Computational protocol: Gene Expression Profiling of the Cephalothorax and Eyestalk in Penaeus Monodon during Ovarian Maturation

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[…] Sample collection. Wild-caught adult female P. monodon (116.75 ± 17.99 g) were obtained from commercial hatchery suppliers in Innisfail, Queensland and air freighted to the Queensland Department of Primary Industries and Fisheries (QDPIF), Bribie Island Aquaculture Research Centre (BIARC), Woorim, Queensland. Captive-reared adult female P. monodon (114.5 ± 19.6 g) were obtained from the Australian Institute of Marine Science (AIMS), Townsville, Queensland, following grow-out (pond) culture at BIARC. Animals were stocked in 5 tonne tanks with flow-through seawater heated to 26oC and acclimated for 7 days whilst fed fresh diet (squid and mussels) twice daily. All animals were moult staged according to extent of epidermal retraction. For wild-caught animals, tissue samples were collected at various ovarian maturation stages based on in vivo observation of maturing ovaries, as described by Duronslet et al., (1975) , for subsequent classification by histological analysis of developing oocytes as follows: whole ovaries, cephalothorax and ESs, (containing the MTXO-SG complex) were collected from un-ablated inter-moult females (immature ovaries) euthanized in saline ice slurry, snap frozen in liquid nitrogen and stored at -80°C until processing. Additionally, seventy inter-moult females were unilaterally eyestalk ablated to induce ovarian maturation, eye- and carapace-tagged and maintained for a further 7 days as above. Captive-reared animals were sampled across several moult cycles after ablation. In vivo ovarian maturation stage, moulting, mortality and other behaviour were recorded daily. Animals were sampled at 2 h and 24 h post ablation (immature ovaries) and further sampled during this period with tissues of interest collected as outlined above (including remaining eyestalk) from random animals representing each of 4 in vivo ovarian maturation stages: immature, early maturing, late maturing and mature. Gonadosomatic index (GSI) was also calculated (ovarian weight expressed as a percentage of total body weight) for all samples. All wild-caught animals were sampled during the first moult cycle after ablation.Histological characterisation of ovarian samples. Small pieces (100 mg) of the middle ovarian lobes from specimens at selected ovarian maturation stages were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) solution (pH 7.2) overnight, washed with PBS at room temperature, dehydrated in ethanol series, embedded in paraffin, sectioned (6µm) and stained with either haematoxylin and eosin for detection of acidophilic and basophilic substances, Periodic Acid Schiff (PAS) for detection of carbohydrates (glycoproteins) or Luxol Blue for detection of phospholipids, or alternatively frozen and cut with cryostat and stained with Oil Red O for detection of simple lipids, as described by Bell & Lightner (1988) . Ovarian stages used in the present study were characterized largely as determined by Tan Fermin & Pudadera (1989) with some modifications as follows: previtellogenic (P) stage - the ovary contains only oogonia and basophilic previtellogenic oocytes at chromatin nucleolus and perinucleolus stage (GSI 1.7-2.9); vitellogenic (V) stage - in addition to the presence of oogonia and basophilic previtellogenic oocytes, the ovary contains yolk accumulating oocytes, the ooplasm of which is full of eosinophilic (acidophilic) yolk substances and also stains positive to PAS and Luxol blue indicating presence of glycoproteins and phospholipids respectively. Large globules in the ooplasm are also notable which stain positive with Oil Red O indicating simple lipids (GSI 3.5-6.5); round cortical rod (R) stage - staining affinities of oocytes are similar to those described for V stage ovaries with the addition of the appearance of round cortical rods (CRs) developing radially at the peripheral cortex of those oocytes containing yolk substances (GSI 7.7-10.5); elongated cortical rod (E) stage - staining affinities of oocytes are similar to those described for R stage ovaries except CRs are elongated and extended towards the nucleus (GSI 6.0-14.0).RNA isolation. Total RNA was isolated from small pieces (100mg) of the middle ovarian lobes, whole cephalothoraxes and whole eyestalks (initially ground under liquid nitrogen using mortar and pestle) from prawns of interest, using TRIZOL reagent as recommended