Im 31

The larvae from 2 inbred populations (GG♂ × GG♀, SD♂ × SD♀) were injected with KD, YN, XZ or QH fungal isolates of O. sinensis. An aliquot of 4 µL blastospore suspension containing 1.2 × 10⁴ blastospores was injected into each 6th instar larva by a microinjection system (IM-31; Narishige, Tokyo, Japan). One hundred and eighty larvae were used for each replicate, and three replicates were set for each injection. Larvae injected with PBS buffer or without any injection were set as controls. The injected larvae were reared at 4 °C for one week and then transferred to a culture room at 13 °C. After 90 days, about 10 µL of hemolymph of each injected larva (6th instar) was sampled to confirm the presence of the growing blastospores stained by Calcofluor White (Sigma, Kanagawa, Japan) and observed by a fluorescence microscope (IX73; Olympus, Tokyo, Japan). The injected larvae were reared at 13 °C until the larvae became stiff and were coated with growing mycelia. The mummified larvae with head upward were then planted into soil of 55–60% humidity to induce the formation of stroma at 4 °C. The survival and mummification of the injected larvae were monthly checked. Data on larval infection of the hybrid populations could not be gathered due to an insufficient number of larvae available for fungal injection.

In utero electroporation was performed as described previously [17] (link). Briefly, pregnant ICR mice carrying E12.5 embryos (Shimizu Laboratory Supplies Co., Kyoto, Japan) were deeply anesthetized with pentobarbital (50 mg/kg) prior to electroporation. Plasmid DNA was introduced into the central canal of the spinal cord of the embryos by a microinjector (IM-31; Narishige, Tokyo, Japan). Half-ring-type electrodes were attached to the uterus, and 5 electric pulses (35 V, 50 ms) were applied with an electroporator (CUY21SC; Nepagene, Ichikawa, Japan). All animal experiments were approved by the Animal Experimentation Committee of Kansai Medical University.

Zebrafish embryos were harvested and incubated at 28 °C for the first 48 h. Al 48 hpf, embryos were anesthetized with 0.003% tricaine (Sigma). The human colorectal carcinoma cells with 80% confluence were trypsinized and harvested (approximately one million cells). Cells were labelled with Dil lipophilic dye and concentrated in 10 µL of phosphate-buffered saline (PBS) containing 2% polyvinylpyrrolidone 40 (PVP40) to prevent cell aggregation. They injected 200–300 cells in circulating embryos using a micromanipulador and an electric microijector IM-31 (Narishige). They were used to inject borosilicate glass capillary needles (1 mm O.D. × 0.75 mm I.D.; World Precision Instruments, Sarasota, FL, USA) with an outlet pressure of 34 kPa and an injection time of 30 ms. The embryos were incubated for four days after injection (hpi) al 34 °C in Petri dishes. At 24 hpi the embryos were imaged and the compound JHOR11 was added to the water. The concentration used was 80 µM. For in vivo monitoring of cells within the embryo, images were also taken at 96 hpi.