Supplementary MaterialsFigure S1-S3 rspb20160580supp1. is a globally important agricultural pest . As in other whiteflies (family Aleyrodidae), the bacteriocyte symbiont is Portiera aleyrodidarum (henceforth bacteriocytes bear a second bacterial symbiont, the identity of which varies among species; the MEAM1 studied here bears , facilitating their identification and enumeration. We reveal that the whitefly bacteriocyte is a remarkably dynamic cell type that changes from a non-motile, adherent condition in nymphs to motile cells that continue to proliferate through adulthood, generating a continuous Rabbit Polyclonal to RPC3 supply of cells for vertical transmission. These changes in cell behaviour are underpinned by major differences in the expression of genes for cell adhesion and mobility, providing the molecular basis for the transmission process. 2.?Material and methods (a) Insects The whitefly Suvorexant inhibition MEAM1 cultures were maintained in climate-controlled chambers at 271C with 14 L : 10 D regime. (b) Bacteriocyte dynamics The number of bacteriocytes was scored in 10 individuals dissected in phosphate-buffered saline (PBS) at pH 7.4 for 1- to 5-day-old eggs, third instar nymphs, early stage of fourth instar nymphs (prior to detectable eye pigmentation), late stage of fourth instar nymphs (with red eyes, known as pupae) and female adults at 0C35 Suvorexant inhibition days after emergence. The diameter of bacteriocytes in each sample was determined using an eye-piece graticule in a Leica stereo microscope and cell volume was calculated as 4/3and in whiteflies was investigated by fluorescence hybridization (FISH) with the probes BTP1-Cy3 (5-Cy3-TGTCAGTGTCAGCCCAGAAG) for and BTH-Cy5 (5-Cy5-CCAGATTCCCAGACTTTACTCA) for . Stained samples were viewed under a Zeiss LSM780 confocal microscope. To visualize the cytoskeleton, bacteriocytes and ovarioles were fixed and permeabilized. For the first assay with nymph bacteriocytes and adult bacteriocytes, the samples were blocked and then incubated sequentially with mouse anti–tubulin monoclonal antibody (Sigma), goat anti-mouse antibody conjugated to Alexa Fluor 488 (Sigma), and, finally, with phalloidin-Alexa Fluor 568 (Thermo Scientific) and Hoechst 33342 (Thermo Scientific). Microtubule and actin intensities were quantified by ImageJ. For the second assay with ovarioles and adult bacteriocytes, only actin was studied by incubation of samples with phalloidin-Alexa Fluor 488 and 568, respectively. To visualize the membranes in ovarioles with internalized bacteriocytes, ovarioles were incubated in Grace’s Insect Medium (Sigma) with FM 4-64 (Thermo Scientific) and Hoechst 33342. Images were collected and analysed on a Zeiss LSM700 confocal Suvorexant inhibition microscope. (d) RNA-seq analysis Approximately 20 000 bacteriocytes were dissected from each of approximately 4000 fourth-instar nymphs and approximately 3000 female adult whiteflflies at 7 days after emergence, using fine pins and a dissecting microscope. RNA isolation, library preparation and sequencing were conducted in accordance with Luan . We used the pipeline of Luan  for transcriptome assemblies and differential expression gene analyses. Web gene ontology annotation plot was used to investigate the distribution of gene functions for two samples. (e) Statistical analysis Statistical significance was evaluated using one-way ANOVA at a 0.05 level. Fisher’s least significant difference tests were followed for the number of bacteriocyte and egg and KruskalCWallis test for bacteriocyte volume. All data analyses were conducted using the software Statistica v. 6.1 (StatSoft, Inc., Tulsa, USA). See electronic supplementary material, text S1 for complete details on material and methods. 3.?Results (a) Bacteriocyte dynamics in the nymph and adult host Suvorexant inhibition To test our hypothesis that the cellular behaviour of bacteriocytes differs between the adult host (in which bacteriocytes are transmitted to the ovaries) and nymphal host (with no transmission), we quantified the number and size of bacteriocytes through development of the host. This analysis was facilitated by the distinctive greenCyellow colour of the carotenoid pigments expressed exclusively in bacteriocytes at all stages of the host life cycle (figure?1(red) and (green) in different developmental stages of 0.0001 for the whole body and 0.0001 for body cavity only, respectively. ( 0.001. ( 0.0001 for volume per bacteriocyte. The data in ( 0.001), declining more than five-fold from 11.6 1.87 10?5 mm3 (mean s.e.m., 10 replicates) in third-instar nymphs to 2.1 0.22 10?5 mm3 in newly emerged adult females, but Suvorexant inhibition did not vary significantly through adulthood to day 21 post-emergence (figure?1 0.001, 0.001) and adult bacteriocytes ( 0.001); and a significant difference was also obtained for actin in nymph bacteriocytes ( 0.005) but not for adult bacteriocytes ( 0.05) (five replicates per test). The.