Nanofil syringe

To assess the effect of DWV infection on argK expression, we artificially infected NEB with semi-pure DWV and maintained them under controlled conditions. NEB were injected with a DWV lysate prepared as described elsewhere [86 (link)]. Briefly, adult bees with symptomatic DWV infection were homogenized in 500 μl PBS in 1.5 ml microcentrifuge tubes. Tubes were centrifuged at 14,000 g for 10 min at 4 °C, and supernatant was clarified by adding an equal volume of chloroform. Samples were vortexed for 30 s, centrifuged at 10,000 g for 10 min at 4 °C, and supernatants were filtered through 0.22-μm sterile syringe filters (VWR, Radnor, Pennsylvania, USA). Lysates were stored in 40% glycerol at – 80 °C until use. RNA isolated from a subsample of the viral lysates was evaluated for DWV load by RT-qPCR, as described below, and for the presence of other common bee viruses by PCR, as described elsewhere [87 (link)]. NEB were cold-anesthetized at – 20 °C for 3 min and then either injected (or not) with 2.5 μl of 1X PBS containing 10⁴ DWV copies, under a stereomicroscope (SteREO Discovery V8, Zeiss, Jena, Germany). The viral lysates were injected between the 4th and 5th abdominal segments using a beveled 33G NanoFil Needle (NF33BV-2) mounted on a 10-μl NanoFil syringe (World Precision Instruments, Berlin, Germany). After injection, each bee was manually fed 5 μl of honey bee gut homogenate, using a micropipette, to establish the same characteristic gut community in experimental individuals, as described elsewhere [88 (link)–90 (link)]. This step is important because emerging bees lack their gut microbiome. In a hive, this is quickly supplied by interaction with older worker bees [88 (link)], but in laboratory settings, this needs to be supplied experimentally, to avoid gut dysbiosis with a possible negative impact on their health and unclear (and avoidable) disturbing factor during the bioassay experiment. The freshly prepared gut homogenates were obtained by pulling the intestines from four nurse bees, homogenizing these in 500 μl 1X PBS, and diluting the homogenate 1:1 with 50% filtered sucrose/water solution. DWV-injected and non-injected controls were confined in separated disposable plastic cages (28 × 11x11 cm) and provided with pollen paste (90% w/w fresh corbicular pollen with water) and 50% filtered sucrose/water solution (w/v) ad libitum, inside an incubator at the abovementioned conditions. After 3 days, the experimental bees were sampled and stored at −80 °C until dissection.

During surgery, VGaT-IRES::Cre mice were continuously anesthetized with 1–2% isoflurane gas while secured in the stereotactic instrument. Mice were injected with AAV1-hSyn-FLEX-GCaMP6m (Addgene) unilaterally into the BNST (5 × 1012 titer, 350 nL volume; 100 nl/min rate; +0.3 mm anteroposterior, +0.6 mm mediolateral, −4.1 mm dorsoventral coordinates from bregma) using an UltraMicroPump, Nanofil syringes, and 35-gauge needles (Micro4; World Precision Instruments, Sarasota, FL). For DREADD experiments, VGaT-IRES::Cre mice were bilaterally injected with either AAV8-hSyn-DIO-mCherry, AAV8-hSyn-DIO-hM4Di-mCherry, or AAV8-hSyn-DIO-hM3Dq-mCherry using the same coordinates as previously described. Syringes were left in place for 10 min following injections before being slowly withdrawn. For fiber photometry, after the syringe was withdrawn, an optic fiber (400μm core diameter, 0.66 NA, Doric Lenses) was implanted slightly dorsal to the BNST (+0.3 mm anteroposterior, ±0.6 mm mediolateral, −3.9 mm dorsoventral coordinates from bregma). All implants were secured with skull screws and dental cement. Mice were given 3 days of postoperative care with daily carprofen (5 mg/kg, I.P.) and allowed 3–4 weeks of recovery before experimentation.