Tetracycline hydrochloride

Tetracycline hydrochloride (TC), decane, calcium dichloride, magnesium dichloride, octanol, pig stomach mucin, and 8 M NaOH were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). OxyTetracycline hydrochloride (OTC), minocycline hydrochloride (MINO), and doxycycline hyclate (DOXY) were purchased from TCI (Tokyo, Japan). 2-Morpholinoethanesulfonic acid (MES) was purchased from Dojindo laboratories (Tokyo, Japan). Demeclocycline hydrochloride (DMCTC) and chlorTetracycline hydrochloride (CTC) were purchased from LKT Labs, Inc (MN, USA). Soybean lecithin (SLP-white) was provided by Tsuji Oil Mills co., Ltd (Mie, Japan).

Fifteen mice were divided into five groups: 1) subcutaneously PBS-injected group immediately after intravenous PBS injection from the tail vein (negative control) (n = 3), 2) alizarin complexone (alizarin; Dojindo, Kumamoto, Japan) -injected group (n = 3), 3) calcein (Sigma-Aldrich) -injected group (n = 3), 4) tetracycline hydrochloride (tetracycline; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan)-injected group (n = 3), and 5) demeclocycline hydrochloride (demeclocycline; Sigma-Aldrich, St. Luis, MO, USA) -injected group (n = 3) (Figure 1A). Later four groups received subcutaneous injections of fluorescent reagents using a 27 G needle and a 1 ml syringe (Terumo Corporation, Tokyo, Japan) at a dose of 20 mg/kg immediately after intravenous S. mutans inoculation to the tail vein. All mice were sedated and then sacrificed by cervical dislocation 24 hrs after intravenous PBS injection or S. mutans inoculation. The mice were immersed in 70% ethyl alcohol for several seconds, and then right femurs and liver were dissected for bacteriological analyses. The comparison between alizarin and PBS subcutaneous injections on the colony formation was shown in the Supplementary Section.

We generated a Wolbachia uninfected line using antibiotics. Freshly-laid eggs were surface-sterilized in 2.7% sodium hypochlorite (Sigma-Aldrich, St. Louis, MO) for 2 min and twice in 70% ethanol for 2 min. Following two 1 min rinses in distilled water, they were raised on a normal cornmeal, molasses, and yeast food containing 50 µg/mL tetracycline hydrochloride (Wako Pure Chemical Industries, Osaka, Japan). After tetracycline treatment for three generations, a single female was transferred to a vial to establish isofemale lines. After laying eggs, each female was assayed for the presence or absence of Wolbachia infections by PCR (see Supplementary Methods). After selection of the vial derived from an uninfected female, flies were maintained on tetracycline-free normal food. Because antibiotic treatment can eliminate other fly microorganisms besides Wolbachia such as beneficial gut-associated microbes, these were restored by backcrossing Wolbachia uninfected tetracycline-treated females with control infected males as described previously^{33 (link)}.