Used planarians belong to the species Schmidtea mediterranea (ClW4). Planarians were maintained as previously described in autoclaved water at 20°C and fed once per week with calf liver. Animals were starved for at least 2 weeks prior to the experiments.
Staphylococcus aureus (ATCC25923) was grown on blood agar plates (BioMerieux SA) at 37°C.
Worm feeding with bacteria
S. mediterranea were fed with S. aureus (1×10e9 bacteria) using a protocol adapted from a dsRNA feeding method as previously described. Briefly, S. aureus were suspended in homogenized liver, mixed with ultra-low-gelling-temperature agar and red food coloring, and allowed to solidify on ice. Room temperature solidified food was fed to planarians. After 2 h (defined as day 0) of feeding, the planarians were extensively washed, and then collected or kept at 20°C in function of the experiments. Each experiment has been made in triplicate, for each experiment there were 10 worms per time point.
As previously described, S. mediterranea were collected and homogenized in PBS. The lysate was passed 5 times through a sterile syringe with a 29G needle to disrupt planarian tissue clumps, and CFUs were counted after plating of 10 μl of a serial dilution of planarian lysates onto blood agar plates (BioMerieux SA) followed by an incubation of 24 h at 37°C.
Smed-LTA4H has been identified using PlanMine transcriptomes database (http://planmine.mpi-cbg.de). Protein sequence of the Hs-LTA4H (NP_005082.1) has been blast via blastp against planmine transcriptomes dd_Smed_v6 using default parameters (e-value 1e-5). We identified the sequences producing a significant alignment with Hs-LTA4H. The top BLAST hit was used to predict Smed-LTA4H via FGENESH+ (http://www.softberry.com/) using default parameters. The homology at the protein level between predicted Smed-LTA4H and Hs-LTA4H (NP_005082.1) was analyzed using blastx. The conservation scoring was performed by PRALINE (http://www.ibi.vu.nl/ programs/pralinewww/) using default parameters. The results are color coded for amino acid conservation and the scoring scheme works from 0, for the least conserved alignment position, to 10, for the most conserved alignment position.
To generate the Smed-LTA4H RNAi, cDNA from S. mediterranea was amplified via PCR designed with Primer3 (http://primer3.sourceforge.net/) and containing attB recombination sequences, and then the obtained PCR product was cloned as described elsewhere. The dsRNA in silico accuracy prediction was defined as follows. Targeted transcript sequences were extracted between the 3' end of the 5’ primer and the 5' end of the 3’ primer used for cloning. The extracted sequences were then cut into 21 mers using a sliding window of 1 nucleotide. All possible RNAi sequences were then generated, and each putative RNAi sequence was aligned to the planarian transcriptome using BLAST with a word size of 21; only perfect matches were considered. For each transcript for which an RNAi was designed, a theoretical target accuracy was calculated based on the number of RNAi sequences matching the target divided by the total number of generated RNAi sequences. Smed-LTA4H primers (left primer CTCGTTCCGGTTCTTGTCGA, right primer GCAGGCGTGTGATTTGATCG), attB recombination sequences (CATTACCATCCCG).
Delivery of dsRNAs
dsRNAs were delivered to S. mediterranea as previously described. The quality of Smed-LTA4H knock down was controlled via real-time RTqPCR as described elsewhere. Primers used for real-time RTqPCR were for Smed-LTA4H (left primer TATGCCACTACCGCGAACTG, right primer CGGGCTATCTTGGCATGGAA ). Results were normalized by the expression of the control housekeeping gene Smed-ef2.
Total RNA (1 animal per sample, and 3 animals per experimental time point) were extracted from planarians using the Trizol method, as recommended by the manufacturer (Invitrogen). Experiments were made in triplicates.
The results are expressed as means ± SD and were analyzed using the nonparametric Mann-Whitney U test. Differences were considered significant at p <0.05.
Nucleotide sequence and protein sequence of predicted Smed-LTA4H
>FGENESH: predicted Smed-LTA4H 617 aa MALSLNDPVSYANSEDYVTEHLDFDWIIDFDRKLIEGSVNLQLKNLTKKQADLKLDTKYLDIKKVSVKGTETRFDIVDCKIEALGSCLKIPIGISDEKFSVCIQYATTANCTALQWLIPEATVGKKHPYLFSQCQAIHARSLFPCQDSPCVKSTYSAKVKAPNQFNVLMSAIKNEDPTKEGNHLIHKFEQKVNISSYLVAIVCGNLAGKKLSNRSTVWSEPEMVEKAAHEFADTENLLSIAESICGEYVWGIYDLLVLPPSFPYGGMENPCLTFVTPTLLVGDKSLVFVVAHEITHSWTGNLVTNKTWEDFWLNEGHTRYIERLIMEKYYDSELIRHLLISIGIAELQESLNAYGEGHKFNQLVPVLVECDPDDSFSRVPYEKGSNLLFHLENICGKDQMLRWLKCYFEHYKYKSLTTDEWKAFFYKYMIDKENISKAKLDNVDWKKWFYGRWKIPVDHVLKRDHGDASERLADKWINLNPEISDSDQKDLLLEYESFLPHQKYYFLQVLNDFKEAKLHSKVIETMGNLYQYTTSSNSEILFGWVMLCIRSNHTPAIDQIFDFLNSQGRLKYTKPIYRNLANWPNDHIKRLTVKNFLKNENTMHPITAKSIRLLLCL