assembling_Ixodes_ricinus_RRL

assembling_Ixodes_ricinus_RR

output_read2snps_cov4_10_Q30

output data generated by Read2snps tool. This file contain only SNPs sequences with SNPs with a depth sequencing between 4 and 10 for a minimum quality of 30. This file have been used to select and to design primers

Oviposition site choice requires IR76b and bitter taste neurons.

<p>(A) Schematic drawing of the oviposition assay setup (bottom) and a sample plate used to calculate the oviposition preference (top). <i>D</i>. <i>melanogaster</i> evaluates polyamine levels during egg-laying choices. The egg-laying plate halves contain 1% low melting agarose alone or agarose supplemented with a specific polyamine (purple/green boxes) in all cases with the exception of Fig 3D. (B–J) Box plots show oviposition PI of flies. <i>y</i>-axis value of 0 indicates indifference, while positive values indicate the degree of attraction and negative values indicate aversion. (B) Oviposition assay using plain agarose versus agarose + different polyamines at 1 mM. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀/trial). (C) Same assay as in B with single females showing group-independent decision-making to polyamines. (<i>n</i> = 30, 1 ♀ flies/trial). (D) Polyamines increase attractiveness of fruit. Oviposition PI of females for putrescine or cadaverine (grey plate) versus apple juice (red plate). While apple juice is more attractive than plain putrescine or cadaverine, apple juice supplemented with polyamine is more attractive than apple juice alone. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀/trial). (E) Oviposition assay (agarose versus putrescine or cadaverine) with females missing either antennae, wings, different tarsae (legs), or labellum compared to intact flies (control). Flies missing the labellum show no preference, while all other ablations had no effect on the PI. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀/trial). (F) Oviposition PI of loss of function <i>Poxn</i> females (<i>Poxn</i>^<i>-/-</i>) and <i>Poxn</i> rescue construct (SuperA-158 (53)) for putrescine and cadaverine. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀/trial). (G) Expression of GR66a (<i>GR66a-Gal4;UAS-mCD8GFP</i>, green) and IR76b (<i>IR76b-QF;QUAS-mtdTomato-3xHA</i>, magenta) in the AL, SEZ, labellum and legs. GR66a and IR76b are not expressed in the same taste neurons but innervate neighboring areas in the SEZ (arrow). (H) <i>IR76</i>b mutants lose their preference behavior to polyamine taste (<i>IR76b</i>^<i>05</i>, <i>IR76b</i>^{<i>MB00216</i>}, <i>IR76b</i>^<i>1</i>, <i>IR76b</i>^<i>2</i>), <i>IR76b-Gal4;UAS-Kir2</i>.<i>1</i>, and appropriate genetic controls. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀/trial). (I) Two taste receptors mediate oviposition preference. Oviposition PI of silenced sweet tasting GRs (<i>GR5a-Gal4;UAS-Kir2</i>.<i>1</i> and <i>GR64f-Gal4;UAS-Kir2</i>.<i>1</i>), and inactivated bitter tasting receptor neurons (<i>GR66a-Gal4;UAS-Kir2</i>.<i>1</i>) and appropriate controls. Silencing of bitter neurons makes polyamines attractive, while silencing sweet neurons has no effect. This attractiveness is dependent on the activity of IR76b neurons as <i>GR66a-Gal4</i>,<i>IR76b-Gal4;UAS-Kir2</i>.<i>1</i> flies show no preference behavior. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀/trial). (J) IR76b is required to mediate the behavioral response to polyamine odor. IR76b was re-expressed in the <i>IR76b</i> mutant background using <i>IR76b-Gal4</i>, <i>IR41a-Gal4 or GR66a-Gal4</i>. While <i>IR76b</i> mutants show no preference behavior to putrescine, re-expression of IR76b in IR76b but not in IR41a or GR66a neurons fully rescued this defect. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀/trial). All <i>p</i>-values were calculated via two-way ANOVA with the Bonferroni multiple comparison posthoc test (ns > 0.05, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001). In all figures, asterisks above bars refer to <i>p</i>-values of comparison to wild type control (first bar of the panel). Lines joining two bars or groups of bars denote all other comparisons.</p

IR76b neurons respond to polyamines in gustatory processing.

<p>(A) Schematic drawing of SEZ calcium imaging setup and position of ROIs within the SEZ, which were used for quantification of relative changes in GCaMP-fluorescence (%ΔF/F). (B) Representative images of SEZ imaging of <i>IR76b-Gal4;UAS-GCaMP6f</i> female flies stimulated with distilled water or increasing concentrations of putrescine. (C) Quantification of GCaMP6f-fluorescence peak responses (in %ΔF/F) in the ROI 1 and ROI 2 area, respectively, when female flies were stimulated with increasing concentrations of putrescine (<i>n</i> = 11 ± SEM). (D) Average response trace of ROI 1 area to putrescine (<i>n</i> = 11 ± SEM). The gray bar shows the stimulation period. Dark colored line in the middle presents the average value, and the light shade presents the SEM. (E) Average response trace of ROI 2 to putrescine (<i>n</i> = 11 ± SEM). (F) Representative images of SEZ of <i>IR76b-Gal4</i>,<i>UAS-GCaMP6f; IR76b</i>^<i>1</i> and heterozygous control female flies stimulated with distilled water or increasing concentrations of putrescine. (G) Quantification of peak responses (in %ΔF/F) in <i>IR76b</i> mutant and control. Responses in the ROI 1 and ROI 2 are calculated separately with increasing concentrations of putrescine (<i>n</i> = 6 ± SEM). Boxes show median and upper/lower quartiles, and whiskers show minimum/maximum values. (H) Average response trace of ROI 1 in <i>IR76b</i> mutant and control (<i>n</i> = 6 ± SEM). (I) Average response trace of ROI 2 in <i>IR76b</i> mutant and control (<i>n</i> = 6 ± SEM). (J) Scheme of the <i>Drosophila</i> labellum with different types of sensilla. IR76b <i>(IR76b-Gal4;UASmCD8GFP)</i> is expressed in peg taste sensilla on the labellum. Filled star indicates peg taste neuron, open star indicates L-type sensillum. (K) Electrophysiological recording of S-type and L-type sensilla to putrescine at different concentrations (0 mM–100 mM, <i>n</i> = 8 ± SEM). (L) Quantification of the response of S-type and L-type sensilla to putrescine in <i>IR76b</i> mutant and wild-type control. (<i>n</i> = 8 ± SEM). All <i>p</i>-values were calculated via Student’s <i>t</i> test (ns > 0.05, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001).</p

