph. After that, a 18L:6D photoperiod with a light intensity of 200 lux was established. Temperature and salinity were 19.0ºC and 37.6, respectively. Water was filtered through sand filters and
D
10 µm and 3 µm nominal retention cartridges to maintain the water quality. Water was
TE
kept stagnant until 7 dph followed by a daily water exchange of 40%. Tanks were provided with a central draining pipe with a 250 µm mesh and gentle aeration.
AC CE P
Two different treatments consisting of rotifers enriched with two marine oil emulsions were tested. Emulsions were formulated with 2 g 100 g-1 wet weight Tween 80 (Panreac Quimica S.A., Castellar de Vallès, Spain), 0.1 g 100g-1 wet weight vitamin E (Vitamin E acetate 97%, Alfa Aesar, Karlsruhe, Germany) and 0.5 g 100g-1 wet weight vitamin C (L-Ascorbic acid 6-palmitate 95%, Sigma Aldrich, Steinheim, Germany) as constant ingredients and two different oil bases: krill oil (AkerBiomarine, Oslo, Norway) and fish oil (Sopropêche, Wimille, France) at 25 g 100g-1 emulsion wet weight. In basis to total lipid and TAG contents, fish oil-based diets were named as high TAG diet (HTAG) and krill oil-based diet as low TAG diet (LTAG). Enrichments were conducted at a density of 2x105 rotifers L-1, at 20ºC for 3 h, adding 0.31 g emulsion wet weight per liter of rotifers culture. Larvae were fed twice a day, between 11:00 and 12:00 am and 5:00 and 6:00 pm. Final concentration of rotifers in the tank was 1x104 preys L-1. To maintain constant rotifer concentration within each experimental tank, three water samples (10 mL) from each tank were sampled before supplying new food. Estimated average number of prey in each sample was used to adjust total prey concentrations in the tank.
5
ACCEPTED MANUSCRIPT Rotifers samples for lipid analysis (n=4) were collected throughout the experimental period. Samples were washed with clean seawater and ammonium formiate solution (1% w/w), frozen in liquid nitrogen and kept at -80ºC until analysis. Larvae were
PT
sampled at 3, 6, and 9 dph for lipid analysis whereas for histological and molecular analyses only at the end of the experiment. Samplings were carried out at 11:00 a.m.,
RI
before supplying new food (when needed). For RNA isolation, three pools of larvae (one per tank including 50 larvae) were randomly collected using a 350-µm-mesh net,
SC
washed with DEPC water, frozen in liquid nitrogen and stored at -80ºC until RNA isolation and further analysis. For lipid determination, three pools of larvae (300 larvae
NU
in each larval pool) from each tank were randomly collected, washed with clean seawater and ammonium formate solution (1% w/w), frozen in liquid nitrogen and kept
MA
at -80ºC until analysis. For histological analysis, 30 larvae of each tank were randomly collected, euthanized with an overdose of tricaine methane sulphonate (MS-222) and fixed in buffered formaldehyde at 4ºC overnight and preserved in ethanol 70% until
D
histological processing.
TE
2.2 Total lipids, lipid classes and fatty acids analyses
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Total lipids (TL) were extracted with chloroform:methanol (2:1 v/v) containing 0.01% of butylated hydroxytoluene (BHT) as antioxidant (Christie, 2003). The organic solvent was evaporated under a stream of nitrogen and the lipid content was determined gravimetrically. Lipid classes were separated by one dimensional double development high
performance
thin
layer
chromatography
(HPTLC)
using
methyl
acetate/isopropanol/chloroform/methanol/0.25% (w/v) KCl (25:25:25:10:9 by vol.), as the polar solvent system and hexane/diethyl ether/glacial acetic acid (80:20:2 by vol.), as the neutral solvent system. Lipid classes were visualized by charring at 160ºC for 20 minutes after dipping in cupric acetate in 3% phosphoric acid (Olsen and Henderson, 1989). Final quantification was made by densitometry in a CAMAG scanner at a wavelength of 325 nm, and by comparison with external standard (Sigma-Aldrich) (Morillo-Velarde et al., 2013). TL extracts were subjected to acid-catalyzed transmethylation for 16 h at 50°C, using 1 ml of toluene and 2 ml of 1% sulphuric acid (v/v) in methanol. The resultant FA methyl esters (FAME) and dimethylacetals (DMA) formed from ether lipids were purified by thin layer chromatography (TLC), and visualized by spraying with 1% (w/v) iodine in CHCl3 (Christie, 2003). FAME were separated and quantified by using a Shimadzu GC 2010-Plus gas chromatograph 6
ACCEPTED MANUSCRIPT equipped with a flame ionization detector (280 °C) and a fused silica capillary column SUPRAWAX-280 (15 m×0.1 mm I.D.). Hydrogen was used as carrier gas and the oven initial temperature was 100°C for 0.5 min., followed by an increase at a rate of 20 °C
PT
min−1 to a final temperature of 250°C for 8 min. Individual FAME and DMA were identified by reference to authentic standards and to a well-characterized fish oil.
