<p> 1</p><p>1 Electronic Supplementary Information for</p><p>2 Queen contact and among-worker interactions dually suppress worker brain dopamine as a </p><p>3 potential regulator of reproduction in an ant</p><p>4 </p><p>5 Hiroyuki Shimoji1,4,*, Hitoshi Aonuma2,3, Toru Miura1, Kazuki Tsuji4, Ken Sasaki5 and Yasukazu </p><p>6 Okada6,*</p><p>7</p><p>8 1. Laboratory of Ecological Genetics, Graduate School of Environmental Science, Hokkaido</p><p>9 University, Hokkaido, Japan</p><p>10 2. Research Center of Mathematics for Social Creativity, Research Institute for Electronic</p><p>11 Science, Hokkaido University, Hokkaido, Japan</p><p>12 3. CREST, Japan Science and Technology Agency</p><p>13 4. Department of Agro-Environmental Sciences, Faculty of Agriculture, University of the</p><p>14 Ryukyus, Okinawa Japan</p><p>15 5. Graduate School of Agriculture, Tamagawa University, Tokyo, Japan</p><p>16 5. Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan </p><p>17</p><p>18 * Correspondence:</p><p>19 H. Shimoji, Department of Agro-Environmental Sciences, Faculty of Agriculture, University of</p><p>20 the Ryukyus, 1, Senbaru Nishihara, Okinawa, 903-0213, Japan. </p><p>21 Y. Okada, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-</p><p>22 ku, Tokyo 153-8902, Japan.</p><p>23 E-mail address: [email protected] (H. Shimoji), [email protected] (Y.</p><p>24 Okada)</p><p>25 1.</p><p>2 3</p><p>26 Materials and Methods</p><p>27</p><p>28 Amine measurement</p><p>29 Brains were homogenized in 50 µl of ice-cold 0.1 M perchloric acid containing 12.5 ng/ml 3,4-</p><p>30 dihydroxyphenylacetic acid (DHBA) as an internal standard. Then, samples were centrifuged at</p><p>31 15,000 × g for 30 min at 4 °C and supernatants were used in HPLC-ECD analysis following</p><p>32 Aonuma and Watanabe (2012) as below. </p><p>33 The HPLC-ECD system consisted of a solvent delivery pump, a refrigerated automatic</p><p>34 injector and a C18 reverse-phase column (UG 120, Shiseido, Japan) maintained at 35 °C in a</p><p>35 column oven. An electrochemical detector (ECD-300, EICOM, Japan) with a glassy carbon</p><p>36 electrode was set at 0.82 V against an Ag/AgCl reference electrode. Signals from the</p><p>37 electrochemical detector were recorded and integrated using analysis software PowerChrom</p><p>38 (ADInstrument, Australia). The mobile phase contained 0.18 M of monochloroacetic acid and 40</p><p>39 µM of Na2EDTA (Wako) adjusted to pH 3.6 with NaOH (Wako). Sodium-1-octanesulfonate (1.62</p><p>40 mM) (Nacalai Tesque, Kyoto, Japan) and CH3CN (final concentration 7.4%, v/v, Nacalai) were</p><p>41 added to this solution. A constant flow rate of 0.7 ml/min was employed. External standards</p><p>42 (octopamine, dopamine, serotonin, tyramine and DHBA, (Sigma, St. Louis, USA) were used for the</p><p>43 chemical identification and quantification. Each biogenic amine peak was identified by comparing</p><p>44 the retention time and hydrodynamic voltamogram with those of the standards and uncertain peaks</p><p>45 were eliminated from the analysis. Measurements based on the peak area of the chromatograms</p><p>46 were obtained by calculating the ratio of the peak area of a substance to the peak area of the</p><p>47 external standard. The same method of HPLC-ECD analysis was applied in the following</p><p>48 experiments.</p><p>49</p><p>50 Analyses of Genes encoding dopaminergic system</p><p>51 Diadopr1 and Diadopr2 are categorized as D1-like excitatory receptors from their sequences</p><p>4 5</p><p>52 (Okada et al. 2015). Diaddc encodes an enzyme that synthesize DA from DOPA (Miyazaki et al.</p><p>53 2014), and this was quantified as indicatives of dopamine biosynthetic activity in brain (Table S1).</p><p>54 Brain RNA was individually extracted by RNAqueous micro (Life Technologies, Tokyo, Japan) and</p><p>55 stored in -80 °C until RNA extraction. Extracted RNA was subjected to DNAse treatment (DNAse,</p><p>56 Invitrogen) and reverse transcription (High-capacity cDNA Reverse Transcription kit, Applied</p><p>57 Biosystems, Foster City, CA). Fast SYBR Green Mater Mix (Applied Biosystems) and ABI PRISM</p><p>58 7500 (Applied Biosystems) with the condition of 95°C for 20 s, 95°C for 3 min, and 60°C for 30</p><p>59 min for 40 cycles were used in realtime qPCR. We used gapdh (Glyceraldehyde 3-phosphate</p><p>60 dehydrogenas) as an internal control gene (Okada et al. 2015).</p><p>61</p><p>62</p><p>6 7</p><p>63 References</p><p>64 Aonuma H, Watanabe T (2012) Changes in the content of brain biogenic amine associated with</p><p>65 early colony establishment in the queen of the ant, Formica japonica. PloS One, 7: e43377 (doi:</p><p>66 10.1371/journal.pone.0043377)</p><p>67 Miyazaki S, Okada Y, Miyakawa H, Tokuda G, Cornette R, Koshikawa S, Maekawa K, Toru M </p><p>68 (2014) Sexually dimorphic body color is regulated by sex-specific expression of Yellow gene in </p><p>69 Ponerine ant, Diacamma sp. PloS One, 9: e92875 (doi: 10.1371/journal.pone.0092875)</p><p>70 Okada Y, Sasaki K, Miyazaki M, Shimoji H, Tsuji K, Miura T (2015) Social dominance and </p><p>71 reproductive differentiation mediated by the dopaminergic signaling in a queenless ant. J Exp </p><p>72 Biol 218: 1091–1098</p><p>73</p><p>8 9</p><p>74 Figure legend</p><p>75 Figure S1. Dual suppression systems of worker reproduction in Diacamma sp. Q, Dom and Sub</p><p>76 indicate queen, dominant and subordinate workers, respectively. Ranks of dominants are in</p><p>77 parentheses. Solid and dotted lines indicate queen contact stimulus and among-worker dominance</p><p>78 interactions, respectively.</p><p>79</p><p>80 Figure S2. Effect of queen contact stimulus on gene expression level of internal standard</p><p>81 (Diagapdh).</p><p>82</p><p>10 11</p><p>83 Figure S1</p><p>84</p><p>85</p><p>86</p><p>87</p><p>88</p><p>89</p><p>90</p><p>91</p><p>92</p><p>93</p><p>94</p><p>95</p><p>96</p><p>97</p><p>98</p><p>99</p><p>100</p><p>101</p><p>102</p><p>103</p><p>104</p><p>105</p><p>106</p><p>107</p><p>108</p><p>12 13</p><p>109 Figure S2</p><p>110</p><p>111</p><p>14 15</p><p>112 Table S1. Information of primers used in real-time qPCR</p><p>113</p><p>114</p><p>115</p><p>116</p><p>117</p><p>118</p><p>119</p><p>120</p><p>121</p><p>122</p><p>123</p><p>124</p><p>125</p><p>126</p><p>127</p><p>16</p>