Microinjections

A. gambiae mosquitoes were maintained in standard insectary conditions (28°, 75–80% humidity, 12-hr/12-hr light/dark cycle). Larvae were raised in deionized water and fed finely ground TetraMin fish food. Embryo microinjection was performed essentially as described (Fuchs et al. 2013 (link); Pondeville et al. 2014 (link)). Freshly laid eggs were directly aligned against the edge of a nitrocellulose membrane kept wet with overlaying filter paper soaked with demineralized water. A mix of plasmids totaling 400 ng/µl of DNA (0, 1 mM NaHPO₄ buffer pH 6.8, 5 mM KCl, 60 ng/µl helper plasmid, and generally 85 ng/µl of each of four distinct transgenesis plasmids) was injected under a Nikon Eclipse TE2000-S inverted microscope using an Eppendorf Femtojet injector and TransferMan NK2 micromanipulator. Injections were performed using the compensation pressure of the device, which was kept at 6000 hPa to promote a constant moderate flow of the DNA solution out of the quartz capillary. Microinjected eggs were left undisturbed on the injection slides, which were placed diagonally in a container with 1-cm-deep demineralized water, the part of the filter paper most distant from the eggs was dipped in water so that eggs remained wet by capillarity (Figure 1). Adult mosquitoes that survived microinjection were separated according to sex and crossed en masse to an excess of fresh wild-type adults. Neonate progeny larvae from several successive gonotrophic cycles were screened by spotting groups of 50–80 onto the wells of a 24-well teflon-coated diagnostic slide (Erie Scientific, Menzel GmbH, Braunschweig, Germany) under a Zeiss Axiovert 200M fluorescence microscope. When a fluorescent larva was detected, it was carefully isolated from the remainder larvae with the cut tip of a P200 pipette.

The reproductive organs were individually dissected from the newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population, and the relative transcript levels of clip-domain serine protease genes and PPO were examined by RT-qPCR assays. The male reproductive organs were also examined to determine the conversion of PPO to PO in western blot assays using PPO and histone H3 antibodies (0.5 μg/μl). A pool of 30 RGDV-positive males was used for each replicate in RT-qPCR and western blot assays, respectively. The experiment was conducted in at least three replicates for RT-qPCR and western blot assays. To analyze effect of RGDV infection on PO activity, the reproductive organs dissected from approximate 100 newly emerged males were homogenized with the His-Mg buffer (0.1 M histidine, 0.01 M MgCl₂, pH 6.2) buffer in liquid nitrogen. The supernatant was gently mixed with 1 mM dopamine in 10 mM Tris-HCl buffer (pH 8.0) in a 96-well plate at room temperature for 5 min. Enzyme activity was measured using the phenoloxidase kit (Geruisi, G0146W) according to the manufacturer’s protocol. To analyze the effect of M. luteus infection on PO activity, freeze-dried M. luteus was dissolved in water, and then microinjected in dose of ~23 ng/leafhopper into newly emerged males. At 24-h post microinjection, the reproductive organs of approximate 100 RGDV-infected or M. luteus-treated males were dissected and tested for PO activity.
We then tested the effect of knockdown of PPO or HongrES1 expression on PO activity and RGDV infection. The newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population were microinjected with dsGFP, dsPPO or dsHongrES1 (~200 ng/leafhopper). The male reproductive organs of these tested leafhoppers were individually collected and dissected for RT-qPCR and western blot assays to determine the effect of dsRNAs on the expression levels of HongrES1, PPO, or RGDV P8, and the conversion of PPO to active PO, as well as PO activity. A pool of 30 males was used for each replicate in RT-qPCR and western blot assays, respectively. A pool of 100 males was tested for each replicate in PO activity. The experiment was conducted in three replicates for RT-qPCR and western blot assays, as well as PO activity tests.

All mice were bred in specific pathogen–free conditions, and experiments were approved by the Institutional Animal Care and Use Committee of Osaka University. Littermate controls were used for all experiments when feasible. C57BL/6J mice were purchased from CLEA Japan. CAG-EGFP mice (RBRC 00267) were obtained from Riken BioResource Research Center (Okabe et al., 1997 (link)), Mac-Gata6 KO was described previously (Okabe and Medzhitov, 2014 (link)), and these mice were backcrossed to B6. Postn-DTR mice were generated by CRISPR/Cas-mediated gene targeting in C57BL/6 zygotes. In brief, CRISPR RNA: 5′-ACG​GAG​CTC​AGG​GCT​GAA​GA-3′ was used as targeting guide (Integrated DNA technologies) and mutated DTR sequence was inserted by microinjection (Saito et al., 2001 (link); Furukawa et al., 2006 (link)).