by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA, USA). The samples were used for synthesis of complementary DNA (cDNA), creation of cDNA libraries and for construction and screening of microarrays. Concentration and purity of the RNA were determined using a spectrophotometer (GeneQuant Pro, GE Healthcare UK Ltd., Buckinghamshire, England) with 260 and 280 nm readings. RNA quality was assessed for all samples by visualisation on denaturing formaldehyde RNA gels (protocol recommended by Qiagen, Valencia, CA, USA) using ethidium bromide staining.cDNA library creation and sequence analysis. Three P. monodon cDNA libraries were created in our laboratory from total RNA isolated from ovary, eyestalk (containing X-organ/Sinus gland complex) and whole cephalothorax (containing target organs including hepatopancreas, Y-organ and mandibular organ) collected from wild-caught animals. Briefly, reverse transcription of total RNA (pooled in equal part from three animals from each ovarian maturation stage) was conducted using the SMART IV Polymerase Chain Reaction (PCR) cDNA synthesis kit (Clonetech Laboratories, Inc., Mountain View, CA, USA). First strand cDNA synthesis was conducted using Deoxyribonucleotide triphosphate (dNTP) mix, SMART IV oligonucleotide and CDS III/3' PCR primer with Powerscript reverse transcriptase. After determining optimal number of cycles in order to reduce the likelihood of producing redundant cDNA libraries, single stranded cDNA served as template for PCR based cDNA amplification using dNTP mix, 5' PCR primer, CDS III/3' PCR primer with Advantage II polymerase. Complementary DNA was then purified using the QIAquick™ PCR purification Kit (Qiagen, Valencia, CA, USA). To ensure efficient ligation into pGEM-T easy vector (Promega, Madison, WI, USA) a standard A-tailing procedure was conducted. Complementary DNA size fractionation was then conducted to separate larger cDNA species using CHROMA SPIN™ columns (Clontech Laboratories, Inc., MountainView, CA, U.S.A) according to the manufacturer's protocol. Ligations were then conducted and subsequent transformation into XL10 Gold ultracompetent cells (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol.Clones were sequenced using the 5' SMART PCR primer as sequencing primer. Sequences were edited (removal of poor sequence, vector and poly-A sequence) and then assembled into contiguous over-lapping sequence alignments (contigs) using Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA). The sequences were annotated with the name of the highest basic local alignment search tool (BLAST) score from an analysis of GenBank entries by the BLASTx and BLASTn procedures. Further putative functional annotation was assigned to each of the sequences by conducting a BLASTx similarity search using the GOanna annotation tool (http://agbase.msstate.edu/GOAnna.html). Protein domains were identified for selected sequences from the Pfam database (http://pfam.sanger.ac.uk).Microarray construction. A total of 601 sequences were selected from the three cDNA libraries created for this study together with an additional 387 sequences obtained from P. monodon Expressed Sequence Tag (EST) collections created from ovary, hepatopancrease and eyestalk sourced from publicly available databases (GenBank http://www.ncbi.nlm.nih.gov) for inclusion as probes on the microarrays. These included key transcripts encoding known eyestalk neuropeptides including several crustacean hyperglycemic hormone (CHH) family members from P. monodon where available and from other penaeids where not, as follows: P. monodon CHH-1 (AY346378.1) -2 (AF104931.1), Litopenaeus vannamei moult-inhibiting hormone (MIH) (S73824.1), P. monodon sinus gland peptide (PmSGP)-I (AF104386.1) -II (AF104387.1) -III (AF104388.1) -IV (AF104389.1) -V (AF104390.1), L. vannamei pigment-dispersing hormone (PDH)-1 (Y11723.1) -2 (Y11722.1) and Marsupenaeus japonicus PDH-3 (AB247562.1). Oligonucleotide microarrays were produced by CombiMatrix corporation (Mukilteo, USA) using the CustomArray™ 4 × 2K platform which contains four identical, independent 2240-feature microarrays on each slide. Between two and five oligonucleotide probes were designed and incorporated on each microarray for each of the selected transcripts.Target preparation and microarray hybridisation. Ovarian RNA samples from nine wild-caught animals representing six ovarian maturation stages (P, 2, 24, V, R, E) were used in microarray hybridisations. Similarly, RNA samples from three captive-reared animals representing four maturation stages (P, 24, V, E) were