Isoamyl acetate

Fructose (99%) and tannic acid (95%) were purchased from J.T. Baker (Avantor; Radnor, PA, USA). Tartaric acid (99.7%) was provided by Chem-Lab (Eernegem, West-Vlaanderen, Belgium), and caffeine (99.2%) by ACEF (Piacenza, Italy). 2-Phenylethanol (≥99%), citral (95%), linalool (97%), 1-octen-3-one (96%), cis-3-hexen-1-ol (≥98%), ethyl butyrate (≥99%), damascenone (1.1–1.4 wt.%), benzaldehyde (≥99.5%), isoamyl acetate (≥95%), gamma-dodecalactone (≥97%), sotolone (≥97%), 4-ethylguaiacol (≥98%), 4-ethylphenol (99%), eucalyptol (99%), furaneol (≥98%), ethyl caproate (≥99%), eugenol (≥98%), citronellol (95%), phenylacetaldehyde (≥95%), furfuryl acetate (≥98%), 2,4,6-trichloroanisole (99%), 2-methyl-1-propanol (99.5%), methanethiol (≥98%) were all provided by Sigma-Aldrich (St. Louis, MO, USA). Ethanol (food grade, 70%) was supplied by ITW Reagents (Milano, Italy). Tanin VR colour, Tanin VR grape, Tanin plus, Tanin galalcool were all purchased from Laffort (Bordeaux, France).

Olfactory stimuli were delivered with a custom-built olfactometer comprising eight odor channels and controlled via a custom-made LabVIEW (National Instruments, Austin, TX, USA) interface, which synchronized stimulation protocol with the imaging acquisition [21 ]. For this study, we used six ecologically relevant odorants known to elicit distinct responses in the antennal lobe glomeruli: 1-hexanol (1HEX), 3-hexanol (3HEX), 1-nonanol (1NON), isoamyl acetate (ISOA), acetophenone (ACTP), and benzaldehyde (BZDA) (all from Sigma-Aldrich) [2 (link),9 (link)]. Additionally, they provide different degrees of structural and functional variability: 1-hexanol and 3-hexanol have the same carbon length but vary for the position of the hydroxyl group; 1-hexanol and 1-nonanol are primary alcohols with different chain lengths; acetophenone and benzaldehyde both have a benzene ring, but with a ketone and an aldehyde group, respectively; isoamyl acetate is the major component of the honey bee alarm pheromone [27 (link)], while all other odorants are typical floral scent components. Stimuli were presented in a 1 s ON/9 s OFF protocol and each odorant was delivered for 30 consecutive trials. All odorants were diluted 1:200 in mineral oil (Sigma-Aldrich). These concentrations were chosen to be well above the receptor sensitivity threshold and below the saturation level [5 (link)].

The leukocytes (1×10⁶ cells mL^-1) from the head kidney on circular coverslips were stimulated with PMA (100 ng mL^-1), E. coli, E.tarda, or S. aureus (1×10⁸ CFU mL^-1) for 3 h respectively, and then fixed with 2.5% glutaraldehyde (Hushi, Shanghai, China) at 4 °C in the dark for 2 h. Next, the samples were dehydrated by adding ethanol (Hushi, Shanghai, China) by a graded series (30%, 50%, 70%, 80%, 90%, 100%) for 15 min of each at room temperature. Samples were treated with isoamyl acetate (Sigma, St. Louis, Mo, USA) for 20 min, subjected to critical point drying for 3 h (Hitachi-HCP, Hitachi, Tokyo, Japan), coated with gold (MC1000, Hitachi, Tokyo, Japan) and viewed under SEM (S-3400N, Hitachi, Tokyo, Japan).

Octan-2-ol (97%), 1-hexanol (99%), cis-3-hexenol (98%), trans-3-hexenol (97%), vanillin (99%), 2,6-dimethoxyphenol (99%), linalool (97%), terpinen-4-ol (≥95%), α-terpineol (90%), nerol (≥97%), geraniol (98%), linalool oxide (≥97%), β-citronellol (95%), p-cymene (99%), terpinolene (≥85%), γ-terpinene (≥97%), limonene (97%), 1,8-cineole (99%), 1,4-cineole(≥98.5%), β-damascenone (≥98%), isoamyl alcohol (98%), benzyl alcohol (≥99%), 2-phenylethanol (≥99%), ethyl acetate (99%), ethyl butanoate (99%), ethyl 3-methyl butanoate (≥98%), isoamyl acetate (≥95%), ethyl hexanoate (≥95%), phenylethyl acetate (99%), n-hexyl acetate (≥98%), ethyl lactate (≥98%), ethyl octanoate (≥98%), ethyl decanoate (≥98%), hexanoic acid (≥99%), octanoic acid (≥98%), α-phellandrene (95%), p-menthane-1,8-diol (97%), 3-methylbutanoic acid (99%), α-ionone (90%), 1-pentanol (99%), 1-butanol (≥99%), 2-butanol (≥99%), ethyl guaiacol (≥99%), vinyl guaiacol (≥98%), methyl-vanillate (99%), ethyl vanillate (99%), were supplied by Sigma Aldrich (Milan, Italy). Dichloromethane (≥99.8%) and methanol (≥99.8%), were provided by Honeywell (Seelze, Germany). Sodium chloride (≥99.5%) was supplied by Sigma Aldrich (Milan, Italy).

Three of the aroma compounds (isoamyl acetate (> 95%), 2,3-butandione (diacetyl) (97%) and dimethyl trisulfide (> 98%)) were purchased from Sigma Aldrich, U.S., and methanethiol (methyl mercaptan) (1 mg/mL in H₂O) was purchased from Fisher Scientific UK Ltd.