RI
2.3 Histological and histochemical procedures
SC
The samples were dehydrated through ethanol series, infiltrated and embedded in paraffin blocks. Sagittal sections were cut at 5 µm thick and stained with
NU
Haematoxylin/eosin (H-E). Oil red O stainning method for identification of NL was carried out on cryopreserved larvae embedded in OCT (Tissue-Tek 4583). Cryosections
MA
were cut at 8 µm and stained according to Pearse (1985). Histological images were obtained by light microscopy and analyzed using the digital image analysis software ImageJ 1.47t (National Institutes of Health, USA; http://imagej.nih.gov/ij).
D
To estimate the number of zymogens in the pancreas, four larvae per tank were
TE
used. Photographs of different pancreatic sections of pancreas stained with H-E were taken at 100x magnification. In general, photos spanning 500-2000 µm2 of pancreas
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area over which there would exist 30-200 zymogen granules were processed. Histological images were obtained with a light microscope (LEICA DM5500B) and analyzed with digital image analysis software (Image J). 2.4 RNA isolation, RNA-seq libraries preparation and sequencing analysis Homogenization of the larval pools (50 mg) was carried out in the Fast-prep FG120 instrument (Bio101) using Lysing Matrix D (Q-Bio- Gene) for 40 s at speed setting 6. Total RNA was isolated using the RNeasy Mini Kit (Qiagen). All RNA isolation procedures were carried out in accordance with the manufacturer’s protocol. In all cases, total RNA was treated twice with DNase I using the RNase-Free DNase kit (Qiagen) for 30 min. RNA sample quality was checked on an agarose gel, and quantification was determined spectrophotometrically using the Nanodrop ND-8000. RNA integrity was further investigated using the Bioanalyzer 2100 (Agilent Technologies) before RNA-seq analysis. Illumina libraries were constructed at the Centre Nacional d'Anàlisi Genòmica (Barcelona, Spain). Libraries were prepared using the mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801) according to
7
ACCEPTED MANUSCRIPT the manufacturer’s protocol. Briefly, 0.5 µg of total RNA was used for poly-A based mRNA enrichment selection using oligo-dT magnetic beads followed by fragmentation by divalent cations at elevated temperature resulting into fragments of 80-250 nt, with
PT
the major peak at 130 nt. First strand cDNA synthesis by random hexamers and reverse transcriptase was followed by the second strand cDNA synthesis performed using
RI
RNAseH and DNA Pol I. Double stranded cDNA was end repaired, 3´adenylated and the 3´-“T” nucleotide at the Illumina adaptor was used for the adaptor ligation. The
SC
ligation product was amplified with 15 cycles of PCR. Each library was sequenced using TrusSeq SBS Kit v3-HS, in paired end mode, 2 x 76 bp, in a fraction of a lane of a
NU
HiSeq sequencing system (Illumina, Inc) following the manufacturer’s protocol, generating minimally 15 million paired-end reads for each sample. Images from the
MA
instrument were processed using the manufacturer’s software to generate FASTQ sequence files.
