coli cultures or Phosphate Buffered Saline (PBS) as control. All supplements were allowed to dry for 20 min under laminar flow. Two first-instar Manduca sexta neonate larvae were placed on each food block (12 larvae per treatment) and after 7 days incubation at 25°C the weight of each caterpillar was recorded. Tobacco hornworm M. sexta larvae were maintained on a wheat

germ-based artificial diet [35] at 25°C with a photoperiod of 17 h light: 7 h dark. G. mellonella larvae were supplied by Livefood (UK). For the assessment of symbiosis, infective juvenile (IJ) H. bacteriophora were propagated on a lawn of either TT01rif or TT01pam cells constitutively producing Depsipeptide solubility dmso GFP. The proportion of IJs transmitting GFP-labeled bacteria was determined by fluorescence microscopy, as previously described [36]. Transmission electron microscopy and hemolymph attachment assays For transmission electron microscopy (TEM), bacterial colonies grown on LB plates

were fixed in 2.5% glutaraldehyde for 2 h, washed in PBS and post-fixed in 1% osmium tetroxide for 1 h. Samples were dehydrated in acetone and Afatinib embedded in Spurr’s resin (TAAB, Premix). Sections were cut with an ultra-microtome (Leica, Reichert Ultracut E) and stained with uranyl acetate and lead citrate before examination with a JEOL JEM 1200 EXII transmission electron microscope (JEOL Tokyo, Japan). For labeling Pam at the ultra-structural Selleck LY2606368 level, samples were dehydrated in ethanol and embedded in LR White acrylic resin before sectioning and incubation with the primary (anti-Pam) and secondary antibodies (goat anti-rabbit IgG conjugated with 10 nm gold particles). For analysis of attachment in 5th instar M. sexta plasma (filtered hemolymph), overnight cultures of TT01rif wild-type and TT01pam were diluted to 0.05 OD600 and incubated at 28°C for 8 h without agitation in 24-well tissue culture plates on sterile circular glass coverslips. After 8 h the planktonic L-gulonolactone oxidase bacteria were gently aspirated and the coverslips were washed 3 × with PBS. The coverslips were fixed with 300 μl of 4% PBS-paraformaldehyde at 4°C for 24 h and stained

with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were mounted with Miowiol (Calbiochem) and analyzed with fluorescent microscopy (Nikon Eclipse 90i, Japan). Cells count at 8 h was the average in 12 fields of vision at 60× magnification from triplicate samples for the TT01rif and the same sample size for the TT01pam mutant. Physicochemical methods Surface plasmon resonance (SPR) data were acquired using the Autolab ESPRIT (Eco Chemie, BV, The Netherlands). Measurements were made by following the variation in the reflected light minimum angle with time, which is indicative of the change in optical properties of the interface as bacteria attach. Glass disks with a thin gold coating (50 nm thick) were placed on a hemispherical prism using index match fluid (RI = 1.