Thionins

The unicellular green alga C. reinhardtii strain CC-125 cells (1.5 × 10⁶) were cultured in 50 mL liquid tris-acetate-phosphate (TAP) media in 250 mL flasks (Harris, 1989 ) by shaking at 25°C and 140 rpm under constant white light of 90–100 μmol photons m⁻² s⁻¹. In addition to the wild type (WT), two transgenic C. reinhardtii lines (AtTHI-OE1 and AtTHI-OE2) harboring the Arabidopsis thaliana Thionin 2.1 gene, which encodes antibacterial peptides, thionins, were used to investigate genotype-dependent radiation-sensitivity. Cells were harvested at 800 × g, dispensed, and subjected to X- or γ-irradiation as described below. The subsequent post-irradiation cultivations of the cells were performed in 10 mL fresh TAP media in 50 mL conical tubes, with an initial optical density of 0.05 at 750 nm for mock (or control) under the same culture conditions. The basal TAP medium was supplemented with 5 or 10 mM NaHCO₃ to evaluate the synergistic effect of X-rays and sodium bicarbonate on cell growth.

Queenright Strumigenys colonies were collected from the field in various sites in Taiwan (locality information in Table 1 and Table 2). They were transferred to artificial nests in the laboratory that consisted of a round plastic tray with a diameter of 21.5 cm and a floor cover made of plaster of Paris to provide moisture. The colonies were reared under a 12:12 (L:D) photoperiod at 24 ± 2 °C in an incubation room. Springtails (Cyphoderus albinus) were provided as food ad libitum while a glass tube with water and cotton was provided to keep sufficient humidity. The trays were covered with a plastic lid to prevent escape by the ants and the springtails. We assured that alate queens (F1 generation) were virgin by raising them in the strict absence of males in the source colonies (F0 generation). Experimental colonies were created, each consisting of 1 alate queen (F1 queen) and 15 nestmate workers. Colony development (Table 1) was recorded twice a week during an experimental period of 28 weeks to monitor whether virgin queens can lay eggs that thelytokously develop into workers and/or queens (for S. membranifera, the total experimental period was 202 weeks). The virgin condition of all alate queens was verified by dissecting them at the end of the experimental period, which confirmed that their spermatheca was empty. To obtain additional evidence of whether mated queens occur, we dissected field-collected dealate queens for examination of their ovaries and spermatheca contents (Table 2).
Histological and ultrastructural examination was performed on queens of the thelytokous S. emmae, S. liukueiensis, S. rogeri and S. solifontis, as well as on the non-thelytokous S. lacunosa (Lugu Township, Nantou County, Taiwan), S. nanzanensis (Lanyu Township, Taitung County, Taiwan), S. sauteri (Lugu Township, Nantou County, Taiwan) and S. sydorata (Bogor, Indonesia) for comparison. The posterior part of the gaster was cut off and fixed in 2% glutaraldehyde (Agar Scientific, Stansted, UK) buffered with 50 mM Na-cacodylate (Agar Scientific, Stansted, UK) and 150 mM saccharose (pH 7.3). Postfixation was carried out in 2% osmium tetroxide (Agar Scientific, Stansted, UK) in the same buffer and was followed by dehydration in a graded acetone series and embedding in Araldite (Polysciences, Warrington, PA, USA and Merck, Darmstadt, Germany). Longitudinal serial semithin sections of 1 µm thickness were made with a Leica EM UC6 ultramicrotome (Leica, Wetzlar, Germany). They were stained with methylene blue and thionin (Merck, Darmstadt, Germany) and examined with an Olympus BX-51 microscope (Olympus, Tokyo, Japan). Thin sections of 70 nm were double-stained with lead citrate (Merck, Darmstadt, Germany) and uranyl acetate (Polysciences, Warrington, PA, USA) and were examined with a Zeiss EM900 electron microscope (Zeiss, Oberkochen, Germany).

After the marmosets had performed all the experiments, including the pre- and post-lesion sessions, they were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). The fixed brains were removed from the skull, postfixed in the same fresh fixative overnight at 4°C, and placed into 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. The brains were then cut along the coronal plane into 50 μm thickness slices using a freezing microtome. One section out of six was immediately mounted for thionin staining. For immunohistochemistry, adjacent sections were incubated with a mouse monoclonal antibody for glial fibrillary acidic protein (1:1,500 dilution; Sigma-Aldrich, St. Louis, MO, USA) or a rabbit polyclonal antibody for Iba-1 (1:4,000 dilution; WAKO Pure Chemical Industries, Osaka, Japan). Secondary biotinylated anti-mouse (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) or biotinylated anti-rabbit (1:200 dilution; Vector Laboratories) antibodies were also used. Immunoreactive signals were visualized using the ABC Staining Kit (Vector Laboratories) with 3,3'-diaminobenzidine. All stained images were acquired using an inverted microscope (BZ-X700, Keyence, Osaka, Japan).