For RNA-seq analysis, Illumina short-reads were pre-processed using
D
SeqTrimNext pipeline (http://www.scbi.uma.es/seqtrimnext; Falgueras et al., 2010)
TE
available at the Plataforma Andaluza de Bioinformática (University of Málaga, Spain) using the specific configuration parameters for illumina data. Filtered-reads were onto
the
reference
AC CE P
mapped
transcriptome
of
S.
senegalensis
v4.1
(http://www.juntadeandalucia.es/agriculturaypesca/ifapa/soleadb_ifapa/) using Bowtie2 (Langmead and Salzberg, 2012). Total number of transcript counts was extracted using Sam2count.py (https://github.com/vsbuffalo/sam2counts). Finally, differential gene expression analysis was carried out using edgeR with a p-value cut-off of 0.05 and using the Benjamini-Hochberg method for multiple testing correction as implemented in the Robina software (Lohse et al., 2012). The raw data was normalized according to the default procedure of the differential expression analysis package used. RNA-seq reads have been deposited at the NCBI short read archive (SRA; BioProject ID: PRJNA241068).
2.5 Functional analysis Transcript annotations and GO information were downloaded from SoleaDB (http://www.juntadeandalucia.es/agriculturaypesca/ifapa/soleadb_ifapa/).
These
annotations were done using Sma3s, Autofact and FullLengtherNext (Koski et al., 2005; Lara et al., 2007; Muñoz-Merida et al., 2014). KEGG analysis was performed by 8
ACCEPTED MANUSCRIPT importing annotation data for the complete set of differentially expressed transcripts (DET; p
S1744-117X(14)00051-3 doi: 10.1016/j.cbd.2014.09.005 CBD 345
To appear in:
Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics
Received date: Revised date: Accepted date:
5 July 2014 29 September 2014 30 September 2014
Please cite this article as: Hachero-Cruzado, I., Rodr´ıguez-Rua, A., Roman-Padilla, J., Ponce, M., Fern´andez-D´ıaz, C., Manchado, M., Characterization of the genomic responses in early Senegalese sole larvae fed diets with different dietary triacylglycerol and total lipids levels, Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics (2014), doi: 10.1016/j.cbd.2014.09.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Characterization of the genomic responses in early Senegalese sole larvae fed diets
PT
with different dietary triacylglycerol and total lipids levels
I. Hachero-Cruzado*#; A. Rodríguez-Rua; J. Roman-Padilla; M. Ponce; C. Fernández-
NU
SC
RI
Díaz and M. Manchado#
IFAPA Centro El Toruño, Junta de Andalucía, Camino Tiro Pichón s/n, 11500 El
D
MA
Puerto de Santa María, Cádiz
AC CE P
dietary triacylglycerol.
TE
Running title: Genomic responses in Senegalese sole larvae fed diets with different
# Theses authors contributed equally to the study ms 107 pages, 6 figures, 5 tables, 4 suppl. files
*Address for Correspondence: Ismael Hachero Cruzado. IFAPA Centro El Toruño. Camino Tiro de Pichón s/n. 11500 El Puerto de Santa María (Cádiz), Spain. Tel: +34 956011334. Fax: +34 956011324. Email: [email protected]
Abbreviations: CM, chylomicrons; DPH, days post hatch; DEPC, diethyl pyrocarbonate; DET, differentially expressed transcripts; DMA, dimethylacetal; FA, fatty acids; GO, gene ontology; HDL, high-density lipoproteins; HTAG, high triacylglycerol diet; KEGG, Kyoto Encyclopedia of Genes and Genomes; LDL, low-density lipoproteins; LTAG, low triacylglycerol diet; LV, lipid vacuoles; NGS, Next Generation Sequencing; NL, neutral lipids; PI: posterior intestine; PL, phospholipids; rRNA, ribosomal RNA; SD, standard deviation; SNI: supranuclear inclusions TAG, triacylglycerol; VLDL, very lowdensity lipoproteins, ZG, zymogen granules
1
1
ACCEPTED MANUSCRIPT Abstract The aim of this work was to evaluate the genomic responses of premetamorphic sole larvae (9 days post-hatching, dph) fed diets with different lipid and triacylglycerol
PT
(TAG) content. For this purpose, two diets with high (rotifers enriched with a fish oilbased emulsion; referred to as HTAG) and low (rotifers enriched with a krill oil-based
RI
emulsion; LTAG) levels of total lipids and TAG were evaluated. Lipid class and fatty
SC
acid (FA) profiles, histological characterization of intestine, liver and pancreas and expression patterns using RNA-seq were determined. Discriminant analysis results showed that larvae could be clearly differentiated on the basis of their FA profile as a
NU
function of the diet supplied until 9 dph although no difference in growth was observed. RNA-seq analysis showed that larvae fed HTAG activated coordinately the transcription
MA
of apolipoproteins (apob, apoa4, apoc2, apoe, and apobec2) and other related transcripts involved in chylomicron formation, likely to facilitate proper lipid absorption and delivery. In contrast, larvae fed LTAG showed higher mRNA levels of several
D
pancreatic enzymes (try1a, try2, cela1, cela3, cela4, chym1, chym2, amy2a and pnlip)
TE
and appetite modulators (agrp1) and some intra- and extracellular lipases. Moreover, KEGG analysis also showed that several transcripts related to lipid metabolism and
AC CE P
glycolysis were differentially expressed with a higher abundance in larvae fed LTAG diet. All these data suggest that early larvae were able to establish compensatory mechanisms for energy homeostasis regulating key molecules for FA and TAG biosynthesis, FA uptake and intracellular management of TAG and FA to warrant optimal growth rates.
