RNA from dermis was extracted using Trizol reagent (Invitrogen).
Total RNA was extracted from nodules and the remaining roots were extracted using TRIzol® reagent (Thermo Fisher Scientific, Waltham, MA, Cat. 15596018).
A half of parasitic nematodes sampled from each flea was preserved for subsequent DNA extraction, and another half was used for morphological analysis.
Morphological data from seedlings and mature plants was extracted from images using FIJI21.
Total RNA was extracted from tissues, using phenol.
Each flea was crushed with a sterile pestle, and DNA was extracted by lysis with 0.7 M ammonium hydroxide.
RNA was extracted from the cells using TRIzol reagent (Thermo Fisher).
DNA from these fleas, as well as negative controls from uninfected lice maintained as colonies in our laboratory, were extracted by using a QIAmp Tissue Kit (QIAGEN, Hilden, Germany), as described (5 ).
DNA was extracted from each of the 110 flea pools by using a standard silica cartridge method (QiaAmp DNA mini kit, QIAGEN Ltd., Crawley, West Sussex, U.K). using the manufacturer's instructions for tissue DNA extraction.
Briefly, gDNA was extracted from Rickettsia-uninfected, unfed adult cat fleas (Heska Corporation, Loveland, CO) using Qiagen DNeasy DNA Extraction Kit and digested with five restriction enzymes (6 µg gDNA/enzyme): Eag I, Pst I, Xba I, Xho I (New England BioLabs, Ipswich, MA), and EcoR I (Promega, Madison, WI).
Bacterial DNA was extracted from 277 fleas of 5 species: 233 P. irritans, 24 S. fonquerniei, 9 X. cheopis, 10 E. gallinacea, and 1 C. canis.
