References 1. Coates, J. & de Bono, M. Antagonistic pathways in neurons exposed to body fluid regulate social feeding in Caenorhabditis elegans. Nature 419, 925-929 % Oxygen (2002). 2. Oka, T., Toyomura, T., Honjo, K., Wada, Y. & Futai, M. Four subunit a 22 20 18 16 isoforms of Caenorhabditis elegans vacuolar H+-ATPase. Cell-specific expression during development. J. Biol. Chem. 276, 33079-33085 (2001). 3. Yu, S., Avery, L., Baude, E. & Garbers, D. L. Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors. Proc. Natl. Acad. Sci. U S A 94, 3384-3387 (1997). 21% to 17.4% O 2 21% to 19.2% O 2 4. Okkema, P. G., Harrison, S. W., Plunger, V., Aryana, A. & Fire, A. Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics 135, 385-404 (1993). 5. Mello, C. C., Kramer, J. M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959-3970 (1991). 6. Brunori., Eraldo Antonini and Maurizio. Frontiers of Biology (lsevier, New York, North-Holland, Amsterdam, 1971). 7. Kerr, R. A. & Schafer, W. R. Intracellular Ca2+ imaging in C. elegans. Methods Mol. Biol. 351, 253-264 (2006). 8. Bennett-Lovsey, R. M., Herbert, A. D., Sternberg, M. J. & Kelley, L. A. Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70, 611-625 (2008). 9. Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. 0 60 120 180 240 300 360 420 480 Bioinformatics 22, 195-201 (2006). Supplementary Figures Time (secs) Supplementary Figure 1. O 2 stimuli used in behavioural experiments. The graphs show O 2 levels in the behavioural arena when O 2 is switched from 21% to 19.2% (blue) or 17.4% (red). Measurements were made using fibre-optic O 2 microsensor probes (PreSens). The bars under the graph show the time intervals during which the speed of animals was measured. 7 1
265 recombinants 34 recombinants 2 recombinants V unc-46 dpy-11 kb 5098 5521 5558 5566 5795 6515 Copy 1 Copy 2 Recomb 1 Recomb 2 kb 5558 5559 5560 5561 5562 5563 5564 5565 5566 Polymorphisms 5557739 5559017 5559070 5559363 5559580 5559581 5559740 5559911 5560380 5561117 5561378 5562722-5563486 5566156 Supplementary Figure 2. Fine genetic mapping and molecular identification of glb- 5(Haw) By genotyping and/or phenotyping 265 Dpy-non-Unc and Unc-non-Dpy recombinants between unc-46 and dpy-11, we mapped the locus conferring Hawaiianlike responses to an 8 kb interval between the SNP at 5566156 and the SNP at 5557739. Recomb 1 and 2 are the two recombinants that narrow the position of the naturally varying locus to this 8 kb interval. Arrows indicate where CB4856 sequences begin in each recombinant chromosome. This interval contains 11 polymorphisms between N2 and CB4856. Ten of these are SNPs not found in other wild strains that show Hawaiian-like O 2 responses (see Supplementary Table 2). The remaining polymorphism was a partial duplication found in glb-5(bri) but not in glb- 5(Haw) allele. The unduplicated Haw allele was found in all strains that display Hawaiian-like O 2 responses (see Supplementary Table 1). Horizontal numbers refer to position, in kb, along chromosome V. Slanting numbers indicate the positions of SNPs in the region between N2 and CB4856. Shown in blue and red are the two copies of the duplication found in the N2 glb-5 allele. Boxes represent exons, lines introns. Arrowheads point direction of transcription. 2