Keywords: Senegalese sole; apolipoproteins; proteases; dietary triacylglycerol; expression
2
ACCEPTED MANUSCRIPT 1. Introduction Lipids represent a structurally and functionally diverse class of metabolites that, along with proteins, are the major constituents of cells. They play different important
PT
functional roles including the formation and maintenance of cell membranes, energy storage and cellular signalling (Sargent et al., 2002). Among all lipid classes, neutral
RI
glycerolipids and phospholipids (PL) play a pivotal role during larval development
SC
promoting cell proliferation and providing the energy required for an active anabolic metabolism during exponential growth (Tocher, 2003). Absorption of dietary lipids implies their active hydrolization by pancreatic lipases in the gut lumen. Released fatty
NU
acids (FA) are later taken up and re-esterified into triacylglycerols (TAG) and PL in the endoplasmatic reticulum of enterocytes. To be transported in the blood, lipids are
MA
assembled into lipoproteins, macromolecular complexes formed by specific carrier proteins, apolipoproteins, with varying amounts of PL, cholesterol esters, and TAG. As a result, four main types of lipoproteins with different density and size can be
D
synthetized including chylomicrons (CM), very low-density lipoproteins (VLDL), low-
TE
density lipoproteins (LDL) and high-density lipoproteins (HDL). Apolipoproteins are major components of these particles and they can be classified into five major classes, A
AC CE P
through E, according to their structure and function, similarly to mammals (Chapman, 1980). Apolipoproteins not only act as lipid transporters but can also target lipoproteins to specific tissues throughout specific binding to lipoprotein receptors and activation of lipolytic enzymes (Gursky, 2005). Mechanisms behind lipid digestion, absorption and transport have not been extensively studied in fish and they are generally assumed to be similar to those described in mammals (Tocher, 2003; Tocher et al., 2008). Nevertheless, a better knowledge of the mechanisms modulating lipid digestion and transport is required to produce high-quality larvae. Lipids and amino acids are the main energy sources during larval development although TAGs are considered as the most relevant lipid class for energy provision (Hamre et al., 2013; Mourente and Vázquez, 1996; Tocher et al., 2008). However, dietary TAG levels must be provided to larvae within an optimum range since several authors have reported detrimental effects on larval growth and development when they are provided in excess (Gawlicka et al., 2002; Izquierdo et al., 2000; Olsen et al., 2000; Pousão-Ferreira et al., 1999). Larvae fed high levels of TAG exhibit a high fat accumulation in the enterocytes that interfere with nutrients traffic throughout the gut 3
ACCEPTED MANUSCRIPT and, consequently, an inadequate absorption of protein and other essential nutrients can occur (Gisbert et al., 2008; Hamre et al., 2013; Izquierdo et al., 2000; Morais et al., 2007). Moreover, a reduced capacity of Senegalese sole (Solea senegalensis) larvae to
PT
digest and absorb FA esterified to TAG compared to PL has been suggested probably associated to a deficient emulsification of TAG and a lower activity of neutral lipases
RI
versus phospholipases (Morais et al., 2005a).
