S0389

All animals were sacrificed by a deadly ip injection of ketamine and xylazine and the tibial anterior muscles removed, cleansed of contiguous soft and fat tissue, cut in half in the mid-belly region, and fixated in a solution containing 4% paraformaldehyde (P6148; Sigma-Aldrich, St. Louis, MO, USA), 2.5% sucrose (S0389; Sigma-Aldrich, St. Louis, MO, USA), and 0.1% glutaraldehyde (G5882; Sigma-Aldrich, St. Louis, MO, USA) in PBS (pH 7.2) at 4 °C for 24 h. After fixation, both halves were dehydrated through graded-ethanol solutions, cleared in xylene, and embedded in paraffin blocks. Transverse 5 μm thick sections from each muscle part were cut on a Leica 2125 rotary microtome (Leica Microsystems, Wetzlar, Germany). The created slides were then used for muscle histomorphometry, evaluation of fibrotic-tissue accretion, and immunohistochemistry analysis.

Animals were anaesthetized in 0.1% MS222 (Sigma, A5040). Their hearts were excised, washed in 70% PBS with heparin (100 units ml⁻¹; Sigma, H4784) and fixed with 4% paraformaldehyde for 2 h at room temperature (RT) or overnight at 4 °C (Santa Cruz, CAS 30525-89-4) for immunostaining and rinsed three times before being exposed to 10, 20 and 30% sucrose (Sigma, S0389) solutions at 4 °C. The tissues were then equilibrated to O.C.T. compound (Tissue-Tek, 4583) by immersing them in a 1:1 mixture of 30% sucrose and O.C.T., followed by 100% O.C.T. and finally frozen. Sections (10–14 µm) were prepared on a cryostat at −14 °C (Thermo, Cryostar NX70) in the frontal plane and stored at −80 °C.
To obtain fresh frozen tissue sections, hearts were mounted in 7% tragacanth (Sigma, G1128), dipped in pre-cooled 2-metyhlbutane (Sigma, 277258) and frozen in liquid nitrogen.

Sucrose preference test was performed to assess hedonic behaviour towards a sweet gustatory stimulus^{11 (link)}. Mice were given access to two water bottles (50 ml conical tubes with sipper tops) for 24 h for habituation. Then, one water bottle was exchanged with a bottle containing 1% sucrose (Sigma, S0389) in drinking water. After 24 h, the bottle positions were swapped to prevent position bias. After another 24 h, sucrose preference was assessed as follows (based on weight of bottles): (sucrose (g)/total fluid (g)) × 100%.

In brief, ovaries were fixed with 4% PFA at 4°C overnight, incubated in 30% sucrose (#S0389, Sigma-Aldrich) at 4°C for three days, frozen, sectioned, incubated with PBS containing 0.25% Triton X-100 for 15 min, and blocked with PBST containing 3% BSA for 30 min. Sections were incubated with a rabbit anti-VASA (DDX4) antibody (1∶200; #ab13840, Abcam, Cambridge, UK) for 1 h at room temperature, rinsed with PBS, and incubated with a goat anti-rabbit IgG secondary antibody labeled with Alex Fluor 594 (1∶300). Nuclei were stained with DAPI for 3 min. All sections were mounted with fluoroshield mounting medium (#ab104135, Abcam) on glass slides and observed using a confocal microscope (Leica TCS SP5, Wetzlar, Germany).

Following the habituation phase, all animals were deeply anesthetized with an intraperitoneal injection of pentobarbital (Morbital, Biowet, Poland; 2 mL/kg body weight), and after cessation of breathing, immediately perfused transcardially with saline (0.9%) followed by 4% paraformaldehyde (pH 7.4; 1040051000, Sigma Aldrich, Taufkirchen, Germany) in phosphate-buffered saline (PBS; P5493, Sigma Aldrich, Taufkirchen, Germany). After perfusion, brains were carefully dissected from the skulls and post-fixed by immersion in the same fixative for 24 h, washed twice in 0.1 M phosphate buffer (pH = 7.4, 4 °C), and then stored for 3–5 days in graded solutions (19% and 30%) of sucrose (S0389, Sigma Aldrich, Saint Louis, MO, USA) in 1×PBS at 4 °C until full saturation. Finally, the brains were frozen and then coronally sectioned at a thickness of 10 μm using a cryostat (HM525 Zeiss, Germany). The sections were mounted on object slides and stored at −80 °C until further processing.

We sampled feral honey bee foragers (A. mellifera) in the 8 urban and 8 rural sites during June 2015, which corresponds to the peak bee foraging period and many of the plant species visited by honey bees are flowering in both urban and rural sites during this period. Also, ambient temperatures resulting from urban heat islands are more pronounced during the summer months (Imhoff et al., 2010 (link)). Three randomly selected sites were visited each day under sunny conditions to collect bees from each site between 10:00–14:00, which corresponds to the bee activity period with the lowest temperature variation. We minimized environmental and procedural effects by collecting two bees from each site each day it was visited until reaching at least six individuals (6–12) to test for CT_min and at least six individuals (6–12) to test for CT_max. Honey bees were collected with 250 ml plastic containers and transported to the laboratory at 15 °C to reduce their movement. After being captured, individuals were kept with sucrose (Sigma-Aldrich, S0389) and distilled water (50/50) until the beginning of the experiments (see below). Honey bee collection and treatment followed regulations in Mexico with approval granted from SEMARNAT (License No. FAUT-0174).