a Copy 1 Copy 2 GACCTTTGAGTGCAATTAACGAGGAAATTCGTGAATACGATCAATTGAGC AGAGAACTTGAAGTTtga Glu Val Stop N b Copy 1 Copy 2 N ATTTGTgt...agacatca Cys Met Stop tgcatgtga GGTTGGCAATTCGGGGATGCTTCCTAACAATGTTAGAAAAGTATCCACAA GTTCGTCCAATTTGGGGGTTTGGAAAAAGAATTGAGGGAAGGGGTGATGA GACATGGAAGCCTGAGATCGTGGAGGATTTTTATTTTAGgtaagacgggt atcttaagtttgaagttaagagtatcatcggagagcattttttctatatt gtattacatcatgctagaaaatcataataaaacactccaaacacttttca gacttgtcaaaaatttcagtcaaagttttggcaaattgccgaattttcca aaaatgtagactatctgagtaatttttatcatagggtttaacgcctacaa aattgaaaaaaaaaatcataaaaaattttctgaaaagtcttactggcata tttggtcattttagcactatcggggcaattttaagcatttatcccgctga cgacactcactttcaataggtctagaaatttaaactggaaaaatttcaac aaataagtaactaaaaataataagataacgatttagccaacgtgttttac atggttcaattaattttaacttttctgatgttaactttttttaaactgaa agggtattgactttttaaaactaaactttttgcgcagaacattttatact tggaaaatttctagacatcattgcgcatccctccaggcagctttgaatat GATAATTCAAAACAAAGACGACAAAAGTGGAATGCGGCGGATGCTCAACG AAATGGGAGCTCATCACTTTTTCTACGATGCATGTGAACCACATTTTGAA GTTTTTCAAGACAGTCTCCTAGAATCAATGAAGCTTGTATTAAATGGTGG TGACTCGTTGGATGATGATATTGAGCAATCTTGGATTTGTgtgagtgaaa agtttggagaacactttgaaatttaaagtgctgttgtgatcatcggagag cattttttctatattgtattacatcatgctagaaaatcataataaaacac tccaaacacttttcagacttgtcaaaaatttcagtcaaagttttggcaaa ttgccgaattttccaaaaatgtagactatctgagtaatttttatcatagg gtttaacgcctacaaaattgaaaaaaaaaatcataaaaaattttctgaaa agtcttactggcatatttggtcattttagcactatcggggcaattttaag catttatcccgctgacgacactcactttcaataggtctagaaatttaaac tggaaaaatttcaacaaataagtaactaaaaataataagataacgattta gccaacgtgttttacatggttcaattaattttaacttttctgatgttaac tttttttaaactgaaagggtattgactttttaaaactaaactttttgcgc agaacattttatacttggaaaatttctagacatcattgcgcatccctcca ggcagctttgaatatgataattcaaaacaaagacgacaaaagtggaatgc ggcggatgctcaacgaaatgggagctcatcactttttctacgatgcatgt gaaccacattttgaagtttttcaagacagtctcctagaatcaatgaagct tgtattaaatggtggtgactcgttggatgatgatattgagcaatcttgga tttgtgtgagtgaaaagtttggagaacactttgaaatttaaagtgctgtt gtgatttcaaaaaacggtacttttaatttgaattacatgaagaacagact acttgtactaccaaaaaataaatttccataacttttcgaattgcagctgc TCCAAACAATCCGACTACATATGGGAGAAGGAATCGAAATTCAAAGAGCC AACTATCTAACGCAATGCTTGATTCCAAAAGAAATGGAAGAAGTACGTGC GAATTGGATGCAAGTCGAAAATTACGGGTTTCGAAAAGCCGGGCTTTTAT c Copy 1 Copy 2 N Supplementary Figure 3 The glb-5(bri) allele generates several mrna products a-c. RT-PCR analysis of glb-5 from N2 identifies multiple alternately spliced cdna products (a-c), whereas the glb-5(haw) allele generates one major product, corresponding to (c). Different exons are given different colours for clarity. Transcripts in (a) and (b) introduce premature stop codons into the glb-5 open reading frame. In (b), arrows point to the start of each copy of the glb-5(bri) duplication; the arrowhead marks splicing to an exon in the duplication, which results in a premature stop (underlined). 3
a A B C glb-5 -----------------RIVDDDFELARTHWIQLQKSNKQGLAIRGCFLTML-EKYPQVR 42 Rice nshb -----------------SFSEEQEALVLKSWAILKKDSANIALRFFLKIFE---VAPSAS 52 neuroglobin ------------------MERPEPELIRQSWRAVSRSPLEHGTVLFARLFA---LEPDLL 39 cytoglobin -----------------ELSEAERKAVQATWARLYANCEDVGVAILVRFFV---NFPSAK 57 D.melanogaster Hb ------------------MNSDEVQLIKKTWEIPVATPTDSGAAILTQFFN---RFPSNL 39 SwMb -----------------VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFK---SHPETL 41 C D E glb-5 PIWGF -GKRIEGRGDETWKPEIVEDFYFRHHCASLQAALNMIIQN----KDDKSGMRRM 94 Rice nshb QM -FSFLRNSD-VPLEKN-------PKLKTHAMSVFVMTCEA-AAQLRKAGKVTVRDTT 100 neuroglobin PL--FQYNCRQFSSPEDCL-----SSPEFLDHIRKVMLVIDAA----VTNVEDLSSLEEY 86 cytoglobin QY -FSQFRHME-DPLEME-----RSPQLRKHACRVMGALNTV----VENLHDPDKVSSV 103 D.melanogaster Hb EK--FPFRDVP -LEELSG------NARFRAHAGRIIRVFDES---IQVLGQDGDLEKLD 86 SwMb EK -FDRFKHLK-TEAEMK-----ASEDLKKHGVTVLTALGAI-------LKKKGHHEAE 88 F G H glb-5 LNEMGAHHFFYDACEPHFEVFQDSLLESMKLVLNGGDSLDDDIEQSWICLLQTIRLHM 146 Rice nshb LKRLGATHLKY-GVGDAHFEVVKFALLDTIKEEVPADMWSPAMKSAWSEAYDHLVAAI 152 neuroglobin LASLGRKHRAVGVKLSSFSTVGESLLYMLEKCL-GPAFT-PATRAAWSQYGAVVQAMS 138 cytoglobin LALVGKAHALKHKVEPMYFKILSGVILEVIAEEFA-ND-FPVETQKAWAKLRGLIYSH 160 D.melanogaster Hb EIWTKIAVSHIPRTVSKESYNQLKGVILDVLTAA--CSLD-ESQAATWAKLVDHVYGIIF 140 SwMb --LKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRH-PGDFGADAQGAMNKALELFRKDI 141 H glb-5 ------------------------------- 155 Rice nshb KQEMKPAE------------------------166 neuroglobin RGWDGE------------------------- 151 cytoglobin VTAAYKE--VGWVQQVPNTTTPPATLPSSGP 190 D.melanogaster Hb KAIDDDGNAK----------------------153 SwMb AAKYKEL-----GYQG--------------- 154 b Supplementary Figure 4. Alignment of the glb-5 globin domain with other globins and best-fit structural prediction. a. Alignment of the glb-5 globin domain with non-symbiotic plant hemoglobin from rice (PDB ID: 1D8U), neuroglobin (H. sapiens), cytoglobin (M. musculus), globin 1 (D. melanogaster), and myoglobin (sperm whale, P. catodon) using the ClustalW2 program. Above the sequences are indicated predicted helices. Conserved residues are in gray. b. Model of the structure of the GLB-5 globin domain. The 3D-Pssm and Phyre programs 8 were used to find proteins with a similar fold. The best match was the rice non-symbiotic hemoglobin. This was used as a template to generate a 3D model of the glb-5 globin domain in the SWISS-MODEL program 9. The model generated is aligned with the 1D8U (rice) to highlight the hexacoordinated heme. The rice globin is colored in green and glb-5 in magenta. 4
a b kda ** * 102 76 52 38 c 0.5 0.4 His 6 - GLB-5 Ferric Ferrous Absorbance 0.3 0.2 0.1 0 375 475 575 675 Wavelength (nm) Supplementary Figure 5. GLB-5 biochemistry a. GLB-5 tagged with maltose binding protein expressed and purified from E. coli is a soluble red-brown protein. b. Recombinant His-tagged GLB-5 expressed and purified from E. coli and run on an analytical SDS-PAGE gel. The single asterisk marks the predicted monomer and the double asterisk potential multimers of GLB-5. c. Oxidized and reduced GLB-5 protein exhibits absorbance spectra characteristic of hexa-coordinated globins. Spectra in the visible range showing the characteristic twin peaks of a hexa-coordinated globin. The recombinant protein shown is N-terminally His-tagged GLB-5. Although this protein localized to E. coli inclusion bodies it could be refolded in vitro with hemin. Its spectral properties were similar to those of MBPtagged GLB-5, which could be expressed as a soluble protein in E. coli (see Figure 2). 5
O 2 Concentration (%) 25 20 15 10 5 0 0 5 10 15 Time (seconds) Supplementary Figure 6. Rapid oxygen switches in the microfluidic device. The time taken to switch from 7% to 21% O 2 in the microfluidic device under our recording conditions was less than 3 seconds. Measurements were made using a small optode sensor spot (PreSens, GmBH) placed in the agar under the device. 6