the pelagic stage
SC
Senegalese sole is an important species in aquaculture with high growth rates in and negligible mortalities until complete metamorphosis
(approximately three weeks after hatching) (Parra and Yúfera, 2001; Yufera et al.,
NU
1999). This high performance during early development has been associated to an early maturation of the digestive capacity as well as to an adequate management of energy
MA
reserves to accomplish successfully the metamorphic process. Studies on pancreatic enzymes showed that the activation of the expression and activity of zymogens occurs during development, including pancreatic proteases, amylases and lipases (Conceição et
D
al., 2007; Gamboa-Delgado et al., 2011; Manchado et al., 2008) making feasible the
TE
early assimilation of lipids and proteins at similar rates (Yufera et al., 1999). Moreover, some studies have evaluated lipid requirements and the role of essential FA, PL and
AC CE P
neutral lipids (NL) in sole larvae, providing new relevant information on the effects of these lipids on the digestive capacity, lipid absorption, FA composition and their effects on sole larvae development (Boglino et al., 2012; Morais et al., 2006; Morais et al., 2005a; Morais et al., 2005b; Mourente and Vázquez, 1996; Navarro-Guillen et al., 2014; Parra et al., 1999; Vazquez et al., 1994; Villalta et al., 2005a; Villalta et al., 2005b). Nevertheless, little is known about the overall physiological and genomic responses associated to dietary lipid components. To address this question, Next Generation Sequencing (NGS) technologies offer important advantages to evaluate wide transcriptomic responses in non-model species in a cost-effective manner (Cerda and Manchado, 2013). The aim of this work was to evaluate the physiological, metabolic and molecular responses induced by two diets differing in their TAG and total lipid contents in young sole larvae at 9 days post-hatching (dph). Lipid classes, FA profiles and wide-gene expression patterns using RNA-seq were determined. Histological characterization of the intestine, liver and pancreas and RT-qPCR were also carried out to evaluate the results. 4
ACCEPTED MANUSCRIPT 2. Material and methods 2.1 Larval rearing and experimental diets
PT
Fertilized eggs were obtained from naturally spawning Senegalese sole broodstock (IFAPA Centro El Toruño). Eggs were collected early in the morning (9:00 a.m.) and
RI
transferred to a 1,000 ml measuring cylinder to separate buoyant (viable) from nonbuoyant (non-viable) eggs. The number of eggs in each fraction was estimated using
SC
volumetric methods (1,100 eggs ml–1). Eggs were incubated at a density of 2,000 embryos L-1 in 300 L cylinder-conical tanks with gentle aeration and complete water
NU
exchange every two hours. Newly hatched larvae (one day post-hatch (dph)) were then transferred to six 300 L tanks (three replicates per treatment) at an initial density of 30
MA
larvae L-1. Lights were kept off until the onset of external feeding at 3 dph. After that, a 18L:6D photoperiod with a light intensity of 200 lux was established. Temperature and salinity were 19.0ºC and 37.6, respectively. Water was filtered through sand filters and
D
10 µm and 3 µm nominal retention cartridges to maintain the water quality. Water was
TE
kept stagnant until 7 dph followed by a daily water exchange of 40%. Tanks were provided with a central draining pipe with a 250 µm mesh and gentle aeration.
AC CE P
Two different treatments consisting of rotifers enriched with two marine oil emulsions were tested. Emulsions were formulated with 2 g 100 g-1 wet weight Tween 80 (Panreac Quimica S.A., Castellar de Vallès, Spain), 0.1 g 100g-1 wet weight vitamin E (Vitamin E acetate 97%, Alfa Aesar, Karlsruhe, Germany) and 0.5 g 100g-1 wet weight vitamin C (L-Ascorbic acid 6-palmitate 95%, Sigma Aldrich, Steinheim, Germany) as constant ingredients and two different oil bases: krill oil (AkerBiomarine, Oslo, Norway) and fish oil (Sopropêche, Wimille, France) at 25 g 100g-1 emulsion wet weight. In basis to total lipid and TAG contents, fish oil-based diets were named as high TAG diet (HTAG) and krill oil-based diet as low TAG diet (LTAG). Enrichments were conducted at a density of 2x105 rotifers L-1, at 20ºC for 3 h, adding 0.31 g emulsion wet weight per liter of rotifers culture. Larvae were fed twice a day, between 11:00 and 12:00 am and 5:00 and 6:00 pm. Final concentration of rotifers in the tank was 1x104 preys L-1. To maintain constant rotifer concentration within each experimental tank, three water samples (10 mL) from each tank were sampled before supplying new food. Estimated average number of prey in each sample was used to adjust total prey concentrations in the tank.
