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