used in microarray hybridisations (Fig. A). For wild-caught animals, samples from each ovarian maturation stage were pooled into groups of four and five, enabling two hybridisations. For captive-reared animals, samples from each ovarian maturation stage from all three animals were pooled enabling one hybridisation for each stage. Importantly, as the four stages for captive-reared animals were (1) pre-ablation pre-vitellogenic (P), (2) post-ablation pre-vitellogenic (2 for 2 h post ablation, 24 for 24 h post ablation), (3) post-ablation vitellogenic (V), (4) post-ablation vitellogenic with cortical rods (E), this arrangement allowed for 2 samples of captive-reared pre-vitellogenic and 2 samples of captive-reared vitellogenic, thereby enabling t-tests between samples, while also allowing analysis across the whole 4 stages via cluster analysis. All hybridisations were single channel hybridisations conducted using equal amounts of RNA pooled from each individual.Reverse transcription of total RNA, RNA amplification by in vitro transcription and amplified antisense RNA (aRNA) labelling were conducted using the RNA ampULSe amplification and labelling kit (KREATECH Biotechnologies, Amsterdam, Netherlands) according to manufacturer's instructions. Briefly, reverse transcription reactions were performed using T7 Oligo(dT) primer with Arrayscript reverse transcriptase. Second strand synthesis was then performed and cDNA purified using cDNA filter cartridges and used as template for aRNA synthesis in in vitro transcription reactions. aRNA was then purified using aRNA filter cartridges and yield and quality of aRNA assessed by measuring the absorbance at 260 nm and 280 nm using a spectrophotometer (GeneQuant Pro, GE Healthcare UK Ltd., Buckinghamshire, England). 1 µg of non-modified aRNA was labelled non-enzymatically using the Universal Linkage System (ULS) technology coupled with Cy5 fluorochrome. Unbound ULS label was removed using included KREApure columns and aRNA fragmentation conducted using RNA fragmentation reagents (Applied Biosystems/Ambion, Austin, TX, USA) according to manufacturer's instructions. Labelled aRNA was then hybridised to the microarray slides according to manufacturer's instructions. Briefly, slides were assembled with supplied hybridisation caps in hybridisation clamps and re-hydrated prior to use by adding nuclease-free water and incubating at 65°C for 10 min. Pre-hybridisation steps (30 min) and hybridisations (16 h) were conducted in 30µl volumes using hybridisation solution containing 6× saline-sodium phosphate-EDTA (SSPE), 0.05% Tween-20, 20mM EDTA, 5×Denhardt's solution, 100ng/µl denatured salmon sperm DNA, 0.05% SDS, and 6×SSPE, 0.05% Tween-20, 20mM EDTA, 25% deionized formamide, 100 ng/µl denatured salmon sperm DNA, 0.04% SDS and 1 µg of target aRNA respectively, with microarrays loaded onto a rotisserie in a hybridisation oven and incubated at 45°C. Microarrays were then washed according to manufacturer's instructions and scanned wet using supplied imaging solution with LifterSlips™ (Thermo Fisher Scientific Inc., Waltham, MA, USA) applied. After imaging, microarrays were stripped for subsequent re-hybridisation up to three times using the CombiMatrix CustomArray stripping kit (CombiMatrix Corporation, Mukilteo, USA) according to manufacturer's instructions.Microarray data analysis. Microarray slides were scanned using a fluorescent microarray scanner (GenePix 4000B, MDS Analytical Technologies, Toronto, Canada). Microarray Imager (CombiMatrix Corporation, Mukilteo, USA) was then used to create spot intensity reports, generate gene ID mapping files and assign gene identification. Final intensity reports were retrieved as raw spot intensities in tab-delimited files. The data set is deposited in the Gene Expression Omnibus database (accession nos. GSE31862) at the following site: http://www.ncbi.nlm.nih.gov/geo. Final intensity reports were then used for further data analysis using data mining software Acuity 4.0 (MDS Analytical Technologies, Toronto, Canada). Data were pre-processed and normalised by applying background and floor correction, combining replicates (means) and global means normalisation. Data was grouped and classified using hierarchical and non-hierarchical clustering analyses. Genes displaying significant differential expression were identified via Two-Sample Students t-Tests. […]

Pipeline specifications

Software tools Sequencher, BLASTN, BLASTX, Goanna
Databases GEO Pfam AgBase
Application Amino acid sequence alignment
Organisms Penaeus monodon