The whole-body thyroid hormones levels were measured in larvae
at stage G42. Thyroid hormones were extracted and measured
using Rat fT4 (Free Thyroxine) ELISA kit (Elabscience) and Rat fT3
(Free Triiodothyronine) ELISA kit (Elabscience). Approximately
5–6 larvae (1 g) for each treatment were minced to small pieces
which will be homogenized in 9 mL PBS buffer with a glass homogenizer
on ice. PBS buffer was prepared freshly. To further break the
samples, we sonicated the suspension with a spasmodic ultrasonic
disrupter or subject it to freeze–thaw cycles. Then centrifuge the
homogenized samples at 5000g for 15 min at 4 C. After centrifugation
at 5000g for 15 min at 4 C, the supernatants were collected
for thyroid hormones measurement according to the manufacture’s
instructions. The detection limit was 0.94 ng/mL. Thyroid
hormones levels are expressed as nanogram hormone per gram
body weight. Intra-assay and inter-assay variations were below
10% in our study. No significant cross-reactivity or interference
was observed for each kit.