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ACCEPTED MANUSCRIPT Rotifers samples for lipid analysis (n=4) were collected throughout the experimental period. Samples were washed with clean seawater and ammonium formiate solution (1% w/w), frozen in liquid nitrogen and kept at -80ºC until analysis. Larvae were
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sampled at 3, 6, and 9 dph for lipid analysis whereas for histological and molecular analyses only at the end of the experiment. Samplings were carried out at 11:00 a.m.,
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before supplying new food (when needed). For RNA isolation, three pools of larvae (one per tank including 50 larvae) were randomly collected using a 350-µm-mesh net,
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washed with DEPC water, frozen in liquid nitrogen and stored at -80ºC until RNA isolation and further analysis. For lipid determination, three pools of larvae (300 larvae
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in each larval pool) from each tank were randomly collected, washed with clean seawater and ammonium formate solution (1% w/w), frozen in liquid nitrogen and kept
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at -80ºC until analysis. For histological analysis, 30 larvae of each tank were randomly collected, euthanized with an overdose of tricaine methane sulphonate (MS-222) and fixed in buffered formaldehyde at 4ºC overnight and preserved in ethanol 70% until
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histological processing.
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2.2 Total lipids, lipid classes and fatty acids analyses
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Total lipids (TL) were extracted with chloroform:methanol (2:1 v/v) containing 0.01% of butylated hydroxytoluene (BHT) as antioxidant (Christie, 2003). The organic solvent was evaporated under a stream of nitrogen and the lipid content was determined gravimetrically. Lipid classes were separated by one dimensional double development high
performance
thin
layer
chromatography
(HPTLC)
using
methyl
acetate/isopropanol/chloroform/methanol/0.25% (w/v) KCl (25:25:25:10:9 by vol.), as the polar solvent system and hexane/diethyl ether/glacial acetic acid (80:20:2 by vol.), as the neutral solvent system. Lipid classes were visualized by charring at 160ºC for 20 minutes after dipping in cupric acetate in 3% phosphoric acid (Olsen and Henderson, 1989). Final quantification was made by densitometry in a CAMAG scanner at a wavelength of 325 nm, and by comparison with external standard (Sigma-Aldrich) (Morillo-Velarde et al., 2013). TL extracts were subjected to acid-catalyzed transmethylation for 16 h at 50°C, using 1 ml of toluene and 2 ml of 1% sulphuric acid (v/v) in methanol. The resultant FA methyl esters (FAME) and dimethylacetals (DMA) formed from ether lipids were purified by thin layer chromatography (TLC), and visualized by spraying with 1% (w/v) iodine in CHCl3 (Christie, 2003). FAME were separated and quantified by using a Shimadzu GC 2010-Plus gas chromatograph 6
ACCEPTED MANUSCRIPT equipped with a flame ionization detector (280 °C) and a fused silica capillary column SUPRAWAX-280 (15 m×0.1 mm I.D.). Hydrogen was used as carrier gas and the oven initial temperature was 100°C for 0.5 min., followed by an increase at a rate of 20 °C
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min−1 to a final temperature of 250°C for 8 min. Individual FAME and DMA were identified by reference to authentic standards and to a well-characterized fish oil.
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2.3 Histological and histochemical procedures
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The samples were dehydrated through ethanol series, infiltrated and embedded in paraffin blocks. Sagittal sections were cut at 5 µm thick and stained with
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Haematoxylin/eosin (H-E). Oil red O stainning method for identification of NL was carried out on cryopreserved larvae embedded in OCT (Tissue-Tek 4583). Cryosections
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were cut at 8 µm and stained according to Pearse (1985). Histological images were obtained by light microscopy and analyzed using the digital image analysis software ImageJ 1.47t (National Institutes of Health, USA; http://imagej.nih.gov/ij).