a 250 21% O 2 Speed (μm/s) 200 150 100 50 17.4% O 2 b 0 CB4856 BW288 PS1185 PS1186 VT847 EM464 PB227 PB228 PB229 SB129 PS1010 RGD1 RGD2 JU727 Cel Cbri Crem Cbre Csp3 Cspn Vancouver (1) Oregon (4) (1) Madison (1) (1) (9) (4) Hawaii (1) California Madeira (1) England (2) (1) France (42) Germany (22) glb-5 (Haw);npr-1 215F glb-5 (Bri);npr-1 215F glb-5 (Haw);npr-1 215V glb-5 (Bri);npr-1 215V Kenya(2) South Africa (2) Adelaide (3) Supplementary Figure 7. Distribution of glb-5 and npr-1 alleles in different C. elegans and Caenorhabditis sp isolates. a. Wild isolates from other Caenorhabditis species also reduce their locomotory activity in response to slight declines in ambient O 2 close to 21%. Cel, C. elegans; Cbri, C. briggsae; Crem, C. remanei; Cbre, C. brenneri; Csp3, Caenorhabditis species 3; Cspn, unnamed Caenorhabditis species. b. World map showing occurrence of different allelic combinations of npr-1 and glb-5 in 98 wild isolates of C. elegans. 7
Supplementary Table 1 npr-1 and glb-5 alleles in C. elegans wild strains Strain Area of origin Country Phenotype npr-1 glb-5 AB1 Adelaide Australia Social 215F unduplicated AB2 Adelaide Australia Social 215F unduplicated AB3 Adelaide Australia Social 215F unduplicated CB3196 Altadena California Social 215F unduplicated CB3197 Altadena California Social 215F unduplicated CB3198 Pasadena California Social 215F unduplicated CB3199 Pasadena California Social 215F unduplicated CB4852 Taunton England Social 215F unduplicated CB4853 Altadena California Social 215F unduplicated CB4854 Altadena California Social 215F unduplicated CB4855 Palo Alto California Social 215F unduplicated CB4856 Hawaii Hawaii Social 215F unduplicated CB4857 Claremont California Social 215F unduplicated CB4858 Pasadena California Social 215F unduplicated CB4932 Taunton England Social 215F unduplicated ED3040 Johannesberg S. Africa Social 215F unduplicated ED3049 Ceres S. Africa Social 215F unduplicated ED3054 Limuru Kenya Social 215F unduplicated ED3077 Nairobi Kenya Social 215F unduplicated JU258 Ribeiro Frio Madeira Social 215F unduplicated JU262 Le Blanc France Social 215F unduplicated JU263 Le Blanc France Social 215F unduplicated JU299 Le Blanc France Social 215F unduplicated JU300 Le Blanc France Social 215F unduplicated JU301 Le Blanc France Social 215F unduplicated JU302 Le Blanc France Social 215F unduplicated JU303 Le Blanc France Social 215F unduplicated JU304 Le Blanc France Social 215F unduplicated JU305 Le Blanc France Social 215F unduplicated JU306 Le Blanc France Social 215F unduplicated JU307 Le Blanc France Social 215F unduplicated JU308 Le Blanc France Social 215F unduplicated JU309 Le Blanc France Social 215F unduplicated JU310 Le Blanc France Social 215F unduplicated JU312 Merlet France Social 215F unduplicated JU314 Merlet France Social 215F unduplicated JU315 Merlet France Social 215F unduplicated JU316 Merlet France Social 215F unduplicated JU317 Merlet France Social 215F unduplicated JU318 Merlet France Social 215F unduplicated JU319 Merlet France Social 215F unduplicated JU320 Merlet France Social 215F unduplicated JU321 Merlet France