Supplementary Information

Table 1. – GPS Coordinates of three different egg masses collected from rice fields at the IRRI experimental farm in Los Baños, Philippines

Egg Mass / Collection Point
1 / 14°10'21.5"N 121°15'37.5"E
2 / 14°10'09.1"N 121°15'25.9"E
3 / 14°09'36.6"N 121°16'06.0"E
Table 2. – Thyroid hormone sensitive genes run through two-step validation process - primer information and quality control results (P = Pass, F = Fail).
Cane toad
intestine / Luzon wart frog
intestine
Gene / Gene Function / Forward/Reverse Primer (F/R) / Primer Sequence / Specificity (P/F) / Efficiency (P/F) / Specificity (P/F) / Efficiency (P/F) / Annealing Temp (◦C) / Amplicon
rpl8 / Ribosomal protein L8 (housekeeping gene) / F / CAGGGGACAGAGAAAAGGTG / P / P / P / P / 60 / 270
R / TGAGCTTTCTTGCCACAG
klf9 / Krüppel-like factor 9 / F / CYGCTCAGTGTCTGGTGT / P / P / P / P / 62 / 250
R / ARGGGCCGGTACTTGTTT
thra / Thyroid hormone receptor alpha / F / TGATAAGGCCACAGGRTACCACTA / P / F / P / P / 60 / 141
R / CGGGTGATCTTGTCGATRA
thrb / Thyroid hormone receptor beta / F / CTCATAGAAGAAAACAGAGAAAARAGA / P / P / P / F / 60 / 237
R / GAAGGCTTCTAAGTCCACTTTTCC
mmp2 / Matrix metallopeptidase 2 / F / TACAACAGCTGCACTGAT / P / F / P / F / 62 / 248
R / TCCTTGTCATAGTCTTCTGT
dio2 / Iodothyronine deiodinase type II / R / CCTGGCTCTSTAYGACTC / P / F / P / F / 60 / 250
R / RGCTGATCCRAARTTGAC
thibz / TH-induced basic region leucine zipper containing transcription factor / F / ASCTCCRCAGAAYCAGCA / F / F / F / F / 62 / 354
R / TCACGTACCAGGCCAAAA
actb / Cytoplasmic beta-actin (housekeeping gene) / F / TACAGCTTCACCACCACAG / F / F / F / F / 60 / 478
R / TCCACATCTGCTGGAAGGT
col1a2 / Alpha 2 type 1 collagen (housekeeping gene) / F / CTGGTGGTGGATATGATGGT / F / F / F / F / 60 / 182
R / GAGTCTTAAGTCACGGCAAG

Table 3. Results of mixed effects models testing for the effects of butachlor exposure crossed with competition on activity of Luzon wart frog (Fejervarya vittigera) and cane toad (Rhinella marina) tadpoles. We tested whether activity levels varied throughout the experiment (Date), and across treatments. We accounted for variance in our experimental unit by including tank as a random effect. Degrees of freedom were 1, 12.

Activity Level
F. vittigera / R. marina
Treatment / F / P / F / P
Butachlor / 0.18 / 0.68 / 0.46 / 0.53
Competition / 4.94 / 0.05 / 0.88 / 0.36
Competition X Butachlor / 0.21 / 0.65 / 0.00 / 0.98
Date / 18.7 / <0.001 / 24.8 / <0.001

Fig. 1 Box plots depicting percentage of active Luzon wart frog and cane toad tadpoles by treatment group (n = 15 observations for each tank, and n = 75 observations per treatment) Mean plus lower (25%) and upper (75%) quartiles, where whiskers extend to lower and upper values excluding outliers, which are represented as points. Unique letters depict significant differences (P < 0.05) between treatments. Statistics were obtained from a mixed effects model that also included observation day as a fixed effect, and tank as a random effect. Activity measurements were taken for each tank everyday prior to feeding by the same individual at approximately the same time.