Synorf1

In order to examine the neuropil structures of the A. lucorum brain, immunolabeling with anti-synapsin antibody was performed. The brain was dissected out in Ringer’s saline [in mM, 150 NaCl, 3 CaCl₂, 3 KCl, 25 sucrose, and 10 N-Tris(hydroxymethyl)-methyl-2-amino-ethanesulfonic acid, pH 6.9] on ice and then transferred into 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) to be fixed overnight at 4°C in a refrigerator. Preincubation of the brain with 5% NGS (Sigma, St. Louis, MO, USA) in 0.1 M PBS containing 0.5% Triton X-100 (PBST; 0.1 M, pH 7.4) was performed overnight at 4°C after being rinsed in PBS (4 × 15 min). Then, incubation with the primary antibody, SYNORF1 (Developmental Studies Hybridoma Bank, University of Iowa), at a concentration of 1:100 (with 5% NGS in PBST), was applied at 4°C. After 5 days, the brain was rinsed in PBS, 6 × 20 min. Then, incubation with the secondary antibody, Cy2-conjugated anti-mouse (Invitrogen, Eugene, OR, USA; dilution 1:300 with 1% NGS in PBST), was performed for 3 days at 4°C. Afterward, the brain was washed in PBS, 6 × 20 min, and dehydrated with an ascending ethanol series. Finally, the brain was cleared in methylsalicylate, before being mounted in Permount in a perforated aluminum slide with two glass coverslips.

Microglomeruli were labeled and quantified adapting a published protocol for double staining pre-synaptic and post-synaptic profiles (Groh et al., 2006 (link); Krofczik et al., 2008 (link); Hourcade et al., 2010 (link)). Brains were embedded in 5% low melting point agarose (Agarose II, no. 210–815, AMRESCO) and sectioned in a frontal plane (200 μm) with a vibrating microtome (Leica VT 1000S). Free-floating sections were repeatedly washed (3 times for 10 min) in PBS with 2% Triton X-100 and pre-incubated in PBS with 0.2% Triton X-100 and 2% normal goat serum for 1 h at RT. Preparations were then incubated for 4 days at 4^oC simultaneously in 0.2 U of Alexa Fluor 488 phalloidin (Invitrogen, A-12379) and a monoclonal anti-synapsin I antibody (1:50; SYNORF1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). After repeated washes (5 times for 10 min) in PBS, preparations were incubated in the secondary antibody (Alexa Fluor 546-conjugated goat anti-mouse, Invitrogen: 1:250 in PBS with 1% normal goat serum) for 2 h at RT. Brains sections were washed (5 times for 10 min) in PBS, transferred to 50% glycerol in PBS for 15 min and mounted on coverslips with 80% glycerol/PBS solution.

The neuroanatomy of antennal lobes from homozygous mutant (Orco^−/−) and wild type moths were examined following a protocol previously published [90 (link)]. Brains were firstly dissected in phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde overnight at 4 °C. Glomeruli were marked using the antibody SYNORF1 (Developmental Studies Hybridoma Bank, IA, USA) antibody and visualized with Alexa Fluor 488 goat anti-mouse secondary antibody (Invitrogen). Photos were obtained with a Zeiss LSM710 Meta laser scanning microscope (Zeiss, Oberkochen, Germany). The software AMERA 6.0 (ZIB, Germany) was used for construct brain atlas. Four adults were observed in each group.

