Juvenile sea bream (Sparus auratus) were maintained at the Centre of Marine Science (CCMAR) field station (Ramalhete, Faro, Portugal) in through-flow seawater tanks (2000L) at 17-21°C, 36% salinity and 12 h light and 12 h dark photoperiod for several weeks prior to the start of the experiments. The maintenance of fish and subsequent experiments complied with the Guidelines of the European Union Council (86/609/EU) and was covered by a group 1 licence (Direcção-Geral de Veterinária, Portugal). Behaviour and health of animals was monitored visually each day and no mortality occurred during the experiment.
Sea bream (n = 8 fish/tank, 93.4 ± 3.1 g) were acclimatised for one week to the experimental circuit which consisted of 8 through-flow seawater tanks (500L), with water maintained at 19-21°C, 36% salinity and a 12 h light and 12 h dark photoperiod. Food was withheld from the fasted experimental groups for 1 week (-7 days) prior to removal of the scales which was considered day 0 of the trial (Figure ). The experiment had 3 treatment groups: ST = fasted for duration of experiment; WS = scales removed at time 0; STWS = fasted for duration of the experiment with scales removed at time 0 and the control group (N) with no treatment but subjected to the same anaesthesia/handling as the treatments groups.
Figure 3 Experimental design. Eight groups were generated, four were sampled 3 days after scale removal and the others were sampled on day 7. Four different groups were created: N - control fish in normal conditions; WS - fish fed normal ration levels but with (more ...)
Duplicate tanks for each treatment were prepared (day 3 and day 7) and 8 fish were sampled from one tank 3 days after the scales had been removed and from the second tank 7 days after the scales had been removed. Two tanks contained the control fish and were sampled at the same time as the experimental groups at day 3 and 7. To remove the scales, fish were lightly anaesthetised in 2-phenoxyethanol (1:5000, Sigma-Aldrich, Madrid, Spain) and scales were removed (approximately 60-70%) by wiping fish with a wet paper towel in order to minimize damage. For sampling fish were anaesthetised in 2-phenoxyethanol, as described above, weight and length was measured, blood collected and centrifuged (10,000 rpm for 5 minutes) and the plasma stored at -20°C. Fish were sacrificed by sectioning the spinal cord and skin was collected (approximately 1 cm2) from below the dorsal fin and carefully dissected free of muscle and snap frozen in liquid nitrogen and stored at -80°C. For histology, skin with some adhering muscle was fixed overnight at 4°C in 4% PFA.
To determine the possible effect of treatments on plasma composition but also to evaluate the physiological condition of the experimental and control animals, calcium, phosphorus, glucose and lactate were determined. Duplicate samples of sea bream plasma (10 μl diluted 1/4 in milliQ water), collected from 8 fish/experimental group, 3 days after the removal of scales were measured. Colourimetric tests were performed according to the manufacturers' instructions and absorbance measured with a microplate reader:
• Calcium: Calcium-ο-C v/v kit (ο-cresolphtalein, v/v, colorimetric; Spinreact ref. 1001061, Spain), absorbance at 570 nm.
• Phosphorus: Phosphorus - UV kit (Phosphomolybdate, uv; Spinreact ref. 1001155, Spain), absorbance at 340 nm.
• Glucose: Glucose-TR kit (GOD-POD; Spinreact ref. 1001190/1001191/1001192, Spain), absorbance at 505 nm.
• Lactate: Lactate kit (LO-POD Enzymatic colorimetric; Spinreact ref. 1001330, Spain), absorbance at 505 nm.
Histology of sea bream skin and scales
To characterize sea bream skin and scale morphology, samples fixed in 4% PFA were decalcified overnight in 0.5 M EDTA, pH 8, dehydrated through a graded ethanol series, saturated in xylene and impregnated and embedded in paraffin wax (Merck, Germany). Serial sections (5 μm) of skin were mounted on 3-aminopropyltriethoxysilane (APES; Sigma-Aldrich, Madrid, Spain) coated glass slides. The sections were dried overnight at 37°C, cooled to room temperature and stored or stained. To distinguish between collagen rich and/or mineralized and non-mineralized tissue, sections were stained with Masson's trichrome [83
]. Skin sections were rapidly dehydrated through a graded series of alcohols, cleared in xylene and mounted in DPX mountant (BioChemika, Sigma-Aldrich, Madrid, Spain). Stained sections were analyzed using a microscope (Leica DM2000) coupled to a digital camera (Leica DFC480) and linked to a computer for digital image analysis.
Sea bream microarray
A 4 × 44 k oligo-array developed and validated for the gilthead sea bream by Ferraresso et al.
