Femtojet microinjection system

Transgenic worms were created by microinjection using a Leica DMIRB inverted DIC microscope equipped with an Eppendorf Femtojet microinjection system. Each construct was injected at a concentration of 50 ng/µl together with 1 kb DNA ladder as carrier DNA and 5 ng/µl pCFJ90 (myo-2p::mCherry) or a combination of 5 ng/µl rol-6p::rol-6(d) and glr-3p::mCherry as co-injection marker.

For chick neurons, microinjection pipettes (TW100F-4, World Precision Instruments, Inc. Sarasota, FL) were pulled on a Brown and Flaming horizontal pipette puller. For Aplysia neurons, microinjection pipettes (1B100F-4, World Precision Instruments) were pulled on a Narishige PP830 vertical pipette puller. Pipettes were then back-loaded with 1 mg/ml rhodamine tubulin (Cytoskeleton, Inc., Denver, CO, USA) in injection buffer (100 mM PIPES pH 7.0, 1 mM MgCl₂, 1 mM EGTA) as previously described⁸⁴ . Before injection, tubulin was thawed, spun at 13,000 g for 30 min, kept on ice and back-loaded into pipettes pre-chilled to 4 °C. Microinjection was performed using the NP2 micromanipulator and FemtoJet microinjection system (Eppendorf North America, New York, NY), visualized with a Nikon ECLIPSE TE2000 microscope using phase contrast optics with a 40x objective. Alternately, microinjection was performed using a Narishige hydraulic micromanipulator with injection pressure supplied from a 3 ml luer-lock syringe.

All microinjections were done with siRNAs for Satb1 and Satb2 as well as AllStars Negative Control siRNA purchased from Qiagen. For siRNA sequences, see Supplementary Materials and Methods. Satb1 and Satb2 cDNA (Dharmacon) was cloned into pRN3P as described previously (Zernicka-Goetz et al., 1997 (link)). In vitro transcription was undertaken on linearized cDNA using the mMessage mMachine T3 kit (Ambion) according to the manufacturer's instructions. Microinjection of embryos with siRNA (always at a final concentration of 12 μM) or mRNA [together with Ruby mRNA (200 ng/μl) or Gap43-GFP mRNA (400 ng/μl) as markers of injection] was carried out in M2 covered in oil on a glass depression slide using a Femtojet micro-injection system (Eppendorf). Embryos were cultured in KSOM under paraffin oil at 37.5°C in air enriched with 5% CO₂.

Gram-negative E. coli strain K12 and gram-positive B. subtilis were used to inject S. furcifera. Bacteria were cultured overnight at 37°C on LB agar plates using the streak-plate procedure. A single colony of E. coli or B. subtilis was used to inoculate 10 ml of LB broth in culture flasks, which were then incubated at 37°C with shaking at 200 rpm until optical density at 600 nm reached 0.6 and 0.75, respectively. The bacteria were then collected by centrifugation at 5,000 rpm for 5 min, resuspended in phosphate-buffered saline (PBS, 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.4) to a density of 10⁶ cells/ml, and heat-killed by boiling for 30 min.
Fourth-instar S. furcifera nymphs (day 1) were evenly distributed into three injection groups: E. coli, B. subtilis, and PBS (control). The insects were anesthetized with CO₂ (pressure: 5 mPa) for 10 s and fixed on an agarose gel-casting tray in a neat array with their abdomens facing upwards. Then, 0.5 μl of inactivated bacteria or PBS was injected into the abdomen segment junction between the second and third appendages using a FemtoJet microinjection system (Eppendorf, Germany). The injected nymphs were reared in the incubation chamber, fed with fresh rice, and analyzed for Akirin gene expression by qRT-PCR as described.

Laodelphax striatellus Vg-specific nucleotide conserved sequence was cloned into the pGEM-T Easy vector (Promega). Aequorea victoria green fluorescent protein (GFP) was used as a control. The specific primers used to generate DNA templates are shown in Supplementary Table 1. dsRNA was synthesized, as previously described (Huang et al., 2015 (link)).
Second instar nymphs of infected L. striatellus were used in the RNAi microinjection. Each nymph was anesthetized using carbon dioxide. Approximately 250 ng of dsVg or dsGFP was microinjected into the thorax of each nymph using the FemtoJet Microinjection System (Eppendorf). Microinjected nymphs were reared under laboratory conditions. Relative silencing efficiency of the whole body was calculated by RT-qPCR. Wolbachia titers (ovaries and whole body) were measured by qPCR, as described above.

dsRNA synthesis and injections were performed as previously reported [10 (link),45 (link)]. In brief, dsRNA was synthesized using a T7 RNAi transcription kit (Vazyme) according to the manufacturer’s instructions. The dsRNA primers for the corresponding genes were synthesized with a T7 RNA polymerase promoter at both ends (S1 Table). Microinjection was performed using a FemtoJet microinjection system (Eppendorf). Insects were anesthetized with carbon dioxide for 10–15s before microinjection. After injection, all insects were maintained in transparent polycarbonate jars with fresh rice seedlings. At 2 days after injection, insects (n = 5 for each of three replicates) were collected for RNA extraction, and cDNA synthesis for assessment of RNAi efficiency via qRT-PCR.