The structure of brain neuropil, and specifically the antennal lobe MGC of S. littoralis males, was revealed by using immunostaining with an antibody against the Drosophila vesicle-associated protein synapsin 1 (SYNORF1, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). This staining method has been used previously to reveal AL glomeruli in the same moth species [14 (link),31 (link)]. Briefly, brains from 12 wild type and 12 KO newly emerged virgin and naïve males (non exposed to pheromone, as such exposure has been shown to lead to an increased size of the corresponding glomerulus (Guerrieri, 2012 #2564)) were carefully dissected in phosphate buffer saline (PBS) and fixed overnight in 4% Electron Microscopy-grade formaldehyde solution in PBS at 4 °C. After rinsing in PBS, the brains were pre-incubated in PBS with 2% Normal Goat Serum (NGS) and 0.5% Triton X 100 and then incubated with the synapsin 1 antibody (1:25 in PBS with 0.5% Triton X and 2% NGS for 5 days at 4 °C). After rinsing, brains were incubated with the secondary antibody (1:250 in PBS with 1% NGS for 3 days at 4 °C; Alexa-Fluor-488-conjugated anti-mouse; Invitrogen, Abingdon, UK). Brains were then rinsed again in PBS, dehydrated in a graded ethanol series, and mounted in methyl salicylate on aluminum slides between two microscopic cover glasses.

Brains were dissected in fresh Ringer’s solution (150 mM NaCl, 3 mM CaCl₂, 3 mM KCl, 25 mM Sucrose, and 10 mM N-tris (hydroxymethyl)-methyl-2-amino-ethanesulfonic acid, pH 6.9). In order to visualize the AL glomeruli, immunostaining with anti-synapsin antibody SYNORF1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), was performed. The dissected brains were first fixed in a 4% paraformaldehyde solution in phosphate-buffered saline (PBS: 684 mM NaCl, 13 mM KCl, 50.7 mM Na₂HPO₄, 5 mM KH₂PO₄, pH 7.4) for 2 h at room temperature. After fixation, the brains were washed in PBS 4 × 15 min. To minimize non-specific staining, the brains were pre-incubated in 5% normal goat serum (Sigma, St. Louis, MO, USA) in PBS containing 0.5% Triton X-100 (PBSX; 0.1 M, pH 7.4) for 3 h before being incubated in the primary antibody, SYNORF1, at 1:100 in PBSX at 4°C for 5 days. Then the brains were rinsed in PBS 6 × 20 min, before being incubated in the secondary antibody, Cy2-conjugated anti-mouse (Invitrogen, Eugene, OR, USA; dilution 1:300 in PBSX), at 4°C for 3 days. The brains were finally rinsed 6 × 20 min in PBS, dehydrated with ascending ethanol series, and mounted in methylsalicylate.

For visualizing the neural architecture of the stained preparations, immunostaining with an antibody labeling presynaptic terminals was performed. The antibody SYNORF1 (Developmental Studies Hybridoma Bank, University of Iowa) was used as the primary antibody. The preparations were pre-incubated in 5% normal goat serum (Sigma, St. Louis, MO) in PBS containing 0.5% Triton X-100 (PBSX; 0.1 M, pH 7.4) for 3 h to minimize non-specific staining and then incubated in SYNORF1 at 1∶100 in PBSX at 4°C for 5 days. After rinsing in PBS for 6×20 min, the preparations were incubated in a Cy2-conjugated anti-mouse secondary antibody (Invitrogen, Eugene, OR; dilution 1∶300 in PBSX) at 4°C for 3 days. Finally, the preparations were rinsed 6×20 min in PBS, dehydrated with ascending ethanol series (50%, 70%, 90%, 96%, 2×100%, 10 min each), and mounted in methylsalicylate.

Animals were fixed using the Carnoys fixation protocol [63 (link)] unless otherwise stated. Primary antibody concentrations were used as follows: α -H3P 1:250 (Millipore Cat# 05-817R); α -VC1 1:10,000 (Kind gift of K. Watanabe); anti-α-Ac-Tubulin 1:500 (Sigma, clone 6-11B-1), Smed-6G10 1:1000; SYNORF1 1:100 (Developmental Studies Hybridoma Bank). Secondary antibody concentrations were: Alexa488 (1:400) goat anti-mouse (Invitrogen Cat# 673781), goat-anti-mouse HRP IgG 1:1000 (Life Technologies), and Alexa568 (1:800) goat anti-rabbit (Invitrogen Cat# 11036).