] was used in this study. The array contained 39,379 sea bream oligonucleotide probes covering 19,715 unique transcripts and of these, 19,650 were represented by two non-overlapping probes and 65 were present as a single probe. Owing to the expansion of teleost sequences in the public databases since the publication of Ferraresso et al
], a re-annotation of probes was carried out with Blastx similarity searches against the Uniprot/Swissprot and Uniprot/Trembl databases [37
]. Annotation was assigned for probes with an expected score in excess of 1e-10
Total RNA was extracted from five individual fish using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA quality and integrity were checked using an Agilent 2100 Bioanalyser (Agilent technologies, Palo Alto, CA) and only samples with an RNA integrity index number (RIN) >7.5 were processed for use in microarray analysis. Samples from each treatment group (5 replicates) were labelled with Cy3-dCTP and hybridizations were performed using the Agilent One-Colour Microarray-Based Gene Expression Analysis protocol with the modifications described by Ferraresso et al.
]. The arrays were scanned on an Agilent G2565BA DNA microarray scanner, at a resolution of 5 μm, and at two different sensitivity levels (XDR Hi 100% and XDR Lo 10%). The XDR Hi and XDR Lo images generated per array were analysed together and the data extracted. Background subtraction was performed using the standard procedure in the Agilent Feature Extraction Software 9.5.1. Spike-In Viral RNAs were used to control array hybridization intensities and ensure normalization gave a uniform signal across all microarray slides. The R [84
] limma package [85
] was used for microarray analysis. A factorial design of the treatments were compared by fitting a linear model [86
] with differentially expressed clones selected by a Benjamini and Hochberg [87
] globally adjusted p-value of 0.05 and a minimum two-fold change. The transcripts represented by two non-overlapping probes were only selected when both probes were differentially expressed.
Gene Ontology Annotations and GO enrichment
Accession numbers associated with the probe annotations were used to assign GO and GOSLIM terms [89
]. GO enrichment was determined by a proportion test between the number of clones representing a GO term on the array compared to the number of differentially expressed clones representing the same GO term in a given comparison with a p-value cut-off of 0.05.
Ingenuity pathway Analysis
Cellular networks arising from the gene expression data were identified and established through the use of IPA (Ingenuity Systems, http://www.ingenuity.com
). The sequences of differentially expressed genes from treatments collected at 3 days were all submitted to BLASTN in order to identify human orthologues. The accession numbers were extracted and used as identifiers in IPA together with the fold-changes of the corresponding differentially expressed genes. The Ingenuity knowledge base was used as a reference and direct and indirect relationships were included and no filters were applied. Bio functions, namely molecular and cellular functions and physiological system development and function significantly (p < 0.05) related with the input dataset were identified. Networks were then algorithmically generated based on their connectivity and a score was assigned. The score was used to rank networks according to how relevant they were to the genes in the input dataset.
Microarray validation by real time RT-PCR
Array results were corroborated by real-time RT-PCR using when possible RNA extracted from the same individuals used for array analysis from all the different treatments at the 3 day time point. Ten genes were analysed and primers were designed using Beacon Design software (Premier Biosoft Int., Palo Alto, CA) (Table ). For cDNA synthesis, 1 μg of total RNA was pre-treated with DNA-free Kit (Ambion, UK) to remove genomic DNA and then cDNA synthesis carried out using 250 ng of DNAse-treated total RNA, 200 ng of random hexamers (GE Healthcare, Little Chalfont, UK), 40 U of MMLV reverse transcriptase (RT) (Promega, Southampton, UK) and 5 U of RNAguard Rnase inhibitor (GE Healthcare, Little Chalfont, UK) in a final reaction volume of 20 μl. Q-PCR was performed in duplicate reactions using SYBRgreen chemistry (Power SYBR® Green PCR Master Mix, Applied Biosystems, UK) and the relative standard curve method, using a StepOnePlus qPCR thermocycler and StepOne software v2.0 (Applied Biosystems, UK). PCR cycling conditions were 10 min at 95°C, followed by 55 cycles of 10 sec at 95°C, 20 sec at the optimal temperature for each primer pair (Table ), and 30 seconds at 72°C. A final melting curve was carried out between 60 and 95°C for all genes and each produced single products/dissociation curves. Standard curves relating initial template quantity to amplification cycle were generated using serial dilutions of linearized plasmid DNA containing the gene of interest or of RT-PCR specific product obtained from the same specie and tissue, and the efficiency of qPCR reactions ranged between 82-100%, with the exception of SAPD20351 and SAPD13946 that had efficiencies of 73.1 and 78.6%, respectively, and all gave R2 > 0.985. All amplicons were sequenced to confirm specificity of the PCR reaction. Ribosomal protein S18 (S18) expression was quantified using the same conditions as the other genes. No statistically significant differences were found between experimental groups so it was chosen as an endogenous reference gene to normalize qPCR data as it had a low inter-group variation and a similar level of expression to the analyzed genes. Statistical significance of relative gene expression between groups was analysed by one-way ANOVA using the software SigmaStat v.3.1 (SPSS Inc, Chicago, USA). Pearson correlations between the qPCR relative expression and microarray expression of both probes (average of individuals in each experimental group) were calculated for each gene. Statistical significance was established at p < 0.05.
List of primers used for real time RT-PCR.