Nipagin

Flies used in all experiments came from a long-term laboratory adapted population (the IV population) that was initiated from D. melanogaster collected in 1975 (Charlesworth and Charlesworth 1985 ). All flies were reared and maintained as adults in vials with standard 2% yeast food (water, agar, brewer's yeast, cornmeal, sucrose, and Nipagin [Sigma-Aldrich, Buchs, Switzerland]) on a 12L:12D cycle.

The fly strains used in this study are listed in S3 Table. Flies were maintained on “Iberian” food [70 mg/ml yeast (Brewer’s yeast, MP Biomedicals, 903312), 55 mg/ml glucose (VWR, 10117HV), 7.7 mg/ml agar (SLS, FLY1020), 35 mg/ml organic plain white flour (Doves Farm, UK), 1.2 mg/ml Nipagin (Sigma, H3647), 0.4% propionic acid (Sigma-Aldrich, P5561] at 25°C and 60% humidity with a 12-hour light–12-hour dark cycle.
The transgenic fly lines generated in this study were obtained by phiC31-mediated transgenesis to integrate the relevant constructs into either the attP40 (25C6) or attP2 (68A4) landing sites following embryo injection.

Nipagin, agar, menadione, agarose, phenylmethylsulfonyl fluoride (PMSF), reduced glutathione (GSH), NADP+, NADPH, glucose-6-phosphate, ethylenediaminetetraacetic acid (EDTA), xylenol orange, cumene hydroperoxide, 2,4-dinitrophenylhydrazine (DNPH), N,N,N',N'-tetramethylethylenediamine (TEMED), quercetin, 1-chloro-2,4-dinitrobenzene (CDNB), Tris-HCl, and 5,5′-dithio-bis (2-nitro) benzoic acid (DTNB), were purchased from Sigma-Aldrich Chemie GmbH (Germany). Sucrose was purchased from TM Fluka. All other reagents were of the analytical grade from local suppliers (Ukraine).

Experiments were performed on the wild-type D. melanogaster fruit flies originating from Australia, Canada, and Benin, and the OregonR strain (Bloomington Drosophila Stock Center).
The population from Australia had originally been sourced from Coffs Harbour and was collected in 2010 [33 (link)]. The population from Benin, Africa, was originally collected in 1970, is routinely used for Drosophila genetics research, and is called the Dahomey population [34 (link),35 (link)]. The population from Dundas, Canada, was originally collected in 2005 [36 (link)].
Flies were kept in the laboratory in 100 mL bottles at 20 °C, 25 °C, and 28 °C; the photoperiod was 12/12 h (day/night, respectively) with 60% relative humidity on standard fly medium consisting of brewer’s yeast (80 g/L), sucrose (50 g/L), agar (15 g/L), and Nipagin (Sigma–Aldrich) (8 mL/L). To generate the flies for the experiments, groups of 10 mated females were allowed to lay eggs in 100 mL rearing bottles during a restricted period of 6 h maximum under their common laboratory conditions. No CO₂ anesthesia was used, as it affects metabolic traits [37 (link)].

DrosDel w¹¹¹⁸isogenicD. melanogaster (iso) with wMelCS_b Wolbachia (Wolb⁺) and the matching control without Wolbachia (Wolb⁻) were described elsewhere (19 (link), 27 (link), 68 (link)). Aljezur 1 and W-20 D. melanogaster lines (Wolb⁺ and Wolb⁻) were also described previously (19 (link)). We determined that the Wolbachia variants in both of these lines lack an IS5 transposon insertion in gene WD1310, based on the primers described in Riegler et al. (69 (link)). This insertion is present in all wMelCS-like variants, but not in wMel variants (27 (link), 69 (link)), and therefore Aljezur-1 and W-20 are both wMel-like Wolbachia variants. Stocks were maintained at a constant temperature of 25°C on a diet consisting of: 45 g molasses, 75 g sugar, 70 g cornmeal, 20 g yeast extract, 10 g agar, 1,100 ml water, and 25 ml of 10% Nipagin, with the addition of live yeast (Sigma).

Drosophila flies were raised on ‘standard’ lab diet (SF) or one of two synthetic diets (M0, M1). SF diet contained per liter: 8 g agar, 18 g brewer’s yeast, 10 g soybean, 22 g molasses, 80 g cornmeal, 80 g malt, 6.3 mL propionic acid, and 1.5 g nipagin. For both M0 and M1 diet, we dissolved 10 g of yeast extract (Kerry, Boston, MA, USA), 10 g of glucose (Merck, Branchburg, NJ, USA), and 1 g of UltraPure Agarose (Invitrogen, Waltham, MA, USA) in 100 mL of filtered tap water. The mixture was microwaved until it boiled and then cooled to 65–70 °C. Next, we added a solution of 0.1 g of stigmasterol (Sigma, St. Louis, MO, USA) in 2 mL of 95% ethanol and 1.5 mL of 10% nipagin (Sigma, St. Louis, MO, USA). In the case of M1 medium, we added a solution of 0.1 g beta-carotene (Sigma, St. Louis, MO, USA) in 2 mL of 95% ethanol. After thorough mixing for several minutes, the medium was poured into plastic vials (Genesee Scientific, El Cajon, CA, USA) and then allowed to solidify at room temperature.

All stocks were maintained at 25°C on a 12 hour light: 12 hour dark cycle on standard food containing 57 g/l cornmeal (Bedorf Mühle, Wachtberg-Villip, Germany), 11.5 g/l yeast (Gewürzmühle Brecht, Eggenstein-Leopoldshafen, Germany), 6 g/l agar-agar (Gewürzmühle Brecht), 7% sugar beet molasses (Grafschafter Krautfabrik, Meckenheim, Germany) and 1.4 g/l Nipagin (Sigma-Aldrich, St. Louis, MO). All experiments have been performed with female flies. RU inducible gal4 expressing lines were Tubulin-GeneSwitch (Tub^GS-gal4) [19 (link)] and Ti-GeneSwitch (Ti^GS2-gal4) [22 (link)]. For the overexpression of AMPs we used the following fly strains: UAS-Dro/CyO (UAS-Dro) and UAS-CecA1 (UAS-CecA1) [42 (link)]. The white strain used is white¹¹¹⁸ from Bloomington stock center (line #5905).