Social 215F unduplicated JU322 Merlet France Social 215F unduplicated JU323 Merlet France Social 215F unduplicated JU342 Merlet France Social 215F unduplicated JU343 Merlet France Social 215F unduplicated JU344 Merlet France Social 215F unduplicated JU345 Merlet France Social 215F unduplicated JU346 Merlet France Social 215F unduplicated JU347 Merlet France Social 215F unduplicated JU362 Franconville France Social 215F unduplicated JU365 Franconville France Social 215F unduplicated JU366 Franconville France Social 215F unduplicated JU367 Franconville France Social 215F unduplicated JU368 Franconville France Social 215F unduplicated JU369 Franconville France Social 215F unduplicated 8
JU370 Franconville France Social 215F unduplicated JU371 Franconville France Social 215F unduplicated JU401 Hermanville France Social 215F unduplicated JU406 Hermanville France Social 215F unduplicated KR314 Vancouver Canada Social 215F unduplicated MY19 Roxel Germany Social 215F unduplicated MY1 Lingen Germany Social 215F unduplicated MY10 Roxel Germany Social 215F unduplicated MY11 Roxel Germany Social 215F unduplicated MY12 Roxel Germany Social 215F unduplicated MY13 Roxel Germany Social 215F unduplicated MY16 Mecklenberg Germany Social 215F unpdulicated MY17 Roxel Germany Social 215F unduplicated MY18 Roxel Germany Social 215F unduplicated MY2 Roxel Germany Social 215F unduplicated MY20 Roxel Germany Social 215F unduplicated MY21 Roxel Germany Social 215F unduplicated MY22 Roxel Germany Social 215F unduplicated MY23 Roxel Germany Social 215F unduplicated MY3 Roxel Germany Social 215F unduplicated MY4 Roxel Germany Social 215F unduplicated MY5 Roxel Germany Social 215F unduplicated MY6 Roxel Germany Social 215F unduplicated MY7 Roxel Germany Social 215F unduplicated MY8 Roxel Germany Social 215F unduplicated MY9 Roxel Germany Social 215F unduplicated PB303 n/a Oregon Social 215F unduplicated PB306 n/a Oregon Social 215F unduplicated PX174 Devil's Lake Oregon Social 215F unduplicated PX178 Eugene Oregon Social 215F unduplicated PX179 Eugene Germany Social 215F unduplicated RC301 Freiburg France Social 215F unduplicated CB3191 Altadena California Solitary 215V duplicated CB4507 Palm Canyon California Solitary 215V duplicated CB4555 Palo Alto California Solitary 215V duplicated DH424 El Prieto California Solitary 215V duplicated N2 Bristol England Solitary 215V duplicated PX176 Eugene Oregon Solitary 215V duplicated TR389 Madison Wisconsin Solitary 215V unduplicated TR403 Madison Wisconsin Solitary 215V duplicated 9
Supplementary Table 2. SNPs in the glb-5 interval Name Position Alters CB N2 CB4932 JU263 JU345 MY1 RC301 hw76737 5559017 intron 1 T C C C C C C hw76738 5559070 intron 1 T C C C C C C hw76739 5559363 intron 1 T G G G G G G hw76740 5559580 intron 1 G T T T T T T hw76741 5559581 intron 1 T C C C C C C - 5559740 intron 1 - A A A A A A hw76742 5559911 intron 1 T A A A A A A hw76743 5560380 intron 1 C A A A A A A C18C4(8) 5561117 intron 1 A T T T T T T hw76744 5561378 intron 2 A T T T T T T Supplementary Table 3. The unduplicated glb-5 allele is likely to be the ancestral form of this gene. Genotype Genotype Species Strain npr-1 glb-5 C. brenneri CB5161 215F Unduplicated C. briggsae AF16 215F Unduplicated C. remanei EM464 215F Unduplicated 10