Bead ruptor elite

Ketamine xylazine–anesthetized mice (150 mg/kg of ketamine, 10 mg/kg of xylazine) were intracardially perfused with PBS and organs were collected in tubes containing zirconia beads (TOMY Digital Biology) and 1 ml TRIzol (Invitrogen) and stored at −80°C. To isolate RNA, tissues were thawed on ice and homogenized using the Bead Ruptor Elite (OMNI International) at a speed of 6 m/s (3 cycles of lysis × 30 s). Tissue lysates were then transferred to 1.5-ml Eppendorf tubes and mixed with 230 μl of chloroform and incubated at RT for 2 min. Samples were centrifuged at 12,000g for 10 min at 4°C and recovered supernatants were transferred into clean Eppendorf Tubes and mixed with 70% ethanol (1:1 volume). RNA was purified using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. Viral RNA copies were quantified by qRT-PCR as described above.

Individual tissues from single mosquitoes were placed in 300 μl of TriReagent (Sigma‐Aldrich), or 200 μl for salivary glands, and homogenized on a Bead Ruptor Elite (Omni International, USA) using a 2.8 mm ceramic bead. Total RNA was extracted with the Direct‐zol RNA 96 Magbead Zymo kit (Zymo Research) according to the manufacture's protocol on a MagMAX Express 96 system (Applied biosystems), and RNA was eluted in 50 μl RNase free water. RNA was treated with 5 units of DNase I (Sigma‐aldrich) at room temperature (RT) for 15 min, followed by inactivation with 50 mM EDTA at 70°C for 10 min. To measure Wolbachia DNA, RNA and DNA combination extractions were performed using the column‐based Direct‐zol DNA/RNA Miniprep kit. RNA was eluted in 50 μl RNase free water, followed by DNA elution in 50 μl of Direct‐zol DNA Elution Buffer, and RNA was treated with DNase I (as above).

Tumor, lymph node and spleen samples were stored in 500 μL of RNAlater (Thermofisher Scientific, Foster City, CA, USA) and frozen at −80°C for future analysis. Tissues were lysed and homogenized in TRIzol using a Bead Ruptor Elite (Omni International, Kennesaw, GA, USA). RNA was extracted using Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA, USA) and reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Primer sequences for PCR were created using the IDT RealTime qPCR Tool (https://www.idtdna.com/scitools/Applications/RealTimePCR/, Integrated DNA Technologies, Coralville, IA, USA) (26 (link), 27 (link)). Real time PCR of cDNA samples was performed using the PowerUp SYBR Green Master Mix (Thermofisher Scientific, Foster City, CA, USA) with beta actin (Actb) as a reference gene. Gene transcripts amplified include VegfA, Hif1α, Prf1, Tnf, Ifng, Rorgc, Il1b, Il17, Pdl1, Klrg1, Klrc1, and Arg1.

To test how high temperatures affect gene expression, we exposed adult mosquitoes in their family groups to extreme heat stress (42°C) and then collected whole mosquitoes for RNA extraction and subsequent PCR. We also included expression of the HSPs in age-matched mosquitoes that were not exposed to heat from the base-rearing population used to create the families. While this control does not capture any unique effects of the breeding design on expression, it does provide context for uninduced Hsp gene expression levels in the mosquito line. Adult females representing the 40 families were exposed to 42°C in an environmental chamber in groups of five or six per family for 15 min. This method and length of exposure were based on previous heat shock studies in Drosophila [59 (link)] and in Ae. aegypti pupae and larvae [60 (link)]. After the initial heat shock, mosquitoes were snap-frozen and stored at −80°C until extraction. We subsequently added 300 µl of Trizol reagent (Invitrogen) containing a 2 mm glass bead, and samples were homogenized using a Bead Ruptor Elite (OMNI International, Kennesaw, Georgia). RNA extraction was then performed according to the manufacturer's instructions [61 (link)].

Brain tissue from the 6-week necropsy was thawed on ice and homogenized using a Bead Ruptor Elite (Omni International, 19-2241E, Kennesaw, GA) for three cycles of 8,000 m/s for 1 minute, ice for 5 minutes, and centrifuged at 6,200 g for 3 minutes. DNA was extracted from the larvae and 100 µL of the homogenized brain samples using the Dneasy Blood and Tissue Kit (Qiagen, 69504, Venlo, The Netherlands) per the respective manufacturer’s protocol with final elution volumes of 100 µL for larvae and 400 µL for brain tissue. DNA was extracted from the feces using the QIAamp Fast DNA Stool Mini Kit (Qiagen, 51604) per the manufacturer’s protocol with 100-µL final elution volume. PCR reactions were run on a QuantStudio 5 Real-Time PCR Instrument (A28134, Applied Biosystems, Waltham, MA) with a species-specific multiplex assay, TaqMan Fast Advanced Master Mix (4444965, Applied Biosystems), and the recommended cycling conditions with the exception of using 50 instead of 40 cycles (L. M. Kaluna and S. I. Jarvi, unpublished data). Samples were analyzed using a 0.05 manual threshold and designated as positive based on the cycle threshold (C_T value ≤40), negative (C_T value 0), or equivocal (where replication of a positive PCR result was not demonstrated).