Glycerin

Fish larvae were bleached and transparentized according to previous reports^{51 (link),52 (link)}, with several modifications. Formalin (10% formalin solution), potassium hydroxide (KOH), hydrogen peroxide, Triton-X, phosphate-buffered saline (PBS), HEPES, and glycerin were purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). All other chemicals used in this study were of analytical grade. O. javanicus larvae (exposure group n = 6; negative control n = 2) were fixed in 10% formalin at 4 °C for 3 days. After removal of formalin, larvae were incubated in pre-bleaching solution (0.3% hydrogen peroxide in PBS) at room temperature overnight. Pre-bleaching solution was replaced with bleaching solution (3% hydrogen peroxide in PBS) and incubated at room temperature overnight. After bleaching, the bleaching solution was replaced with a tissue transparency solution (5% formalin, 5% Triton X-100, 1% KOH in PBS) and incubated at 42 °C for 24 h. In O. latipes, which has a less pigmented peritoneum than O. javanicus, bleaching was omitted, i.e., the tissue transparency solution was directly added after fixation, and incubated at 42 °C for 48 h. Transparency of each sample was confirmed under the microscope.

2′‐Deoxy‐5‐fluorouridine (FUdR), 30% hydrogen peroxide (H₂O₂), 4% paraformaldehyde (PFA) phosphate buffer solution, L‐ascorbic acid, bovine serum albumin (BSA), ethylenediaminetetraacetic acid disodium (EDTA‐2Na), FeCl₃･2H₂O, glycerin, hypoxanthine, sodium azide, sodium dodecyl sulfate (SDS), and sodium hypochlorite were purchased from FUJIFILM Wako Pure Chemical Co. Carnosine (Car) and xanthine oxidase were purchased from Sigma‐Aldrich. Aminoguanidine hydrochloride, D‐glucose, monopotassium phosphate (KH₂PO₄), NaCl, and NaOH were purchased from Kanto Chemical Co. 2‐methyl‐6‐p‐methoxyphenylethynyl‐imidazopyrazinone (MPEC) and β‐mercaptoethanol were purchased from ATTO Co. and Nacalai Tesque Inc., respectively. 2′,7′‐dichlorofluorescein diacetate (H₂DCF‐DA; Invitrogen) and dihydroethidium (DHE; FUJIFILM Wako Pure Chemical Co.) were used for ROS visualization. Synthesized Leu–Lys (LK) and Lys–Leu (KL) were purchased from Scrum Inc.

After feeding bioassays, the worms that exhibited the most developed reproductive organs within each group were selected and embedded for histological sectioning. The worms were relaxed in cold 2% (v/v) HCl in 5/8 Holtfreter’s solution⁵⁴ (link) for 5 min and were then fixed in 4% paraformaldehyde and 5% methanol in 5/8 Holtfreter’s solution for 3h at room temperature (RT). The fixed specimens were dehydrated through an ethanol series, cleared in xylene, and embedded in the Paraplast Plus embedding medium (Sigma-Aldrich Co., St. Louis, MO, USA). Embedded specimens were cut into 5 μm thick sections using a microtome (Leica RM2125 RTS, Leica Microsystems Ltd., Wetzlar, Germany). Tissue sections were placed on slide glasses coated with 5 mg/mL egg albumin (Nacalai Tesque, Japan) in 50% glycerin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), stretched at 40°C overnight, and stained with Mayer’s Hematoxylin (No. 3000-2, Muto Pure Chemicals, Tokyo, Japan) and 1% Eosin Y solution (No. 3200-2, Muto Pure Chemicals, Japan).