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To estimate the number of zymogens in the pancreas, four larvae per tank were
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used. Photographs of different pancreatic sections of pancreas stained with H-E were taken at 100x magnification. In general, photos spanning 500-2000 µm2 of pancreas
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area over which there would exist 30-200 zymogen granules were processed. Histological images were obtained with a light microscope (LEICA DM5500B) and analyzed with digital image analysis software (Image J). 2.4 RNA isolation, RNA-seq libraries preparation and sequencing analysis Homogenization of the larval pools (50 mg) was carried out in the Fast-prep FG120 instrument (Bio101) using Lysing Matrix D (Q-Bio- Gene) for 40 s at speed setting 6. Total RNA was isolated using the RNeasy Mini Kit (Qiagen). All RNA isolation procedures were carried out in accordance with the manufacturer’s protocol. In all cases, total RNA was treated twice with DNase I using the RNase-Free DNase kit (Qiagen) for 30 min. RNA sample quality was checked on an agarose gel, and quantification was determined spectrophotometrically using the Nanodrop ND-8000. RNA integrity was further investigated using the Bioanalyzer 2100 (Agilent Technologies) before RNA-seq analysis. Illumina libraries were constructed at the Centre Nacional d'Anàlisi Genòmica (Barcelona, Spain). Libraries were prepared using the mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801) according to
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ACCEPTED MANUSCRIPT the manufacturer’s protocol. Briefly, 0.5 µg of total RNA was used for poly-A based mRNA enrichment selection using oligo-dT magnetic beads followed by fragmentation by divalent cations at elevated temperature resulting into fragments of 80-250 nt, with
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the major peak at 130 nt. First strand cDNA synthesis by random hexamers and reverse transcriptase was followed by the second strand cDNA synthesis performed using
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RNAseH and DNA Pol I. Double stranded cDNA was end repaired, 3´adenylated and the 3´-“T” nucleotide at the Illumina adaptor was used for the adaptor ligation. The
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ligation product was amplified with 15 cycles of PCR. Each library was sequenced using TrusSeq SBS Kit v3-HS, in paired end mode, 2 x 76 bp, in a fraction of a lane of a
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HiSeq sequencing system (Illumina, Inc) following the manufacturer’s protocol, generating minimally 15 million paired-end reads for each sample. Images from the
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instrument were processed using the manufacturer’s software to generate FASTQ sequence files.
For RNA-seq analysis, Illumina short-reads were pre-processed using
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SeqTrimNext pipeline (http://www.scbi.uma.es/seqtrimnext; Falgueras et al., 2010)
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available at the Plataforma Andaluza de Bioinformática (University of Málaga, Spain) using the specific configuration parameters for illumina data. Filtered-reads were onto
the
reference
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mapped
transcriptome
of
S.
senegalensis
v4.1
(http://www.juntadeandalucia.es/agriculturaypesca/ifapa/soleadb_ifapa/) using Bowtie2 (Langmead and Salzberg, 2012). Total number of transcript counts was extracted using Sam2count.py (https://github.com/vsbuffalo/sam2counts). Finally, differential gene expression analysis was carried out using edgeR with a p-value cut-off of 0.05 and using the Benjamini-Hochberg method for multiple testing correction as implemented in the Robina software (Lohse et al., 2012). The raw data was normalized according to the default procedure of the differential expression analysis package used. RNA-seq reads have been deposited at the NCBI short read archive (SRA; BioProject ID: PRJNA241068).
2.5 Functional analysis Transcript annotations and GO information were downloaded from SoleaDB (http://www.juntadeandalucia.es/agriculturaypesca/ifapa/soleadb_ifapa/).
These
annotations were done using Sma3s, Autofact and FullLengtherNext (Koski et al., 2005; Lara et al., 2007; Muñoz-Merida et al., 2014). KEGG analysis was performed by 8
ACCEPTED MANUSCRIPT importing annotation data for the complete set of differentially expressed transcripts (DET; p
