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This procedure does not violate any European convention European Convention for the protection of Vertebrate animals used for experimental and other scientific purposes; CETS , Belgian law Art. This research was approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel Project number and all experiments were performed in accordance with the approved guidelines and regulations. For each proteomic downstream analysis, a minimum of five courting couples was combined this was repeated three times in total. Molecules present in the three types of water were extracted using two kinds of reversed-phase adsorbent resins, viz.

Elution of molecules from these reversed-phase columns was carried out by applying 7. Concentrated samples of courting couples were pooled and loaded onto a Source 5RPC column 4. The order in which the proteins of interest eluted did not change as a result of using a different column or flow rate.

By re-running courtship water on the 15RPC column, we were able to define the entire SPF elution range that had to be screened to identify any potential SPF molecules in male and female water see Supplementary Figure S3. Bands of interest were excised from the blot, destained with methanol and the amino acid sequence of the proteins determined on a Procise cLC protein sequencer Applied Biosystems.

Averaged profile spectra of proteins were obtained using Bruker Daltonics deconvolution software data analysis 4. Signal peptides were predicted using SignalP 4. In case multiple N-glycosylation sites were predicted, additional calculations were made with two or three N-linked glycan trees. Sequences in FASTQ format were obtained after filtering low quality reads, trimming of low quality sequence ends and trimming of adapters.

Apparent successfully cloned colonies were picked randomly and their inserts were amplified with Faststart Taq DNA polymerase. The obtained amplification products were cycle sequenced using the BigDye Terminator v3. Nucleotide sequences were assembled into contiguous sequences contigs with CodonCode Aligner v. Expression values are reported in RPKM reads per kilobase of transcript per million mapped reads. We combined translated amino acid sequences of 13 SPF precursors obtained in the current study from A.

Amino acid sequences were aligned using the automatically assigned L-INS-I algorithm and standard automatic parameters implemented in Mafft v7 ProtTest 2. Bayesian analyses were run with a mixed prior for the AA substitution model and gamma correction for among-site rate heterogeneity in MrBayes 3. Two parallel runs of four Markov chain Monte Carlo were executed for 10,, generations, with trees sampled every 1, th generation and the first 5, generations discarded as burn-in. Convergence of parallel runs was confirmed by split frequency standard deviations less than 0.

Adequate posterior sampling for each run was verified using Tracer 1. How to cite this article : Maex, M. Smith, H. Discovery of the axolotl and its early history in biological research In Developmental Biology of the Axolotl eds Armstrong, J. The history of the oldest self-sustaining laboratory animal: years of axolotl research. Part B Mol. Voss, R.

Ambystoma mexicanum , the axolotl: a versatile amphibian model for regeneration, development and evolution studies. Cold Spring Harb. Eisthen, H. Gasco, F. Les amours des axolotls. Google Scholar. Salthe, S. Courtship patterns and the phylogeny of urodeles.

Copeia , — Houck, L. Sever, D. Park, D. Discrimination of conspecific sex and reproductive condition using chemical cues in axolotls Ambystoma mexicanum.

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Ambystomatidae and Dicamptodontidae. A new vertebrate courtship pheromone, PMF, affects female receptivity in a terrestrial salamander. A recombinant courtship pheromone affects sexual receptivity in a plethodontid salamander. Senses 33, — Van Bocxlaer, I.

Side-by-side secretion of Late Palaeozoic diverged courtship pheromones in an aquatic salamander. B Biol. Wilburn, D. Pheromone isoform composition differentially affects female behaviour in the red-legged salamander, Plethodon shermani. Male courtship pheromones increase female receptivity in a plethodontid salamander. Kikuyama, S. Science , — Janssenswillen, S. Hedges, S. TimeTree: A public knowledge-base of divergence times among organisms. Bioinformatics 22, — Palmer, C.

Evolutionary replacement of components in a salamander pheromone signaling complex: more evidence for phenotypic-molecular decoupling. Evolution 61, — Abascal, F. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, — High pheromone diversity in the male cheek gland of the red-spotted newt Notophthalmus viridescens Salamandridae. BMC Evol. Osikowski, A. Greenbaum, D. Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol. Huang, T. PLoS One 6, e Gasteiger, E.

ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. Petersen, T. SignalP 4. Near the hotel you can find several museums and the European Parliament. The center of Brussels is located about two kilometers from Thon Hotel Bristol Stephanie and is easily accessible from Louise metro station. The hotel has excellent facilities including a sauna and a gym. The rooms are stylishly furnished and have air conditioning and free wireless internet. Hotel Cafe Pacific enjoys a central location in Brussels.

Many attractions are within walking distance of the hotel.

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By underground you can easily reach other attractions in Brussels. The hotel is highly rated by its guests due to the friendly staff, the central location and the clean rooms. All rooms feature free wireless internet. The hotel also has a gym and there is free wireless internet in the entire hotel. By public transport it is not much shorter.

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Other attractions near the hotel include the Toy Museum and City2 Mall. The main train station is almost one kilometer from the hotel. Warwick Hotel Brussels is a hotel with excellent facilities. The hotel is located in the heart of downtown Brussels. Several attractions are in the immediate vicinity such as the City Hall and the Grand Place.

Besides the central location, also the rooms of the Hotel Warwick Brussels are highly appreciated. The rooms are in fact beautifully decorated with luxurious materials. The hotel also has a gym and a sauna. The City Hall and Grand Place are also close to the hotel.

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