Leitz 1512 microtome

After 24 h fixation, mantle samples were transferred to 70% ethanol and then processed for paraffin embedding following an automated program in a Leica AS300S tissue processor. Paraffin blocks were cut at a thickness of 5 μm using a Leitz 1512 microtome (Leica, Vienna, Austria). Sections were then automatically stained with hematoxylin/eosin using a Leica AutostainerXL (Leica) to finally mount the slides with DPX (Sigma-Aldrich, St. Louis, MO, USA) for microscopic visualization. Sex and developmental stage of each animal was identified within any of the six gamete developmental stages (1 = resting, 6 = spawning) described by Seed [23 (link)]. Histological analysis revealed no lesions nor any meaningful infestation or disease.

Samples were fixed in FAA (4% formalin/50% alcohol/5% acetic acid in water to 100%). Samples were washed twice in 50% ethanol, then dehydrated through a graded ethanol series at 2‐hr intervals, then 50:50 ethanol/xylene (2 hr), two changes of xylene (3 hr each), 1:1 wax/xylene (3 hr), and two changes of paraffin wax (12 hr each), and embedded in Paraplast wax (Oxford Labware, www.kendellhq.com) Sections of 10 μm thickness were cut using a Leitz 1512 microtome (Leica.www.leica-microsystems.com), placed on positively charged glass slides and dried overnight in a slide dryer. Sections were dewaxed in two changes of xylene (5 min each), followed by two changes of absolute ethanol and then air‐dried. The sections were stained with Safranin–Fast Green. Sections were photographed using an Olympus Vanox AHT3 microscope (Olympus Optical, www.olympus-global.com). Fruit cell area was measured using the IMAGEJ software (https://imagej.nih.gov/ij/), and 30 cells were measured at each of the three positions of a fruit.