Females detect odor and taste of polyamines during oviposition site selection.

<p>(A) Scheme of the video-tracking position-oviposition assay. (B) Females spend more time on the polyamine-rich site but avoid pure polyamines for egg laying. Position (solid line) and oviposition (dashed line) preference of Canton S to putrescine and cadaverine over time in position–oviposition assay. <i>y</i>-axis shows position and oviposition PI. <i>x</i>-axis shows total time (min) of the assay. The female’s position–oviposition behavior was quantified in 30 min intervals. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀ flies/trial, total time of assay of 3 h. (C) Position and oviposition preference of loss of function mutant of <i>Poxn</i> (<i>Poxn</i>^<i>-/-</i>) and <i>Poxn</i> full rescue (rescue superA) to putrescine in position–oviposition assay. Red lines indicate behavior of <i>Poxn</i>^<i>-/-</i> females, while black lines designate behavior of <i>Poxn</i> rescue SuperA. Loss of sense of taste does affect oviposition but not position preference. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀ flies/trial). (D) Position and oviposition preference of <i>IR41a-Gal4;UAS-Kir2</i>.<i>1</i>, <i>IR76b-Gal4;UAS-Kir2</i>.<i>1</i>, <i>Gr66a-Gal4;UAS-Kir2</i>.<i>1</i>, and <i>Gr66a-Gal4</i>,<i>IR76b-Gal4;UAS-Kir2</i>.<i>1</i> and their respective genetic controls to putrescine over time in position–oviposition assay. Position preference is mediated by IR41a olfactory neurons, while taste neurons trigger oviposition avoidance. Red lines indicate <i>receptor-Gal4;UAS-Kir2</i>.<i>1</i>, while black lines indicate controls. Box plots show median and upper/lower quartiles (<i>n</i> = 8, 60 ♀ flies/trial). All <i>p</i>-values were calculated via two-way ANOVA with the Bonferroni multiple comparison posthoc test (ns > 0.05, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001).</p

<i>Ae</i>. <i>aegypti</i> mosquitoes are attracted to polyamines for egg laying.

<p>(A) Schema of single female mosquito egg-laying assay. Mated, gravid females were given the choice between two small cups containing either pure water or water enriched with different concentrations of polyamines (1 μM to 100 μM). Females were exposed only to the odor of polyamines, and direct contact to polyamines was prevented (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002454#sec014" target="_blank">materials and methods</a>). (B–C) Females were most attracted to 1 and 10 μM of putrescine (purple, upper panels) or cadaverine (green, lower panels). 100 μM polyamine repelled females from laying eggs into the cup. PIs are averaged (<i>n</i> = 8 ± SEM, 1 female/trial). All <i>p</i>-values were calculated via Student’s <i>t</i> test (ns > 0.05, *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01).</p

IR76b is required for the polyamine odor response of IR41a OSNs.

<p>(A) In vivo imaging setup illustration (top). Illustrative confocal image showing the IR41a and IR76b OSN innervating glomeruli pattern (bottom). VC5 is the glomerulus innervated by the polyamine-responding IR41a sensory neurons. (B–D) In vivo calcium imaging of <i>IR41-Gal4;UAS-GCaMP6f</i> flies stimulated with water and 6, 8, and 10 ppm putrescine, respectively. Please note that that based on PID measurements, 10 ppm most closely represented the 1 mM concentration used in behavioral experiments due to the technical differences of odor application. (B) Representative pseudocolor images showing the response to water and increasing doses of putrescine, respectively. (C) Quantification of peak ΔF responses of the VC5 glomerulus (<i>n</i> = 9). (D) Average activity trace of VC5 glomerulus upon polyamine stimulation in %ΔF/F. (E) Prestimulation fluorescence micrograph showing IR76b positive glomeruli. (F–H) In vivo calcium imaging of <i>IR76b-Gal4;UAS-GCaMP6f</i> flies stimulated with water and 6, 8, and 10 ppm putrescine, respectively. (F) Representative pseudocolor images showing the response to increasing doses of putrescine. (G) Quantification of peak ΔF responses of the VC5 glomerulus (<i>n</i> = 6). (H) Average activity trace of VC5 glomerulus in %ΔF/F. (I–K) In vivo calcium imaging of <i>IR76b-Gal4</i>,<i>UAS-GCaMP6f;IR76b</i>^<i>1</i> and heterozygous control flies stimulated with water and 10 ppm putrescine, respectively. (I) Representative pseudocolor images showing the response in the VC5 glomerulus. (J) Quantification of peak ΔF responses in mutant and control flies. Boxes show median and upper/lower quartiles, and whiskers show minimum/maximum values. ***<i>p</i> < 0.001 by unpaired <i>t</i> test with Welch correction (<i>n</i> = 6). (K) Average activity trace of VC5 glomerulus. (D, H, K) The gray column represents the 0.5 second stimulation period. Dark colored line is the average response and the light shade is the standard error of the mean (SEM).</p